program book

Transcription

program book

PROGRAM BOOK
2016
31st Congress of the International
Society for Advancement of Cytometry
June 11 – 15, 2016
Washington State Convention Center
Seattle, Washington, USA
cytoconference.org
isac-net.org
Download the CYTO 2016 Mobile App
To access the CYTO 2016 mobile app online, visit ddut.ch/cyto2016.
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ISAC gratefully acknowledges the following outstanding
sponsors for their generous support of CYTO 2016:
Gold Level _____________________________________________________
Silver Level _____________________________________________________
Bronze Level ___________________________________________________
Mobile App Sponsor
Notepad Sponsor
First Time Attendee
Orientation Sponsor
ISAC Marylou Ingram
Scholars Day Sponsor
ISAC 31st International Congress
Seattle, United States of America
June 11-15, 2016
Dear Colleagues,
On behalf of the CYTO Organizing Committee and ISAC Council, it is our pleasure to welcome you to CYTO 2016,
the 31st Congress of the International Society for Advancement of Cytometry. CYTO 2016 will explore the art and
science of cytometry from Bench to Bedside. Recent developments in flow cytometry, advanced microscopy, data
evaluation and fluorescent reagents enable a new understanding of basic molecular mechanisms and human disease.
With all the exciting advancements in cytometry to explore, we proudly recognize that CYTO is the signature venue
for showcasing the state of the art and science of cytometry.
The CYTO 2016 Program includes elements for scientists working in all areas of cytometry and at all career stages.
The CYTO 2016 Education Program starts on Saturday, June 11, with Scientific Tutorials providing focused updates
on a range of research and clinical topics in flow and image cytometry, as well as shared resource lab (SRL) and core
facility management. Again this year, the tutorials are included in the CYTO 2016 registration fee and are free to all
already registered for CYTO. First-time CYTO attendees are encouraged to attend a welcome and orientation
breakfast Sunday morning.
The CYTO 2016 Scientific Program opens Saturday, June 11 with CYTO Innovation, and the CYTO Innovation
keynote lecture will be given by Brent Gaylord on Advancing the Art and Science of Cytometry through Advanced
Materials. This year’s Hooke Lecture, “Imaging of Induced Pluripotent Stem Cells” will be presented by Rick
Horwitz on Sunday, June 12. This year we are fortunate to have an additional keynote lecture on Tuesday, June 14
given by John Mattick on Regulatory RNAs in Human Development and Cognition. Other scientific highlights will
be the ICCS/ESCCA symposium with several high impact translational talks profiling the application of flow and
imaging techniques to clinical problems, several sessions on exovesicles, and several talks that use the Imaging mass
cytometer introduced last year at CYTO.
The CYTO 2016 scientific program continues with Frontiers and Plenary Sessions featuring cutting edge cytometry
technology and applications, Parallel Sessions with examples of contemporary cytometry from the research lab to the
clinic, and Poster Sessions that provide an opportunity for detailed discussions between authors and delegates. A
diverse program of interactive workshops provides an opportunity for experts and novices alike to discuss and debate
emerging or controversial issues in cytometry. We’d like to thank Peter Lopez, Kathy Muirhead and Joe Tario for all
their hard work in organizing this year’s workshops.
CYTO 2016 will feature educational tracks on Shared Resource Lab management and another on innovation and
entrepreneurship. The SRL track will focus on topics of interest to SRL staff and scientists and includes tutorials,
workshops, posters, and a SRL managers’ forum. On Saturday afternoon, CYTO Innovation spotlights the challenges
and opportunities for the development and commercialization of cell analysis technologies.
The CYTO Commercial Exhibition features more than 60 companies displaying hardware, software, and reagents for
cytometry research. We appreciate the support of all of our exhibitors, and especially our Sponsors, for helping to
make CYTO possible. Help us recognize the many contributions our 2016 award winners have made to cytometry,
science and the Society during the Awards ceremony Wednesday afternoon. CYTO 2016 will end with the Closing
Reception at the Seattle Aquarium on Wednesday, June 15 because all cytometrists appreciate a good party.
We want to express our sincere thanks to the members of the CYTO Organizing Committee and to the 50+ members
of the CYTO Program Committee, including the ISAC Scholars and Shared Resource Lab Emerging Leaders, who
proposed themes and speakers, and assisted with abstract review. Thanks go also to our Course and Tutorial faculty,
Workshop leaders, and Session Chairs for contributing their time and talents. Lastly, we want to thank the FASEB
Office of Scientific Meetings and Conventions team, Kanika Pulliam, ISAC’s former education manager, and
Michelle Butler, ISAC Executive Director, for their tireless work in ensuring that CYTO 2016 is a success.
We encourage all members to participate in ISAC’s efforts to advance cytometry. Please attend the Business Meeting
on Wednesday morning to hear about ISAC’s recent efforts and future plans and share your thoughts on the future of
the Society and cytometry with the ISAC Council and management at any time.
Finally, enjoy CYTO and Seattle!
Paul Wallace
CYTO 2016 Program Chair and ISAC President-Elect
2
Andreas Radbruch
ISAC President
ISAC 2016 Program and Abstracts
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Cytometry Education:
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Featuring Courses, Webinars and Recordings from CYTO
Visit CYTO U Today to Learn More!
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Help Advance Cytometry and Cytometry Education Today by
Donating to ISAC’s Fund for Global Cytometry Education!
ISAC’s Fund for Global Cytometry Education helps further the mission of ISAC and support ISAC’s
educational efforts around the world including:
• CYTO University
• ISAC International Cytometry Workshops
• CYTO Travel Support for Students and
Junior SRL Staff
• ISAC Scholars Program
• FlowRepository
• Shared Resource Lab (SRL) Emerging
Leaders Program
• International Cytometry Certification
Exam (ICCE) Program
• Where Support is Most Needed
Individuals, corporations and foundations who share ISAC’s passion for cytometry education are
encouraged to donate to ISAC’s Fund for Global Cytometry Education. You may choose to support
one of the program areas listed above or designate the donation should go where the support is
most needed. Donations of any size are welcome.
DONATE NOW AT WWW.ISAC-NET.ORG
100% of your donation will be applied to the support area you designate. ISAC will absorb all administrative costs.
By virtue of ISAC’s 501(c)3 status with the IRS, contributions may be tax-deductible for people who pay US taxes.
Thank You to Our Donors
Contributing Donors ($1 to 249)
Robert Auer
Nancy Fisher
Virginia Litwin
Doug Redelman
Claudia Bispo
Robert Hoffman
Richard Meiser
Arturo Vargas
Daniela Damiani
Anthony Huggins
Gerald Pfister
Jelena Vider
Bart Eisfelder
John Kettman
Reif Price
Sustaining Donors ($250 – 499)
Michelle Butler
Eric Chase
Italas George
Paul Wallace
Patron Donors ($1000 – 2499)
Jonni Moore
John Nolan
J. Paul Robinson
And, a special thank you to the Wallace H. Coulter Foundation for
its continuous and generous financial support since 2008!
Donate now at www.isac-net.org! 100 percent of your donation will be applied to the support area you
designate.
ISAC
will
all administrative
By virtue
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Donations
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Table of Contents
Sponsors and Supporters............................................................................................................................................................... 1
Welcome Letter ............................................................................................................................................................................ 2
ISAC 2014 – 2016 Executive Committee and Councilors ............................................................................................................ 14
ISAC Leadership and Congress Organizers ................................................................................................................................. 15
ISAC Committees & Task Forces ................................................................................................................................................. 19
General Information ................................................................................................................................................................... 22
Registration ................................................................................................................................................................. 22
Badges .......................................................................................................................................................................... 22
Bank ............................................................................................................................................................................. 22
Business Center ............................................................................................................................................................ 22
Cell Phones................................................................................................................................................................... 23
Child Care .................................................................................................................................................................... 23
Closing Reception at the Seattle Aquarium ................................................................................................................... 23
Commercial Exhibits – Exhibit Hall Hours .................................................................................................................... 23
Commercial Tutorials ................................................................................................................................................... 23
Companion/Guest Registration..................................................................................................................................... 23
CYTO U Cyber Café .................................................................................................................................................... 23
CMLE ............................................................................................................................................................................ 23
CYTO Innovation ......................................................................................................................................................... 24
Dining Options ............................................................................................................................................................. 24
Disabilities and Special Needs ...................................................................................................................................... 24
Exhibitor Showcase ...................................................................................................................................................... 24
First Aid ....................................................................................................................................................................... 24
ICCE ............................................................................................................................................................................. 24
Internet/Wireless Access .............................................................................................................................................. 24
ISAC Booth ................................................................................................................................................................... 24
Job Board ..................................................................................................................................................................... 24
Mobile APP................................................................................................................................................................... 24
Outstanding Poster Awards .......................................................................................................................................... 25
Poster and Multimedia Presentations ............................................................................................................................ 25
Practical Information for Seattle................................................................................................................................... 25
Recording ..................................................................................................................................................................... 25
Scientific Tutorials ........................................................................................................................................................ 25
Speaker Ready Room .................................................................................................................................................... 25
Transportation .............................................................................................................................................................. 26
City Map ..................................................................................................................................................................................... 27
Washington State Convention Center Floor Plans ....................................................................................................................... 28
Service Location Hours & Telephone Numbers ........................................................................................................................... 31
Committee Meetings ................................................................................................................................................................... 32
Congress Overview ..................................................................................................................................................................... 35
Special Lectures .......................................................................................................................................................................... 38
Daily Program ............................................................................................................................................................................ 40
Saturday, June 11 ......................................................................................................................................................... 40
Sunday, June 12............................................................................................................................................................ 41
Monday, June 13 .......................................................................................................................................................... 45
Tuesday, June 14 .......................................................................................................................................................... 48
Wednesday, June 15 ..................................................................................................................................................... 51
ISAC Marylou Ingram Scholars .................................................................................................................................................. 55
ISAC SRL Emerging Leaders ........................................................................................................................................................ 65
Multimedia & Poster Sessions ..................................................................................................................................................... 69
Commercial Tutorials & Exhibits ................................................................................................................................................. 88
Commercial Tutorials ................................................................................................................................................... 88
Exhibitor Showcase ...................................................................................................................................................... 95
Exhibitor Listing............................................................................................................................................................ 97
Exhibit Hall Floor Plan .................................................................................................................................................. 98
Exhibiting Companies ................................................................................................................................................................. 99
Oral Session Abstracts .............................................................................................................................................................. 109
Poster Session Abstracts............................................................................................................................................................ 158
Speaker/Author Index ............................................................................................................................................................... 267
Program-at-a-Glance ................................................................................................................................................................ 276
ISAC 2014 – 2016 Executive Committee and Councilors
Andreas Radbruch
President
Deutsches RheumaForschungszentrum Berlin
Paul K. Wallace
President-Elect
Roswell Park Cancer Institute
John Nolan
Past President
The Scintillon Institute
Timothy Bushnell
Secretary
University of Rochester
Rachel J. Errington
Treasurer
Cardiff University
Ryan Brinkman
Councilor
British Columbia Cancer
Agency
Joanne Lannigan
Councilor
University of Virginia
Peter A. Lopez
Councilor
New York University
Rui Gardner
Councilor
Instituto Gulbenkian Ciencia
Alfonso Blanco-Fernandez
Councilor
University College of Dublin
Gustavo Rohde
Councilor
Carnegie Mellon University
Monica DeLay
Councilor
Cincinnati Children’s Hospital
Medical Center
David L. Haviland
Councilor
Houston Methodist Hospital
Research Institute
14
Jessica P. Houston
Councilor
New Mexico State University
Adrian Smith
Councilor
University of Sydney
ISAC Leadership and Congress Organizers
ISAC 2014 – 2016 Executive Committee
Andreas Radbruch, President
Deutsches Rheuma-Forschungszentrum Berlin
Paul K. Wallace, President-Elect
John Nolan, Past President
The Scintillon Institute
Timothy Bushnell, Secretary
University of Rochester
Rachel J. Errington, Treasurer
Cardiff University
ISAC 2014 – 2016 Councilors
Ryan Brinkman
British Columbia Cancer Agency
Joanne Lannigan
Peter A. Lopez
New York University
Rui Gardner
Instituto Gulbenkian Ciencia
Gustavo Rohde
Monica DeLay
Cincinnati Children’s Hospital Medical Center
David L. Haviland
Houston Methodist Hospital Research Institute
Jessica P. Houston
Adrian Smith
University of Sydney
CYTO 2016 Organizing Committee
Paul Wallace
Roswell Park Cancer Institute – CYTO 2016 Chair
Bruce Bagwell
Verity Software
Ryan Brinkman
British Columbia Cancer Agency
Timothy Bushnell
University of Rochester Medical Center
Pratip Chattopadhyay
National Institutes of Health
Fiona Craig
University of Pittsburgh Medical Center
Monica DeLay
Cincinnati Children's Hospital Medical Center
Rachel Errington
Cardiff University Dept. of Medical Biochemistry &
Immunology
Rui Gardner
Gulbenkian Institute of Science
Michael Halter
NIST
David Haviland
Houston Methodist Hospital Research Institute
David Hedley
University Health Network Princess Margaret Cancer
Centre
Jessica Houston
Tomáš Kalina
Charles University
Joanne Lannigan
Peter Lopez
New York University
Zofia Maciorowski
Institut Curie
Jonni Moore
University of Pennsylvania
Kathy Muirhead
SciGro, Inc.
Robert Murphy
Alex Nakeff
Caralex
15
John Nolan
Scintillon Institute
Michele Black
University of Washington
Frederic Preffer
Massachusetts General Hospital
Alfonso Blanco Fernandez
University College Dublin
Kylie Price
Malaghan Institute
Marcel Bruchez
Carnegie Mello University
Andreas Radbruch
Deutsches Rheuma-Forschungszentrum Berlin
Hyun-Dong Chang
DRFZ
Diether Recktenwald
Desatoya LLC
Sung Hwn Cho
NanoCellect Biomedical Inc.
J. Paul Robinson
Purdue University
Grace Chojnowski
QIMR Berghofer
Mario Roederer
National Institutes of Health
Andrea Cossarizza
University of Modena and Reggio Emilia
Gustavo Rohde
Scott Cram
Bioconsultants Ltd
Vincent Shankey
Shankey Biotechnology Consulting
Benjamin Daniel
University of Texas Health Science Center
Adrian Smith
University of Sydney Centenary Institute
Derek Davies
The Francis Crick Institute
Paul Smith
Oncotherics
Gelo dela Cruz
Danish Stem Cell Center
Joseph Tario
Monica DeLay
Cincinnati Children's Hospital
Alan Waggoner
Jaroslav Dolezel
Institute of Experimental Botany
Carolina Wählby
Broad Institute
Albert Donnenberg
University of Pittsburgh
CYTO 2016 Program Committee
Vera Donnenberg
University of Pittsburgh
Mehrnoosh Abshari
NIDCR, NIH
Donat Alpar
CeMM
Alireza Ardjmand
The University of Newcastle
Kewal Asosingh
Cleveland Clinic
Jessica Back
Wayne State University/Karmanos Cancer institute
Anna Belkina
Boston University School of Medicine
16
Ryan Duggan
University of Chicago
Bruce Edwards
University of New Mexico
Rachel Errington
Cardiff University
Andrew Filby
Newcastle University
Greg Finak
Fred Hutchinson Cancer Research Center
Nancy Fisher
University of North Carolina
David Galbraith
University of Arizona
Virginia Litwin
Covance
Rui Gardner
Instituto Gulbenkian de Ciencia
Er Liu
Roche Tissue Diagnostics
David Gebhard
Pfizer
Stephen Lockett
National Cancer Institute
Michael Gregory
New York University Medical Center
Yiqing Lu
Macquarie University
David Haviland
Methodist Hospital Research Institute
Hervé Luche
CIPHE
Ann Hoffman
GlaxoSmithKline
Enrico Lugli
Humanitas Clinical and Research Center
Jeannine Holden
Beckman Coulter
Orla Maguire
Jessica Houston
Brian McFarlin
University of North Texas
Xu Huang
University of Glasgow
Hans Minderman
Ruud Hulspas
Cytonome
Jonni Moore
University of Pennsylvania
Jonathan Irish
Vanderbilt University
Barry Moran
Trinity College Dublin
James Jacobberger
Case Western Reserve University
Elisa Nemes
University of Cape Town
Tomáš Kalina
Charles University Hospital
Raluca Niesner
Deutsches Rheuma-Forschungszentrum Berlin (DRFZ)
Pia Kvistborg
Netherlands Cancer Institute
John Nolan
Claude Lambert
University Hospital of Saint-Etienne
Katarzyna Piwocka
Nencki Institute of Experimental Biology
Nina Lane
Fluidigm
Kylie Price
Malaghan Institute of Medical Research
Anis Larbi
Singapore Immunology Network
Andreas Radbruch
Deutsches Rheuma-Forschungszentrum Berlin (DRFZ)
Thomas Laurell
Lund University
Diether Recktenwald
Desatoya LLC
James Leary
Retired
J. Paul Robinson
Purdue University
Silas Leavesley
University of South Alabama
Robert Salomon
Garvan Institute
Michael Lewis
University of Vermont
Frank Schildberg
Harvard Medical School
17
Paul Smith
Cardiff University
Paul Wallace
Josef Spidlen
BC Cancer Agency
Qianjun Zhang
Emerald Bio
Janos Szollosi
University of Debrecen
Michael Zordan
Sony Biotechnology Inc
Attila Tarnok
University of Leipzig
Robert Zucker
USEPA
Laura Teodori
ENEA
ISAC Executive Office
Gergely Toldi
Semmelweis University
Valery Tuchin
Saratov State University
Giacomo Vacca
Kinetic River Corp
Gyorgy Vereb
University of Debrecen
Michelle Butler, Executive Director
9650 Rockville Pike
Bethesda, MD 20814 USA
Tel. 301-634-7454, [email protected]
CYTO Congress Management
Marcella Jackson, Director
Roya Jaseb, Senior Meetings Manager
Janet Kearney, Exhibits Manager
Ying Zhu, Meetings Coordinator
Joni Friedman, Exhibits Coordinator
Josie Leftwich, Registrar
Tel. 301-634-7010, [email protected]
Rachael Walker
Babraham Institute
18
ISAC Committees & Task Forces
Task Forces & Committees
2014 – 2016
Awards Committee
John Nolan, Chair
J. Paul Robinson
Paul Smith
János Szöllõsi
Andreas Radbruch, (ex officio)
Paul Wallace, (ex officio)
Kewal Asosingh
Pratip Chattopadhyay
Awtar Krishan
Peter Lopez
Jonni S. Moore
Gustavo Rohde
Elearning Delivery Task Force
Kevin L. Holmes, Chairmen
Stephen P. Perfetto, Co-Chair
Ben Fontes
Phil Hogarth
Richard F. Konz
Simon Monard
Hank Pletcher
Ingrid Schmid
Robert Wadleym
Pratip Chattopadhyay, Co-Leader
Kewal Asosingh, Co-Leader
Anna Belkina
Ryan Brinkman
Andrew Filby
Michael Gregory
Zosia Maciorowski
Mike Ormerod
Robert Salomon
Joseph Tario
Jennifer Wilshire
Certification Advisory Committee
Flow Cytometry Content Task Force
Bruce Greig, Chair
Joanne Lannigan, Vice Chair
Gelo Dela Cruz
Mike Keeney
Peter Lopez
Mara Neal
Teri Oldaker
Elizabeth Stone
Carina Torres
Jonni Moore, Leader
Grace Chojnowski
Derek Davies
Peter Lopez
Steve McClellan
Phil McCoy
Joe Trotter
Paul Wallace
Jennifer Wilshire
Council of ISAC Associated Societies
Flow Cytometry Data Standards Task
Force
Biosafety Committee
Andrea Cossariza, Chair
Hyun-Dong Chang
Tomáš Kalina
Peter Lopez
Enrique O'Connor
Adrian Smith
Janos Szollosi
CYTO Innovation Steering Committee
Dietner Recktenwald, Chair
Nima Aghaeepour
Jonni S. Moore
Betsy Ohlsson-Wilhelm
Allen Poirson
Paul Smith
Alan Waggoner
Andreas Padbruch, (ex officio)
Education
Zosia Maciorowski, Co-Chair
John Nolan, Co-Chair
Ryan Brinkman, Chair
Jay Almarode
Ernie Anderson
Kim RM Blenman
Chris Bray
Martin Buscher
James Cavenaugh
Michael Goldberg
Phil Hexley
Bill Hyun
David Kripal
Robert Leif
Wayne Moore
David Novo
David Parks
Maciej Simm
Josef Spidlen
Adam Treister
Jim Wood
Michael Zordan
19
Flow Repository Steering Committee
Membership Services
John Nolan, Chair
Ryan Brinkman
Rachel Errington
Jeannine Holden
Wayne Moore
Mario Roederer
Peter Lopez, Co-Chair
Rachael Walker, Co-Chair
David Haviland
Sung Hwan Cho
Rui Gardner
Frank Schildberg
Adrian Smith
Finance
Rachel Errington, Chair
Nima Aghaeepour
Tim Bushnell
Andrea Cossarizza
Monica Delay
Nominating Committee
Image Cytometry Content Task Force
Shared Resource Lab (SRL) Content Task
Force
Gustavo Rohde, Leader
Rachel Errington
Andrew Filby
Michael Halter
Silas Leavesley
Stephen Lockett
Raluca Niesner
John Nolan
Anil Parwani
Bartek Rawja
Shantanu Singh
Karen Thickman
Gyorgy Vereb
ISAC Scholars Program Committee
Alex Nakeff, Chair
J. Paul Robinson, Co-Chair
Stephen Lockett
Mara Neal
Mario Roederer
Live Education Delivery Task Force
Awtar Krishan, Leader
Gulderen Yanikkaya Demirel
Paresh Jain
Tomáš Kalina
H. Krishnamurthy
Zosia Maciorowski
Kovit Pattanapanyasat
Alan Saluk
Vivek Tanavde
Bill Telford
Qianjun Zhang
20
John Nolan, Chair
Ryan Brinkman
Rachel Errington
Joanne Lannigan
Peter Lopez, Chair
Bridget McLaughlin
Patricia Simms
Adrian Smith
Shared Resource Lab (SRL) Services
Oversight Committee
Rui Gardner, Chair
Tim Bushnell
Monica DeLay
David Haviland
Joanne Lannigan
Peter Lopez
Adrian Smith
Paul Wallace
Shared Resource Lab (SRL) Task Force
Joanne Lannigan, Chair
Lora Barsky
Michele Black
Matt Chochrane
Ben Daniel
Monica DeLay
Michael Gregory
Rui Gardner
Desiree Kunkel
James Marvin
Rob Salomon
Carina Torres
Rachael Walker
Scientific Communications Committee
Jessica Houston, Chair
Enrico Lugli
John Nolan
Bartek Rajwa
T. Vince Shankey
Visit the Exhibits & Posters
Sunday, June 12
1600 – 2000
1800 – 2000
Authors Must Place Posters on Assigned Boards
Commercial Exhibits Open
Monday, June 13
800 – 1930
Poster Viewing
1030 – 1100
Coffee Break
1030 – 1930
Commercial Exhibits
1800 – 1900
Poster Session I (Authors of odd numbered boards present)
1830 – 1930
Happy Hour
1830 – 1930
Exhibitor Showcase
Tuesday, June 14
800 – 2000
Poster Viewing
1030 – 1100
Coffee Break
1030 – 2000
Commercial Exhibits
1830 – 1930
Poster Session II (Authors of even numbered boards present)
1900 – 2000
Happy Hour
1900 – 2000
Exhibitor Showcase
Wednesday, June 15
800 – 1415
Poster Viewing
1030 – 1100
Coffee Break
1030 – 1430
Commercial Exhibits
1100 – 1200
Poster Session III (consists of Poster Highlights Tour)
Posters must be removed by 1430 on Wednesday, June 15, 2016.
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General Information
All Congress activities will be held at the Washington State Convention Center (WSCC) and the
Conference Center (TCC) located at 800 Convention Place, Seattle, WA 98101-2350, unless
noted otherwise.
How to Get to TCC Building from WSCC Building?
To access session rooms in the TCC building, you must enter the WSCC building first,
go up to Level 4 to pick up your attendee badge. Enter the Exhibit Hall 4EF next to
Registration, and follow sign to escalator for The Conference Center (TCC) building.
Go down the escalator and you will enter TCC on the Tahoma Level. The Tahoma,
Chelan, Yakima, Skagit rooms are located in the TCC building. To access the TCC
building directly from 8th Avenue or Pike Street, be sure to have your attendee badge.
Participation in CYTO 2016 is limited to registered
delegates. Full congress registration includes
admission to all CYTO sessions such as workshops,
parallels, plenaries, frontiers, state of the arts, Hooke
Lecture, CYTO Innovation, scientific tutorials,
commercial exhibits, poster sessions, commercial
tutorials, refreshment breaks, happy hours, opening
reception and the closing reception.
Congress Management Staff will be located at the
Registration Desk in Hall 4D Skybridge, WSCC Level
4. If you need assistance, please visit the Registration
Desk or call 206-219-4501.
Congress Registration – Hall 4D
Skybridge, WSCC Level 4
Registration for CYTO 2016 will be open during the
following days and hours:
Friday, June 10 ..................................... 1200 – 1700
Saturday, June 11 ................................... 700 – 1830
Sunday, June 12 ..................................... 700 – 1830
Monday, June 13 .................................... 730 – 1830
Tuesday, June 14 .................................... 730 – 1830
Wednesday, June 15............................... 730 – 1700
Refund Policy: No refunds will be issued after May 4,
2016.
Exhibitor Registration – Hall 4D
Skybridge, WSCC Level 4
Exhibitor registration provides admittance into the
Exhibit Hall only. Exhibitor registration will be open
during the following days and hours:
Friday, June 10 ..................................... 800 – 1700
Saturday, June 11 ................................. 800 – 1700
Sunday, June 12 ................................... 800 – 1930
Monday, June 13 .................................. 930 – 1900
Tuesday, June 14 .................................. 930 – 1900
Wednesday, June 15............................. 900 – 1430
22
Badges
Participation in CYTO 2016 is limited to registered
attendees. The official badge is required for
admittance to all sessions, social activities and the
Exhibit Hall. A fee may be charged to reissue lost or
misplaced badges. Please do not place a business
card into the badge holder as identification. If there is
an error on a badge, please have it corrected at the
registration desk.
Bank
Two Automated Teller Machines (ATM) are
conveniently located inside the Washington State
Convention Center for cash withdrawals. You will find
an ATM on Level 1 at the base of the South Galleria
escalators, next to the Massage Bar, and on Level 4
near Tougo’s Coffee. Please check with your local
financial institution to make sure that your card will
function properly abroad.
Business Center
FedEx Office in the Washington State Convention
Center is a full-service business center, offering FedEx
shipping, high-speed duplication, binding, posters, fax
service, lamination, Internet access and free pick-up
and delivery, etc. E-mail your documentation
preparation requests right from your PC. FedEx Office
is located on Level 1 of the WSCC. The FedEx office
opens Monday – Thursday, 700 – 2200, Friday 700 –
2100, and Saturday – Sunday, 900 – 1800. If you
have any questions about the service provided, please
contact the office at 206-467-1767 or
[email protected].
Cell Phones
Please turn off all your cell phone (and other
electronic devices) or turn it to silent (not vibrate)
mode prior to the start of a session. If you must leave
a session early, please use the rear entrance and exit
quietly.
Child Care
Please check with your hotel’s front desk or concierge
service for names of babysitters who can provide care
in your hotel room. Parents and guardians are
required to perform their own reference checks and
arrange child care independently. ISAC is not
responsible for child care or the quality of care
provided.
Closing Reception at the Seattle
Aquarium
The Closing Reception will be held at the Seattle
Aquarium on Wednesday, June 15, 2016, from 1900
to 2300. The Seattle Aquarium is the ninth largest
aquarium in the U.S. by attendance and among the
top five paid visitor attractions in the Puget Sound
region. The region’s premier resource for hands-on
marine experiences and conservation education, the
Seattle Aquarium offers fun and exciting ways to
discover more about the amazing Puget Sound and
our world’s one big ocean and it is a perfect venue for
CYTO attendees to celebrate the last evening of the
Congress. Come enjoy the DJ, dancing, and good
times!
Full Congress registration includes an exchange
coupon for the Closing Reception at the Seattle
Aquarium. The Closing Reception coupon must be
exchanged for an actual ticket on-site at the CYTO
registration desk beginning Friday, June 10, 2016,
until Sunday, June 12, 2016. Coupons will be
exchanged on a first come, first served basis until
maximum capacity is reached. A ticket is required for
admittance to the reception and must be presented at
the entrance. Food will be provided at the event and
each attendee will receive 2 tickets redeemable for a
complimentary beverage (spirits, wine, beer, soda).
Transportation will not be provided. The Seattle
Aquarium is only 0.8 miles from the Washington State
Convention Center. For directions, please visit
http://www.seattleaquarium.org/directions. Be sure to
exchange your coupon early so you don’t miss out on
all the fun!
Commercial Exhibits – Exhibit Hall 4EF,
WSCC Level 4
Visit the commercial exhibits featuring displays by
leading suppliers and vendors. A complete directory
of exhibiting companies as well as the Exhibit Hall
floor plan is located under the Exhibits tab of this
program.
Exhibits will be open during the following days and
hours:
Sunday, June 12.................................... 1030 – 1930
Monday, June 13 .................................. 1800 – 2000
Tuesday, June 14 .................................. 1030 – 2000
Wednesday, June 15 ............................. 1030 – 1430
Note: Children under the age of 16 are not permitted
in the Exhibit Hall without parent or guardian
supervision.
Commercial Tutorials
Eighteen (18) commercial tutorial sessions are offered
from 1245 – 1345 on Sunday, June 12, Monday, June
13, Tuesday, June 14, and Wednesday, June 15.
Please refer to the Commercial Tutorial tab of this
program for a complete list of offerings.
Companion/Guest Registration
Registered attendees of CYTO 2016 may sign up a
spouse/guest as a Companion for$150 USD.
Companion registration allows entrance to the
Opening Reception, Happy Hours and the Closing
Reception only. The Opening Reception is scheduled
to be held Sunday, June 12, 2016, and the Closing
Reception on Wednesday, June 15, 2016.
Companion registrants are not permitted in the session
rooms or the Exhibit Hall at any other time.
CYTO U Cyber Café
For your convenience, ISAC has set up several
computers with free Internet access in the CYTO U
Cyber Café. Attendees may use computers to browse
the Internet and/or to check email or print boarding
passes. In consideration of others, please limit your
use to 15 minutes and printing to 2 pages. The CYTO
U Cyber Café will be open during the Commercial
Exhibit hours in the Exhibit Hall for attendee use
beginning Sunday, June 12.
CMLE
This continuing medical laboratory education activity
is recognized by the American Society for Clinical
Pathology as meeting the criteria for 31.5 hours of
CMLE credit. ASCP CMLE credit hours are acceptable
to meet the continuing education requirement for the
ASCP Board of Registry Certification Maintenance
Program.
If you’re interested in earning CMLE credits, please
follow these steps:
1. Be sure to add your name to the sign-in sheet
which will be located at the back of each
session room (sign in sheets will be present in
every session that is eligible to earn CMLE
credits);
2. Complete an evaluation form for each session
you attend and leave it in the box at the back
of the room, immediately following that
session;
23
3. Use the CMLE form available in the session
rooms and the Registration Desk to track the
sessions you attend. Please follow the
instructions on the form to finalize the
process. You will need to drop off your
completed form at the Registration Desk
before leaving the Congress.
Disabilities and Special Needs
If you have a disability or special need that may have
an impact on your participation in the meeting, please
contact the Congress Management Staff at the
Registration Desk. ISAC cannot ensure the availability
of appropriate accommodations without prior
notification of need.
CMLE certificates will be issued by the ISAC Executive
Office upon request via email to [email protected].
Exhibitor Showcase – Exhibit Hall 4EF,
WSCC Level 4
CYTO Innovation
CYTO 2016 will feature four Exhibitor Showcases on
Monday, June 13, from1830 – 1930, and Tuesday,
June 14, from 1900 – 2000. Each Exhibitor Showcase
includes presentations by several exhibiting
companies.
The advancement of cytometry depends on the
translation of innovative research into useful,
accessible tools that can catalyze new biological
understanding. CYTO Innovation is the forum within
ISAC that explores the challenges and opportunities
for translation of new cell analysis technologies into
commercially viable products and services. CYTO
Innovation provides: an opportunity to learn about the
translation of ideas and discoveries into products and
knowledge; the skills required for progressing from a
research finding to useful products with impact; a
unique platform for showcasing innovation in relevant
industry sectors; and engagement with a
knowledgeable network of entrepreneurs, executives
and other business people. CYTO Innovation is part of
ISAC’s wider mission of advancing cytometry and
aims to serve members and Congress attendees either
engaged in or contemplating the translation of
research into high impact products and services. It is
open to Full Congress Registrants; no additional fees
apply. See page 41 for a full description of CYTO
Innovation 2016.
Dining Options
Concession stand in the back of the Exhibit Hall 4EF
will be open Monday – Wednesday from 1030 – 1430
with a variety of cold and hot foods and refreshments
for purchase. Drink vending machines are available
at various locations throughout the WSCC. There are
several restaurants and coffee shops located in the
WSCC. Espresso Caffé Dior, Taco Del Mar, and
Goldbergs’ 2 Go are located on Level 1 of the WSCC.
The Juicy Café is located on Level 2. Subway and
Tungo Coffee are located on Level 4.
Seattle offers one of America's true regional cuisines,
rooted in fresh seafood, an abundance of small-scale
producers and purveyors, great local wines and an
ever-growing pool of talented chefs. There are
Northwestern-style seafood restaurants and
steakhouses, but also restaurants that draw on
influences from Asia, Latin America, France, Italy, and
beyond. For a list of great places to dine and to make
reservations, visit the Visit Seattle’s website at
www.visitseattle.org/food-drink/
24
First Aid
A First Aid room is located in the back of the Exhibit
Hall 4E. The room is staffed with First Aid
Administrators trained in First Aid Response. They
may be reached by phone at 206-694-5127.
ICCE
ISAC is an approved provider of continuing education
for the ICCE certification. Any one hour of ISAC
educational programming is worth one credit. CYTO
2016 – The 31st Congress of the International Society
for Advancement of Cytometry, is worth 31.5 ICCE
continuing education credits. For more information
on the International Cytometry Certification
Examination and how to become a certified
cytometrist, visit http://cytometrycertification.org.
Attendees can pick up their certificate of attendance
that reflects ICCE credits at registration.
Internet/Wireless Access
Complimentary WIFI in meeting rooms and the
Exhibit Hall. To connect to the Wi-Fi please log into
the “CYTO2016” network and use the password
“CYTOMETRY”.
ISAC Booth
Please visit the ISAC booth to learn more about
society activities and membership.
Job Board
Attendees and exhibitors can post jobs on the job
board which is located in the back of the Exhibit Hall
4EF.
Mobile APP
Download the CYTO 2016 mobile app via
iPhone/iPad and Android native apps or via
Blackberry, Windows Phone, and your desktop
through mobile web. To access the CYTO 2016
mobile site go to ddut.ch/cyto2016.
Outstanding Poster Awards
All poster presenters who are students or postdoctoral
researchers (who have received their doctorate within
the last five years) are eligible. The posters will be
judged at the time they are scheduled to be presented
by the author. Those posters not attended at their
scheduled time will not be considered. The names of
authors selected for this award will be posted on the
mobile app as well as the Message/Announcement
Board in the Registration Area on the morning of
Wednesday, June 15. Poster winners will be
presented a prize and recognized at the Awards
Ceremony from 1630 – 1730 on Wednesday, June 15.
Poster & Multimedia Presentations –
Exhibit Hall 4EF, WSCC Level 4
Over 300 poster presentations will be on display in
the Exhibit Hall. Please refer to the Poster Board Map
on page 98 for the assigned location of presentations.
Please refer to the schedule below for viewing hours.
Sunday, June 12
1600 – 2000
Monday, June 13
800 – 1930
1800 – 1900
Tuesday, June 14
800 – 2000
1830 – 1930
Wednesday, June 15
800 – 1415
1100 – 1200
Authors must set up posters
on assigned board
Poster Viewing
Poster Session 1 (Authors of
odd numbered boards must
be present at their poster to
answer questions and
discuss their presentation)
Poster Viewing
Poster Session 2 (Authors of
even numbered boards must
be present at their poster to
answer questions and
discuss their presentation.)
Poster Viewing
Poster Session 3 (consists of
Poster Highlights Tour)
Practical Information for Seattle
Banking and Foreign Exchange
The official currency in the United States is US
Dollars. The US Dollar ($) is divided into 100 cents;
notes are in denominations of $100, 50, 20, 10, 5,
and 1. Banking hours in the United States are
generally Monday through Friday, 900 – 1700. A few
banks are open on Saturdays as well. You can
withdraw money from ATM bank machines using
your credit card and make purchases in stores and
restaurants.
ATMs are located on level 1 by the down escalator
and on level 4 next to Café Tuago of the Washington
State Convention Center.
Climate
June is a moderately warm month with relatively little
rain in Seattle. Seattle's average daily high
temperature is between 68 and 73 degrees fahrenheit
(20 to 23 degrees celsius) in June with low
temperatures between 52 and 54 °F (11 to 12 °C).
Dialing Codes
The USA country code is +001, while Seattle
landlines start with a 206 area code, followed by a 7
digit number.
Electricity
Electric power is standardized in all states across the
USA. It is set at 110 Volts and 60 cycles. If you bring
any electrical appliance to the USA, you may need an
adaptor to fit the US electrical receptacles. You may
also need a converter to change the voltage from 110
volts to 220 volts.
Language
The official language of the Congress is English.
Translation services will not be provided.
Time Zone
Seattle and the Pacific Northwest are in the Pacific
Daylight Time (PDT) Zone.
Recording
Recording any presentation or session (oral or poster)
by any means including audio taping or videotaping is
prohibited, except by an ISAC authorized agent for
official purposes or by first authors who want to
record their own poster presentations.
Scientific Tutorials
The first day of the Congress is dedicated to the
scientific tutorial program and CYTO Innovation.
Fourteen ninety minute tutorials will be offered.
Admission to Scientific Tutorials is included in CYTO
2016 full congress registration.
Speaker Ready Room – Room 400, WSCC
Level 4
Presenters are encouraged to test their presentations
in the speaker ready room 6 hours prior to their
session. Speakers must arrive in the session room 30
minutes prior to the scheduled start of their session
to allow the operator time to load their presentation
onto the computer. The operator will be seated at the
table next to the stage. ISAC is not responsible for
slides, laptops, or cables left in session rooms.
Speakers are not required to bring a laptop. Session
rooms will be equipped with a data projector, screen,
and a PC computer with open USB ports for flash
25
drives, a laser pointer, as well as a lecture
microphone, table microphone and aisle microphone.
Transportation
By Air
Seattle-Tacoma International Airport, or "Sea-Tac," is
located in the city's southern suburbs. Domestically
it's a major hub for Northwest and West Coast
destinations; internationally, it handles heavily
traveled trans-Pacific routes as well as direct flights to
the major European airports and to Dubai, United
Arab Emirates. Sea-Tac is less than 15 miles from
downtown Seattle. For information on passenger
services, flight status, and news for travelers, visit the
airport’s official website at www.portseattle.org/SeaTac.
By Rail
Amtrak operates daily train service into Seattle from
various cities all over the United States. The King
Street Station is located between South King and
South Jackson Streets and Second and Fourth Avenues
South in the Pioneer Square neighborhood of Seattle,
the station is just south of downtown. It is served by
Amtrak Cascades, Empire Builder and Coast Starlight
trains, and by Sound Transit's Sounder commuter
train. King Street Station is also the Seattle terminus
for the Rocky Mountaineer's luxury excursion train,
the Coastal Passage.
By Road
Seattle is easily accessible via extensive road and
highway network. Washington State Department of
Transportation provides up to date traffic and road
work information. http://www.wsdot.com/traffic
Getting Around in Seattle
Seattle is easy to navigate with a wide variety of smart
transportation options.
Link Light Rail
Link Light Rail makes trips from the airport station to
downtown Seattle between 500 and 100, Monday
through Saturday (the last train departs the airport at
1210), and from 500 to midnight on Sunday (the last
train departs the airport at 2305). Trains arrive every 7
to 15 minutes, depending on the time of day, and take
about 40 minutes to travel between Sea-Tac
International Airport and Westlake Station in
downtown Seattle. One-way fare for adults ranges
from $2 to $3. Schedules and station maps are
available on Sound Transit website at
www.soundtransit.org/Schedules.
26
King County Metro Transit
King County Metro Transit provides bus service in
downtown Seattle and outlying neighborhoods in
King county. Metro buses no longer serve Sea-Tac
Airport. Time-tables and route maps are available at
the ground transportation information booth on the
third floor of the airport parking garage, near Island 2
at the airport, at the Transit Information Center in the
tunnel under Westlake Center at 4th Avenue & Pine
Street, or can be found on the King County Metro
Transit website at http://tripplanner.kingcounty.gov/.
South Lake Union Streetcar
The South Lake Union Streetcar makes 11 stops
through the South Lake Union area. Streetcars arrive
every 10-15 minutes and run from 600 to 2100
(Monday-Thursday), 600 to 2300 (Friday & Saturday),
and 1000 to 1900 (Sunday & holidays). Adult fare is
about $2.50. Schedules and maps are available
www.seattlestreetcar.org.
Seattle Center Monorail
The iconic Seattle Center Monorail travels between
Westlake Center in downtown and Seattle Center at
the base of Queen Anne hill. Regular operating hours
are between 730 and 2300, Monday-Friday, and from
830 to 2300, Saturday & Sunday. Trains depart every
10 minutes and the trip takes only 2 minutes each
way. One-way fare for adults is about $2.25; tickets
are cash-only. Schedules can be found at
www.seattlemonorail.com.
Taxis, Limos and Ride Sharing
At Sea-Tac Airport, taxis are available on the third
floor of the parking garage. One-way rides between
the airport and downtown range from $40-$55. To
arrange for a limo, town car or taxi ride in advance,
use any of the travelers’ information boards in
baggage claim or visit the ground transportation
information booth on the third floor of the parking
garage. In downtown Seattle, ride-sharing companies,
such as Uber, Lyft & Sidecar, offer in-city
transportation from local drivers. All rides can be
scheduled using smartphone apps.
Seattle Travel and Tourism
Seattle is a city of many neighborhoods: Eclectic,
urban, outdoorsy, artsy, gritty, down-to-earth, or
posh—it's all here, from the quirky character of the
Seattle Waterfront and the eccentric "Republic of
Fremont," to hipsters walking baby carriages past
aging mansions on Capitol Hill. There's something for
just about everyone within this vibrant Emerald City.
For Seattle tourism information, please visit
www.visitseattle.org.
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Pike Place Market to CenturyLink Event Center: 1 mi / 1.6 km
Pike Place Market to Space Needle: 1 mi / 1.6 km
Pike Place Market to Convention Center: ½ mi / 800 m
Royal Brougham Way
MAJOR ATTRACTION
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E. Mercer St.
Westlake Ave. N.
6th Ave. N.
EMP MUSEUM
9th Ave. N.
Republican St.
E. Roy St.
Harrison St.
Taylor Ave. N.
CHIHULY
GARDEN
& GLASS
PACIFIC
SCIENCE
CENTER
W.John St.
Dexter Ave. N.
SEATTLE
CENTER
5th Ave. N.
W. Thomas St.
MOHAI
LAKE
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UNION
Mercer St.
BILL & MELINDA
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Queen Anne Ave. N.
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Aurora Ave. N.
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W. Republican St.
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4th Ave. N.
2nd Ave. N.
1st Ave. W.
Warren Ave. N.
1st Ave. N.
2nd Ave. W.
W.Roy St.
Aloha St.
Valley St.
SEATTLE CENTER MONORAIL
INFORMATION CENTER
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How to Get to TCC Building from WSCC Building?
To access session rooms in the TCC building, you must enter the WSCC building first, go up to Level 4
to pick up your attendee badge. &nter the Exhibit Hall 4EF next to Registration, and follow sign to escalator for The Conference Center (TCC) building. Go down the escalator and you will enter TCC on
the Tahoma Level. The Tahoma, Chelan, Yakima, Skagit rooms are located in the TCC building. To
access the TCC building directly from 8th Avenue or Pike Street, be sure to have your attendee badge.
LEVEL 6
Escalator
To/From TCC
LEVEL 5
Hall 4C
Exhibit Hall 4EF
SKYBRIDGE
LEVEL 4
LEVEL 4
TCC Meeting Rooms
LEVEL 3
TAHOMA LEVEL 3
CHELAN LEVEL 2
LEVEL 2
8 AVENUE
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CYTO 2016 Registration and
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The Conference Center
Skagit Lower Level
Yakima Level One
Chelan Level Two
2
3
To/From
Level 1
5
2
4
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To/From
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W
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To/From
Level 3
2
1
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Level 1
2
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Tahoma Level Three
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3
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2
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5
Service Locations & Telephone Numbers
Congress Registration & Congress Management
Hall 4D Skybridge, WSCC Level 4
Phone: (206) 219-4501
Friday, June 10
Saturday, June 11
1200 – 1700
700 – 1830
Sunday, June 12
Monday, June 13
Tuesday, June 14
700 – 1830
730 – 1830
730 – 1830
Wednesday, June 15
730 – 1700
Exhibitor Registration & Information
Phone: (206) 219-4501
Friday, June 10
Saturday, June 11
800 – 1700
800 – 1700
Sunday, June 12
Monday, June 13
Tuesday, June 14
800 – 1930
930 – 1900
930 – 1900
Wednesday, June 15
900 – 1430
Exhibit Management Desk
Phone: (206) 219-4502
Speaker-Ready Room
Room 400, WSCC Level 4
Phone (206) 219-4503
Friday, June 10
1200 – 1700
Saturday, June 11
Sunday, June 12
Monday, June 13
730 – 1700
730 – 1700
730 – 1700
Tuesday, June 14
Wednesday, June 15
730 – 1700
730 – 1700
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31
Committee Meetings
All meetings and events are by invitation only and held at the Washington State Convention
Center (WSCC) and the Conference Center (TCC) unless specified otherwise.
Friday, June 10
ISAC Marylou Ingram Scholars Day
700 – 2000
Madison Ballroom A, Renaissance Hotel
Saturday, June 11
ISAC Council Meeting
715 – 915
Vista Room, Renaissance Hotel
ISAC Marylou Ingram Scholar Program Committee Meeting
930 – 1030
Cytometry Part A Editorial Board Meeting
1200 – 1330
Sunday, June 12
Scientific Communications Committee
700 – 800
Yakima 2, TCC Level 1
Associated Societies Luncheon
1245 – 1400
Chelan 2, TCC Level 2
Biosafety Committee Meeting
1245 – 1400
Skagit 1, TCC Lower Level
Live Education Task Force
1245 – 1400
Finance Committee
1245 – 1400
Monday, June 13
Membership Services Committee Meeting
730 – 830
730 – 830
32
Flow Repository Steering Committee
730 – 830
Flow Cytometry Data Standards
1245 – 1345
Tuesday, June 14
SRL Services Oversight Committee
730 – 830
Education Committee
730 – 830
ISAC Marylou Ingram Scholars and ISAC Shared Resource Lab (SRL) Emerging Leaders Luncheon
1245 – 1400
SRL Content Task Force
1245 – 1400
CYTO Innovation Steering Committee
1245 – 1400
Wednesday, June 15
SRL Task Force
715 – 815
Joint Flow Cytometry Content Task Force and ELearning Delivery Task Force Meeting
715 – 815
CYTO 2017 Planning Meeting
1245 – 1400
33
Closing Reception
Seattle Aquarium
Wednesday, June 15 from 1900 – 2300
The Closing Reception coupon must be exchanged for an actual
ticket at the CYTO registration desk beginning Friday, June 10, 2016
until Sunday, June 12, 2016. Coupons will be exchanged on a first
come, first served basis until maximum capacity is reached. A ticket
is required for admittance to the reception and must be presented at
the entrance. Food will be provided at the event and each attendee
will receive 2 tickets redeemable for a complimentary beverage
(spirits, wine, beer, soda). The Seattle Aquarium is located just 15
minutes walking distance from the Washington State Convention
Center. Transportation will not be provided. For directions, please visit
www.seattleaquarium.org/directions.
Be sure to exchange your coupon early so you
don’t miss out on all the fun!
Congress
Overview
Congress Overview
All Congress activities will be held at the Washington State Convention Center and the Conference Center unless
noted otherwise.
ISAC Marylou Ingram Scholars Day
Exhibitor Registration
Scientific Registration
Renaissance Hotel
Saturday, June 11, 2016
ICCE Exam
Scientific Tutorial Sessions 6-10
1345 – 1515
1530 – 1915
1650 – 1700
CYTO Innovation and Keynote Lecture
Coffee Break
Sunday, June 12, 2016
1415 – 1530
Concurrent Workshops 1-5
1530 – 1545
Coffee Break
1545 – 1700
1715 – 1815
1600 – 2000
1800 – 2000
1830 – 1930
Robert Hooke Lecture
Poster Set-up
Commercial Exhibits
Opening Reception
President's Award for Excellence
Presentations
35
Index
1245 – 1345
Hall 4C, WSCC Level 4
Tahoma 1-5, TCC Level 3,
and Skagit 4, TCC Lower Level
Speaker/Author
Poster Viewing
Frontiers Session 1
Coffee Break
Commercial Exhibits
Concurrent Parallel Sessions 6-10
Poster Session
Abstracts
730 – 1830
800 – 1930
900 – 1030
930 – 1900
1030 – 1100
1030 – 1930
1100 – 1230
Oral Session
Abstracts
Monday, June 13, 2016
Commercial
Tutorials &
Exhibits
Scholars &
Emerging
Leaders
1245 – 1345
Tahoma 4, TCC Level 3
Hall 4C Foyer, WSCC Level 4
Tahoma Foyer, TCC Level 3
and Skagit Foyer, TCC Lower Level
Poster
Session
First Time Attendee Orientation
Opening Remarks and State of the Art Lectures
Coffee Break
Wednesday
15 June
700 – 1830
800 – 1930
745 – 845
900 – 1030
1030 – 1100
1100 – 1230
Tuesday
14 June
1000 – 1200
1200 – 1330
Tahoma 1-4, TCC Level 3
and Yakima 1, TCC Level 1
Monday
13 June
Sunday
12 June
700 – 1830
800 – 1700
930 – 1100
Saturday
11 June
700 – 2000
800 – 1700
1200 – 1700
Special
Lectures
Friday, June 10, 2016
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
1415
– 1505
1505 – 1535
1535 – 1625
1645 – 1800
Plenary Session 1A
Coffee Break
Plenary Session 1B
1800 – 1900
1830 – 1930
1830 – 1930
1930 – 2230
Poster Session 1
Happy Hour
Exhibitor Showcase
Shared Resource Lab Forum
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Speaker/Author Poster Session
Index
Abstracts
Tuesday, June 14, 2016
730 – 1830
800 – 2000
900 – 1030
930 – 1900
1030 – 1100
1030 – 2000
1100 – 1230
Poster Viewing
Frontiers Session 2
Coffee Break
Commercial Exhibits
1245 – 1345
Exceptional Student Award
1245 – 1345
1415 – 1530
1530 – 1600
1600 – 1715
Plenary Session 2
Coffee Break
1730 – 1830
1830 – 1930
1900 – 2000
1900 – 2000
Keynote Lecture
Poster Session 2
Happy Hour
Exhibitor Showcase
Poster
Session
Wednesday
15 June
1245
– 1345
Presentations
Wednesday, June 15, 2016
730 – 1700
800 – 1415
830 – 900
900 – 1030
900 – 1430
1030 – 1430
1030 – 1100
1100 – 1200
1100 – 1230
Poster Viewing
ISAC Business Meeting
Frontiers Session 3
Commercial Exhibits
Coffee Break
Poster Highlight Tour
1245 – 1345
1410 – 1500
1500 – 1530
1530 – 1620
1630 – 1730
1900 – 2300
ICCS/ESCCA Guest Symposium A
Coffee Break
ICCS/ESCCA Guest Symposium B
Awards Ceremony
Closing Reception
36
Seattle Aquarium
(Must present ticket for admittance.)
Congress
Overview
Visit the Exhibits & Posters
Special
Lectures
Sunday, June 12
1600 – 2000
1800 – 2000
Authors Must Place Posters on Assigned Boards
Commercial Exhibits Open
Saturday
11 June
Monday, June 13
Poster Viewing
Coffee Break
Commercial Exhibits
Poster Session II (Authors of even numbered boards present)
Happy Hour
Exhibitor Showcase
Tuesday
14 June
Tuesday, June 14
800 – 2000
1030 – 1100
1030 – 2000
1830 – 1930
1900 – 2000
1900 – 2000
Monday
13 June
Poster Viewing
Coffee Break
Commercial Exhibits
Poster Session I (Authors of odd numbered boards present)
Happy Hour
Exhibitor Showcase
Sunday
12 June
800 – 1930
1030 – 1100
1030 – 1930
1800 – 1900
1830 – 1930
1830 – 1930
Wednesday
15 June
Poster
Session
Wednesday, June 15
800 – 1415
Poster Viewing
1030 – 1100
Coffee Break
1030 – 1430
Commercial Exhibits
1100 – 1200
Poster Session III (consists of Poster Highlights Tour)
Posters must be removed by 1430 on Wednesday, June 15, 2016.
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Don’t forget to exchange your Closing Reception coupon for a ticket to the
Tuesday evening Closing Reception at the Seattle Aquarium!
Oral Session
Abstracts
Coupons will be exchanged on a first come, first served basis until Sunday, June
12, or until the maximum capacity is reached.
Poster Session
Abstracts
(Ticket required for admittance; see page 23 for details.)
Index
37
Speaker/Author
Special Lectures
Saturday,
June 11
1745 – 1845
CYTO Innovation Keynote
Advancing the Art and Science of Flow Cytometry through Advanced Materials
Brent Gaylord, BD Life Sciences, USA
Dr. Gaylord received his Ph.D. in material sciences from the University of California at Santa Barbara (UCSB) for his work in the
Development and Biological Application of Light Emitting Conjugated Polymers. This research ultimately led to the development
of Sirigen's core technology and a broad IP portfolio. After cofounding Sirigen he served as the company’s Chief Scientific Officer
for eight years until the company was acquired in 2012 by Becton Dickinson (BD). He currently serves as a Director of R&D
within BD Biosciences, responsible for the development of life science research reagents and the Sirigen technology
platform. Prior to attending UCSB, he obtained a B.S. in Chemistry-Material Science Engineering and a minor in Mathematics
from the United States Air Force Academy.
Sunday, June 12
1715 – 1815
Robert Hooke Lecture
Imaging of Induced Pluripotent Stem Cells
Rick Horwitz, Allen Institute for Cell Science, USA
Rick Horwitz is the inaugural Executive Director of the Allen Institute for Cell Science in Seattle. Previously, he was a University
Professor and Harrison Distinguished Professor of Cell Biology at the University of Virginia. He also served as the Director of the
Cell Migration Consortium: an NIH-funded multi-institutional, multi-disciplinary collaboration. Previously, he served as the Head
of the nascent Department of Cell and Developmental Biology at the University of Illinois and was a Professor of Biophysics and
Physical Biochemistry at the University of Pennsylvania. Rick earned his B.A. in the Honors Program at UW Madison, majoring in
chemistry (math, physics). He received his Ph.D. in Biophysics at Stanford University and did postdoctoral research in NMR at UC
Berkeley. He has served on numerous strategic planning and advisory committees and organized several international meetings.
Rick is known for his pioneering and leadership roles in cell adhesion, migration, and live-cell imaging.
Index
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Scholars &
Emerging
Leaders
Poster
Session
Wednesday
15 June
Tuesday
14 June
Monday
13 June
Sunday
12 June
Saturday
11 June
Special
Lectures
Congress
Overview
38
Congress
Overview
Tuesday, June 14
1730 – 1830
Keynote Lecture
Regulatory RNAs in Human Development and Cognition
John Mattick, Garvan Institute of Medical Research, Australia
Special
Lectures
Saturday
11 June
Monday
13 June
Professor Mattick is best known for showing that the majority of the mammalian genome produces regulatory RNAs that guide the
epigenetic trajectories of development. His awards include the 2011 International Union of Biochemistry & Molecular Biology
Medal, the 2012 Human Genome Organisation Chen Award for Distinguished Achievement in Human Genetic & Genomic
Research and the 2014 University of Texas MD Anderson Cancer Center Bertner Memorial Award for Distinguished Contributions
to Cancer Research.
Sunday
12 June
John Mattick is the Director of the Garvan Institute of Medical Research in Sydney, Australia. He was educated at the University
of Sydney and Monash University in Melbourne. He worked at Baylor College of Medicine in Houston, the CSIRO Division of
Molecular Biology in Sydney, and the University of Queensland, where he was the Foundation Director of the Australian Genome
Research Facility and the Institute for Molecular Bioscience. He also spent research periods at the Universities of Cambridge,
Oxford, Cologne and Strasbourg.
Tuesday
14 June
Wednesday, June 15
1410 – 1620
ICCS/ESCCA Guest Symposiom
Wednesday
15 June
The ICCS/ESCCA symposium hosted by ISAC features some of the cutting edge technologies from these two Societies. The talks
were selected by both Presidents, Fiona Craig (ICCS) and Katherina Psarra (ESCCA) and the Clinical Cytometry Editor Fred Preffer.
The Session highlights recent developments in translational and clinical cytometry with featured talks by Brent Wood, Ruth de
Tute, Richard Bankert and Greg Behbehani. They will speak about evolving applications for multiple myeloma minimal residual
disease testing, the immuno-regulatory impact of tumor derived exosomes and the application of mass cytometry to leukemia
testing.
Poster
Session
Scholars &
Emerging
Leaders
ICCS and ESCCA are ISAC associated societies that foster the education, know-how, dissemination, standardization of flowcytometry and its implementation in pre-clinical and clinical cell analysis. ICCS founded in 1984 to address the growing need for
an exchange of diagnostic and monitoring information between clinical flow cytometry laboratories. Today besides its Annual
Meeting and Clinical Cytometry B Journal, has very active advocacy, standardization and educational programs. ESCCA represent
more than 45 European countries and in association with numerous European national societies, ESCCA organizes international
conferences and training courses and national education program in local languages. Both ICCS and ESCCA are involved with
ISAC in the ICCE certification program.
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Index
39
Speaker/Author
The number listed following time signifies the
program number. The program number
references the abstract located in the back of
the program book.
Monday
13 June
Sunday
12 June
Saturday
11 June
Special
Lectures
Congress
Overview
DAILY PROGRAM
SATURDAY, JUNE 11, 2016
930 – 1100, Tahoma 1, TCC Level 3
SCIENTIFIC TUTORIAL 1
930
1
Considerations for Integrating Flow Cytometry with
Single Cell Transcriptomics
P.K. Chattopadhyay. NIAID, NIH, U.S.A.
Wednesday
15 June
Tuesday
14 June
930
2
Configuring Accurate Cell Detection in Images Using
CellProfiler
C. Wahlby, L. Kamentsky and A.E. Carpenter. Uppsala
Univ., Sweden and Broad Inst. of Harvard and MIT,
Cambridge, MA, U.S.A.
Index
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Scholars &
Emerging
Leaders
Poster
Session
930 – 1100, Skagit 4, TCC Lower Level
930
3
Shared Resource Management – A Changing
Paradigm for the 21st Century
P. Simms, P. Lopez and M. Black. Loyola Univ.
Chicago, NYU Sch. of Med. and Univ. of Washington,
U.S.A.
930
4
Validation - The Key to Translatable Flow Cytometry
in the 21st Century
V. Litwin, T. Oldaker and C. Green. Covance,
Indianapolis, Genoptix Med. Lab., a Novartis Co.,
Carlsbad, CA and Genentech Inc., San Francisco,
U.S.A.
930 – 1100, Tahoma TCC Level 3
A. Tarnok, V. Donnenberg and A. Murphy. Univ.
Leipzig, Germany and Univ. of Pittsburgh and WileyBlackwell, Hoboken, NJ, U.S.A.
ICCE EXAM
1200 6
Cell Sorting: Fundamentals and Selected Novel
Applications
J. Trotter, G. Osborne and C. Pletcher. BD
Biosciences, San Jose, U.S.A., The Univ. of
Queensland, Australia and Univ. of Pennsylvania Sch.
of Med., U.S.A.
1200 7
Image Cytometry for the Flow Cytometrist: When
Image Really Is Everything
A. Filby and D. Novo. Newcastle Univ., U.K., and De
Novo Software, U.S.A.
1200 8
Laboratory Infrastructure: Moving and Designing a
Flow Cytometry Laboratory
K.L. Holmes, N.C. Fisher and S.P. Perfetto. NIAID,
NIH and Univ. of North Carolina at Chapel Hill,
U.S.A.
1200 9
Forensic Flow Cytometry - Multiparameter
J. Wilshire and T. Baumgartner. Mem. Sloan Kettering
Cancer Ctr., U.S.A. and STEMCELL Technols.,
Vancouver, Canada
1200 – 1330, Tahoma TCC Level 3
1200 10
Founding and Launching a Cytometry Startup
Company
G. Durack. The TEKMILL Inc., U.S.A.
930
5
Writing, Publishing and Reviewing: Advice, Tips and
News from Cytometry A
40
1345 13
Microfluidic Flow Cytometry and Cell Sorting
T. Huang. Penn State, U.S.A.
SUNDAY, JUNE 12, 2016
CYTO FIRST TIME ATTENDEE WELCOME &
ORIENTATION
Index
Speaker/Author
41
Poster Session
Abstracts
Is this your first time attending CYTO? ISAC
Welcomes you to its 31st International Congress –
CYTO 2016!
All first time attendees as well as nonmember
registrants are invited to a Welcome & Orientation.
New attendees are welcome to join the CYTO family
for continental breakfast and to meet new friends. The
session will be informal and friendly with the
opportunity to meet with old timers of the CYTO
conferences as well as other first time attendees.
During the event, you will meet ISAC Council
members, Cytometry Part A Editor-in-Chief, the
Oral Session
Abstracts
COFFEE BREAK
Commercial
Tutorials &
Exhibits
Chairs: Diether Recktnewald, Betsy Ohlsson-Wihelm
and Jonni Moore
The advancement of cytometry depends on the
translation of innovative research into useful,
accessible tools that can catalyze new biological
understanding. CYTO Innovation is the forum within
ISAC that explores the challenges and opportunities
for translation of new cell analysis technologies into
commercially viable products and services. CYTO
Innovation provides: an opportunity to learn about the
translation of ideas and discoveries into products and
knowledge; the skills required for progressing from a
research finding to useful products with impact; a
unique platform for showcasing innovation in relevant
industry sectors; and engagement with a
knowledgeable network of entrepreneurs, executives
and other business people. CYTO Innovation is part of
ISAC’s wider mission of advancing cytometry and
aims to serve members and Congress attendees either
engaged in or contemplating the translation of
research into high impact products and services. It is
1650 – 1700, Hall 4C Foyer, WSCC Level 4
Scholars &
Emerging
Leaders
CYTO INNOVATION AND KEYNOTE LECTURE
1745 15
CYTO Innovation Keynote
Introduction: Allen Poirson
Advancing the Art and Science of Flow Cytometry
through Advanced Materials
B. Gaylord. Becton Dickinson, San Diego, U.S.A.
1830
Networking Session
1915
Session Ends
Poster
Session
1530 – 1915, Hall 4C, WSCC Level 4
Joe Victor, Paul Smith, and David Basiji
1730
Break
Wednesday
15 June
1345 448
Introducing Cytometry through Hands-On Activities
J.P. Hill, A. Conway, D. Callen, R. Weeks, T.
Caraballo, J. Chalupny and S. Friend. NIAID, NIH,
Roswell Park Cancer Inst., Buffalo, Coherent Inc.,
Wilsonville, OR, Shoreline Community Col., Seattle,
Glacier Peak H.S. Shoreline and Amnis/EMD
Millipore, Seattle, U.S.A.
1700
Technology Showcase Presentations
Judges:
Tuesday
14 June
1345 – 1515, Yakima 1, TCC Level 1
Joe Victor, Paul Smith, and David Basiji
1650
Break
Monday
13 June
1610
Technology Showcase Presentations
Judges:
Sunday
12 June
1345 12
Flow Cytometry Biosafety
K. Holmes, H. Pletcher, S. Monard and S.P. Perfetto.
NIAID, NIH, Univ. of Pennsylvania, U.S.A. and
Walter & Eliza Hall Inst. of Med. Res., Australia
1530
Introduction
1540 14
The Journey from Concept to Commercialization: A
Daunting Task with High Rewards for Entrepreneurs
J. Phi. IntelliCyt, Albuquerque, U.S.A.
Saturday
11 June
AGENDA
Special
Lectures
1345 11
Imaging Bioinformatics for High Content Screening
of 3D Cell Culture Models
B. Parvin. Univ. of Nevada, Reno, U.S.A.
open to Full Congress Registrants; no additional fees
apply.
Congress
Overview
1345 – 1515, Tahoma 1 TCC Level 3
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Membership Services Committee (MSC) members,
and other leaders of ISAC. Peter Lopez, ISAC Council
member and chair of the MSC, will make opening
remarks, and representatives from the ISAC Scholars
and Shared Resource Lab Emerging Leaders programs
will speak about their first CYTOs and advice they
have to share. After the formal presentation, attendees
will be encouraged to network with each other and
ISAC Leaders, who will be sitting at different tables
throughout the room.
There will be opportunity to ask questions; find out
the resources available at the conference; bring up
specific topics of interest and request to be put in
contact with specific cytometrists who will also be
attending CYTO 2016. This event is sponsored by
Cytometry Part A, ISAC’s Journal.
OPENING REMARKS & STATE-OF-THE-ART
LECTURES
Chair: John Nolan
Cochair: Andreas Radbruch
900
16
Quantitative Physiology of Living Single-Cells: Subtle
Pleasures and Future Promise
R. Brent. Fred Hutchinson Cancer Res. Ctr., Seattle,
U.S.A.
930
17
High Content Analysis of Pancreatic Cancers Using
Imaging Mass Cytometry
D. Hedley. Princess Margaret Hosp., Toronto, Canada
1000 18
Nanotechnology Platforms for Extracellular Vesicle
Analysis
H. Lee. Massachusetts Gen. Hosp., U.S.A.
COFFEE BREAK
Index
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
PARALLEL 1: RNA FLOW CYTOMETRY
Chair: David Hedley
Cochair: Benjamin Daniel
1100 19
Highly Multiplexed Simultaneous Detection of
Proteins and RNAs in Single Cells
P.F. Gherardini, A.P. Frei, F.A. Bava, E. Zunder,
E.W.Y. Hsieh, S-Y. Chen and G.P. Nolan. Stanford
Univ., Univ. of Virgina and Univ. of Colorado
Denver, U.S.A.
1120 20
Simultaneous Imaging of Proteins and Transcripts in
Cells by Highly Multiplexed Imaging Mass Cytometry
D. Schulz, S. Chevrier, H. Jackson, Z. Varga and B.
Bodenmiller. Univ. of Zurich and University Hosp.
Zurich, Switzerland
42
1140 21
Simultaneous Determination of mRNA and Protein
Expression Profiles of T-bet and Eomes in Stimulated
T Lymphocytes by Flow Cytometry
K.T. Soh, J.D. Tario, Jr. and P.K. Wallace. Roswell
Park Cancer Inst., Buffalo, NY, U.S.A.
1200 32
Detection of HIV RNA Expression in Peripheral
Blood from HIV-Infected Study Participants by
RNAflow Cytometry
C. Baker, A. Tan, J. McCune, E. Sinclair and J. Milush.
UCSF, U.S.A.
PARALLEL 2: MICROFLUIDIC CYTOMETRY
Chair: Timothy Bushnell
Cochair: Alfonso Blanco Fernandez
1100 23
Label-Free Separation of Neuroblastoma Tumor Cells
from Mononuclear Cells Using an AcoustophoresisBased Microfluidic Chip
F. Olm, A. Urbansky, T. Laurell and S. Scheding. Lund
Univ., Sweden
1120 24
Microfluidic Post Geometry Design for HighThroughput (100 ȝL/min) Harvesting of Leukocytes
from Blood Using Deterministic Lateral
Displacement Arrays
J. D'Silva, R. Austin and J. Sturm. Princeton Univ.,
U.S.A.
1140 25
A Time-Resolved Microfluidic Flow Cytometer and
Sorter for FRET-Based Biosensors
B. Fiedler, S. Van Buskirk, A. Palmer and R. Jimenez.
Univ. of Colorado Boulder and JILA/NIST, Boulder,
U.S.A.
1200 26
A High Throughput Bubble Jet Microfluidic FACS
C. Liu, D. Vercruysse, K. de Wijs, A. Dusa, S. Mao, K.
Blaszkiewicz, Y. Li, B. Majeed, X. Rottenberg, D.
Sabuncuoglu Tezcan, P. Deshpande and L. Lagae.
imec, Leuven and Katholieke Univ. Leuven, Belgium
PARALLEL 3: IMMUNOLOGY
Chair: Zofia Maciorowski
Cochair: Anna Belkina
1100 27
Generation of Stem Cell-Like Memory T Cells from
Naive Precursors for Adoptive Cell Transfer
Immunotherapy
A. Roberto, V. Zanon, K. Pilipow and E. Lugli. Human
Clin. and Res. Ctr., Rozzano, Milan, Italy
Poster
Session
1245 – 1345, TCC
COMMERCIAL TUTORIALS
Featured Companies:
Scholars &
Emerging
Leaders
CytekBiosciences – Tahoma 5, TCC Level 3
eBioscience, an Affymetrix Business Unit - Tahoma 3,
TCC Level 3
IntelliCyt – Skagit 4, TCC Lower Level
Sony Biotechnology – Tahoma 1, TCC Level 3
Zellkraftwerk GmbH - Tahoma 4, TCC Level 3
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
WORKSHOP 1
1415 39
Characterization and Standardization of Cytometers
for EV Analysis
J. Lannigan and J.P. Nolan. Univ. of Virginia and
Scintillon Inst., San Diego, U.S.A.
Poster Session
Abstracts
1200 34
Application of Silver Nanoparticles for the Specific
Detection of Cellular Antigens in Mass Cytometry
A.R. Schulz, S. Stanislawiak, S. Baumgart, A.
Radbruch, A. Grützkau and H.E. Mei. German
Rheumatism Res. Ctr., Berlin, Germany
1140 37
Identification and Classification of the Malaria
Parasite, Plasmodium falciparum Using Imaging Flow
Cytometry
Z. Porat, E. Dekel, A. Rivkin and N. Regev.
Weizmann Inst. of Sci., Rehovot, Israel
1200 38
Multiplex Intravital Detection of Germinal Center in
Murine Lymph Node with Wave-Mixing Two-Photon
Microscopy
A. Rakhymzhan, R. Leben, A. Acs, T. Winkler, R.
Guenther, P. Mex, L. Oehme, Z. Cseresnyes, A.
Hauser and R. Niesner. DRFZ, Berlin, NikolausFiebiger-Ctr. for Molec. Med., Erlangen and Charité –
Univ. Hosp., Berlin, Germany
Wednesday
15 June
1140 33
Increasing the Sensitivity of Functional Flow Assays
Bymagnetic Pre-enrichment of Antigen-Specific
Lymphocytes
E. Bergmann-Leitner, K. Beck and F. Farooq. Walter
Reed Army Inst. of Res. and Miltenyi Inc., Silver
Spring, MD, U.S.A.
1120 36
Multi-parametric, Single Cell Measurements by
Imaging Mass Cytometry Improves Classification of
Breast Cancer Patients
H. Jackson, D. Schapiro, Z. Varga and B. Bodenmiller.
Univ. of Zurich and Univ. Hosp. Zurich
Tuesday
14 June
Chair: Monica DeLay
Cochair: Adrian Smith
1100 31
Profiling Cellular Experience of Hypoxic
Environments
P. Smith, M. Wiltshire, K. Pors, L. Patterson and R.
Errington. Cardiff Univ., Biostatus Ltd., Shepshed and
Univ. of Bradford, U.K.
1120 350
Multiplexed Detection of Biological and Chemical
Food Contaminants Using Spark-Induced Breakdown
Spectroscopy
C. Gondhalekar, E. Biela, B. Rajwa, E. Bae, V.
Patsekin, J. Sturgis, H. Kim, I-J. Doh, L. Stanker and P.
Robinson. Purdue Univ. and USDA, Albany, CA,
U.S.A.
1100 35
Multispectral Shape Characterization and
Classification of Cells With Complex Morphology by
Imaging Flow Cytometry
A. Li and S. Vaidyanathan. MilliporeSigma, Seattle,
U.S.A.
Monday
13 June
PARALLEL 4: NEW PROBES AND ASSAYS
Chair: Hans Minderman
Cochair: Andrew Filby
Sunday
12 June
PARALLEL 5: IMAGE CYTOMETRY I
Saturday
11 June
1200 30
Impact of Aging on Calcium Influx and Potassium
Channel Characteristics of T Lymphocytes
G. Toldi, S. Kollár, L. Berta and J. Rigó Jr. Semmelweis
Univ., Hungary and Birmingham Women's Hosp.,
U.K.
1100 – 1230, Skagit 4, TCC Level 3
Special
Lectures
1140 29
Kv1.3 Lymphocyte Potassium Channel Inhibition as a
Potential Novel Therapeutic Target in Acute
Ischemic Stroke
A. Bajnok, A. Folyovich, C. Orban and G. Toldi.
Semmelweis Univ. and Szent János Hosp., Budapest,
Hungary
Congress
Overview
1120 28
The IL-21 Signaling Pathway Is Enhanced in RA B
Cells and Has the Potential to Alter Development
and Cytokine Production in RA B Cells
E. Dam and J. Buckner. Benaroya Res. Inst., Seattle,
U.S.A.
Index
43
Speaker/Author
Congress
Overview
Special
Lectures
WORKSHOP 2
WORKSHOP 8
1415 40
Therapeutic Cell Sorting
G. Chojnowski, C. Choi, and C. Wiwi. Celgene
Cellular Therapeutics, Warren, NJ, U.S.A.
1545 46
Systems for Simplifying Core Management
R. Salomon, R. Gardner and M. Thomson. Garvan
Inst. of Med. Res., Australia, Inst Gulbenkian Ciencia,
Portugal and St Vincent's Inst. of Med. Res., Australia
Sunday
12 June
Saturday
11 June
WORKSHOP 3
1415 41
SRLWorkshop: Communicating Up and Down
M. Black and R. Walker. Univ. of Washington, U.S.A.
and Babraham Inst., Cambridge, U.K.
1415 42
Tumor Heterogeneity
V. Donnenberg, A.D. Donnenberg and B.
Bodenmiller. Univ of Pittsburgh Sch. of Med., U.S.A.
and Univ. of Zurich, Switzerland
1415 – 1530, 6NDJLW7&&/RZHU/HYHO
WORKSHOP 5
1415 43
Mining and Modeling of Biomedical Data: Issues,
Approaches and Building Consunsus
B. Rajwa and T.J. Chen. Purdue Univ. Cytobank Inc.,
Mountain View, CA and Stanford Univ., U.S.A.
Poster
Session
Wednesday
15 June
Tuesday
14 June
Monday
13 June
WORKSHOP 4
1530 – 1545, Tahoma Foyer, TCC Level 3 and Skagit
Foyer, TCC Lower Level
Index
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Scholars &
Emerging
Leaders
COFFEE BREAK
WORKSHOP 6
1545 44
Instrument PMT Voltage Optimization for Maximum
Sensitivity
A. Brooks and S. Monard. Sch. of Biol.. Sci., Univ. of
Auckland, Maurice Wilkins Ctr., Auckland, New
Zealand and Walter & Eliza Hall Inst., Parkville,
Australia
WORKSHOP 9
1545 47
Flow Cytometry for Cohort, Longitudinal and Multicenter Studies
A. Larbi and A. Tarnok. SIgN, A*STAR, Singapore and
Univ. of Leipzig, Germany
1545 – 1700, 6NDJLWTCC Lower Level
WORKSHOP 10
1545 48
First Steps as an Innovator: DIY Assessment of the
Commercial Potential of Ideas and Inventions in
Cytometry
B. Ohlsson-Wilhelm, P.J. Smith, G. Durack and R.
Capocasale. SciGro Inc., Cambridge, MA, U.S.A.,
OncoTherics Ltd., Leicestershire, U.K., Univ. of
Illinois at Urbana-Champaign, U.S.A. FlowMetric Inc.,
Doylestown, PA, U.S.A.
ROBERT HOOKE LECTURE
Chair: Robert Murphy
1715 49
Integrated, Multi-scale and Spatial-Temporal Cell
Biology – The Allen Institute for Cell Science
R. Horwitz. Allen Inst. for Cell Sci., Seattle, U.S.A.
1800 – 2000, Exhibit Hall 4EF, WSCC Level 4
COMMERCIAL EXHIBITS
See pages 88 – 108 for full details.
OPENING RECEPTION
WORKSHOP 7
1545 45
Are We Damaging the Cells We Sort?
P. Lopez and A. Box. New York Univ and Stowers
Inst. for Med. Res., Kansas City, MO, U.S.A.
44
PARALLEL 6: EXTRACELLULAR VESICLES
Scholars &
Emerging
Leaders
1200 59
Highly Parallel Acoustic Flow Cytometer for High
Event Rates and Volumetric Throughput
D. Kalb, F. Fencl, G. Maestas, A. Goudarzi, B.
Edwards, A. Shreve and S. Graves. Univ. of New
Mexico, U.S.A.
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
PARALLEL 8: IMAGE CYTOMETRY II
(SUBCELLULAR)
Chair: Michael Halter
Cochair: Henning Ulrich
Poster Session
Abstracts
1140 54
Effect of ADP- and Collagen-Induced Platelet
Activation on Microvesicles in Platelet-Rich Plasma
Studied with Scanning Flow Cytometry
1140 58
Performing Systems Immunology Using Next
Generation High-Dimensional Single-Cell Cytometry
Systems: Bright Lights and Heavy Metals
T. Ashhurst, A. Lim, L. Duckett, R. Balderas, B.
Fazekas de St Groth, A. Smith and N. King. Univ. of
Sydney, RFHSB, Camperdown, BD Biosciences,
Macquarie Park, NSW and San Jose, CA, U.S.A. and
The Centenary Inst., Camperdown, NSW, Australia
Poster
Session
Chair: Richard Bankert
Co-Chair: Joanne Lannigan
1100 52
Size and Refractive Index Determination of
Submicrometer Particles by Flow Cytometry
E. van der Pol, F.A. Coumans, L. de Rond, E.L. Gool,
A.N. Böing, A. Sturk, R. Nieuwland and T.G. van
Leeuwen. Acad. Med. Ctr., Univ. of Amsterdam,
Netherlands
1120 53
Extracellular Vesicle-Based Rare Event Analysis in
Body Fluids Using High Resolution Flow Cytometry:
Impact of Fluorescence Triggering and Swarm
Detection
S. Libregts, G. Arkesteijn, A. Németh, E. Nolte - 't
Hoen and M. Wauben. Utrecht Univ., Netherlands
and Semmelweis Univ., Hungary
Wednesday
15 June
COMMERCIAL EXHIBITS
Chair: Peter Lopez
Cochair: Svetlana Mazel
1100 56
Identification of Small Molecule Inducers of FOXP3
in Human T Cells Using High-Throughput Flow
Cytometry
R. Jepras. GlaxoSmithKline, Stevenage, U.K.
1120 57
A Novel Approach for the High Throughput Imaging
of 3D Spheroids Using Imaging Mass Cytometry
V. Zanotelli, F. Georgi, A. Yakimovich, R. Catena, H.
Jackson and B. Bodenmiller. Univ. of Zurich,
Switzerland
Tuesday
14 June
COFFEE BREAK
PARALLEL 7: HIGH THROUGHPUT & HIGH
CONTENT
Monday
13 June
Sunday
12 June
Chair: Kathy Muirhead
Cochair: Raluca Niesner
900
50
A Picture Is Worth a Million Numbers: Targeting
Disease by Profiling Cells via Microscopy and Flow
Cytometry
A. Carpenter. Broad Inst., Cambridge, MA, U.S.A.
945
51
Biophysical Cytometry
D. Di Carlo. UCLA, U.S.A.
Saturday
11 June
FRONTIERS SESSION 1: MINING DATA FROM
IMAGE AND MICRO-FLUIDIC CYTOMETRY
1200 55
Quantification of Cancer-Specific Exosomes by High
Sensitivity Flow Cytometry
X. Yan, Y. Tian, L. Ma, G. Su, W. Zhang, C. Chen and
S. Zhu. Xiamen Univ., The First Affil. Hosp. and
NanoFCM, Inc., Xiamen, China
Special
Lectures
POSTER VIEWING
D. Chernova, A. Konokhova, M. Yurkin, D. Strokotov
and V. Maltsev. Voevodsky Inst. of Chem. Kinet. and
Combustion SB RAS, Novosibirsk and Novosibirsk
State Univ., Russia
Congress
Overview
MONDAY, JUNE 13, 2016
Index
45
Speaker/Author
1100 60
Quantitative Analysis of Golgi Structure by Imaging
Flow Cytometry
Z. Porat, I. Wortzel and R. Seger. Weizmann Inst. of
Sci., Rehovot, Israel
1120
61
Flow Cytometry Analysis of T-Cell Receptor Mimic
Antibody
RL6A Internalization by Brain-Derived
Endothelial Cells
J.H. Park , O. Hawkins, N. Chintala, J. Weidanz and
U. Bickel. Texas Tech Univ. Hlth. Sci. Ctr., Amarillo
and Abilene, U.S.A.
1140 62
The Cellular Fate and Transport of Silver
Nanoparticles: Hyperspectral Imaging, Flow
Cytometry and Morphology
R. Zucker, L. Degn, J.N.R. Ortenzio and W. Boyes.
EPA and Oak Ridge Inst. of Sci. and Educ. appointee,
EPA, Research Triangle Park, NC, U.S.A.
1200 63
Computational Image Analysis Pipeline for Creating
Spatiotemporal Maps of Signaling Protein
Distributions during T Cell Stimulation
X. Ruan, K.T. Roybal, T.E. Buck, B-H. Cho, D.J. Clark,
R. Ambler, H.M. Tunbridge, J. Zhang, P. Verkade, C.
Wülfing and R.F. Murphy. Carnegie Mellon Univ.,
U.S.A., Univ. of Bristol, U.K., Univ. of Texas
Southwestern Med. Ctr., U.S.A. and Albert Ludwig
Univ. of Freiburg, Germany
1140 66
Two 16-Color Intracellular Cytokine Staining Panels
Identified Immunogenicity Markers in a Malaria
Vaccine Clinical Trial
G. Moncunill, A. Ayestaran, M. Mpina, A.J.
Nhabomba, K.W. Cohen, C.S. Jairoice, J.J. Campo, J.
Harezlack, H. Sanz, N. Díez-Padrisa, N.A. Williams,
D. Morris, C. Valim, J.J. Aponte, C. Daubenberger, C.
Dobaño, M.J. McElrath and S.C. De Rosa. ISGlobal,
Hosp. Clin., Univ. of Barcelona, Fred Hutchinson
Cancer Res. Ctr., Seattle, WA, U.S.A., Bagamoyo Res.
and Trng. Ctr. of Ifakara Hlth. Inst., Tanzania, CISM,
Mozambique, Antigen Discovery Inc., Irvine, CA,
U.S.A., Indiana Univ. Fairbanks Sch. of Publ. Hlth.,
U.S.A., Michigan State Univ., U.S.A., Univ. of Basel,
Switzerland
1200 67
Modeling Single-Cell Gene Expression Identifies a
Novel Correlate of Protection for an Experimental
Malaria Vaccine
G. Finak, K. Sierra-Davidson, A. Mcdavid, M. Yajima,
P. Darrah, P. Chattopadhyay, M. Roederer, R. Seder
and R. Gottardo. Fred Hutchinson Cancer Res. Ctr.,
Seattle and NIAID, NIH, Bethesda, U.S.A.
PARALLEL 9: CLINICAL IMMUNOLOGY
PARALLEL 10: TUMOR MICROENVIRONMENT
Chair: Virginia Litwin
Cochair: Anne Carpenter
1100 64
Ex Vivo Analysis of Multiple CD4+ T-Cell Responses
Using Combinatorial HLA-II Tetramer Staining to
Evaluate the Response to Flu Vaccine and to
Citrullinated Synovial Ag in RA
J. Buckner, H. Uchtenhagen, C. Rims, G. Blahnik, I-T.
Chow, W. Kwok, P. Linsley and E. James. Benaroya
Res. Inst. at Virginia Mason, Seattle, U.S.A.
Chair: Paul Smith
Cochair: Katarzyna Piwocka
1100 68
Structure and Function of Tunneling Nanotubes –
Direct Cell-Cell Connections Formed between
Leukemia and Stroma Cells
K. Piwocka, M. Walczak, A. Kowalczyk, W. DudkaRuszkowska, P. Podszywalow-Bartnicka, P.
Sunderland and J. Wlodarczyk. Nencki Inst. of Exptl.
Biol., Warsaw, Poland
1120 65
CD19 Is an Important Target for the Treatment of
Autoimmune Diseases with the Involvement of
Pathogenic Antibody Secreting Cells
C. J. Groves, B. Rajan, J. Carrell, C. Morehouse, J.
Wang, R. Rayanki, T. Mustelin, R. Kolbeck, Y. Wang
and R. Herbst. MedImmune LLC, Gaithersburg, MD,
U.S.A.
1120 69
Unraveling the Tumor Microenvironment in Three
Dimensions Using Imaging Mass Cytometry
R. Catena, A. Oezcan, V. Zanotelli, H. Jackson and B.
Bodenmiller. Univ. of Zurich, Switzerland
1140 70
Multicolor Flow Cytometric Analysis of Peritumor
Infiltrating Myeloid Cells in Patients with Colon
Cancer Liver Metastases
F. Colombo, C. Soldani, B. Franceschini, F. Clemente,
M.M. Cimino, G. Torzilli, M. Donadon and A.
Anselmo. Humanitas Clin. and Res. Ctr., Rozzano,
Italy
Index
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Scholars &
Emerging
Leaders
Poster
Session
Wednesday
15 June
Tuesday
14 June
Monday
13 June
Sunday
12 June
Saturday
11 June
Special
Lectures
Congress
Overview
46
WORKSHOP 11
1645 76
Sub-micron and Nano-particle Characterization,
Detection, and Analysis in Flow Cytometry
R. Zucker and P. Poncelet. U.S. EPA, Research
Triangle Park, NC, U.S.A. and Biocytex, Marseille,
France
Saturday
11 June
PRESIDENT'S AWARD FOR EXCELLENCE
PRESENTATIONS
Special
Lectures
1245 – 1345, Hall 4EF, WSCC Level 4
1600 75
The Key Role of Flow Cytometry in Developing
Antibodies for Immuno-oncology
M. Selby. Bristol Myers Squibb, Redwood City, CA,
U.S.A.
Congress
Overview
1200 71
Tumor-Associated Macrophages Exhibit M2a-Like
Phenotype and Drive the Epithelial-to-Mesenchymal
Transition, Tumorigenicity and Invasion in Breast
Cancer
V. Donnenberg, J-J. Zhang, V. Riccelli, P. Basse and
A.D. Donnenberg. Univ of Pittsburgh Sch. of Med.
and Univ. of Norte Dame, U.S.A. and Xiangya Sch. of
Med., China
Sunday
12 June
1245 – 1345, TCC
ACEA Biosciences Inc. – Tahoma 5, TCC Level 3
BD Biosciences – Tahoma 1, TCC Level 3
Bio-Rad Laboratories – Yakima 1, TCC Level 1
FlowJo, LLC – Skagit 4, TCC Lower Level
MilliporeSigma – Tahoma 4, TCC Level 3
1415 77
Malaria Workshop
H. Shapiro, G. Chojnowski and B. Grimberg. Shapiro
Lab., West Newton, MA, U.S.A., QIMR Berghofer
Med. Res. Inst., Brisbane, Australia and Case Western
Reserve Univ., U.S.A.
Miltenyi Biotec GmbH – Tahoma 3, TCC Level PLENARY SESSION 1A: IMMUNOLOGY
Chair: Andreas Radbruch
Cochair: Joseph Tario
1645 79
Imaging Flow Cytometry: Applications and
Challenges
D. Jenner and A. Filby. Defense Sci. & Technol. Lab.,
Salisbury, U.K. and Univ. of Newcastle, U.K.
WORKSHOP 15
1645 80
Detective Workshop: How to Solve the Mystery of
Challenging Receptor Occupancy Assays
V. Litwin, C. Green and J. Stewart. Covance,
Indianapolis, Genentech Inc., South San Francisco
and Flow Contract Site Lab., Kirkland, WA, U.S.A.
Poster Session
Abstracts
1535 74
T Cell Trafficking in Tumor Vessels: Yet Another
Checkpoint for Cancer Immunotherapy
S. Evans. Roswell Park Cancer Inst., Buffalo, NY,
U.S.A.
WORKSHOP 14
Oral Session
Abstracts
PLENARY SESSION 1B: IMMUNOLOGY
Commercial
Tutorials &
Exhibits
COFFEE BREAK
1645 78
SRL Equipment Management: Future-Proof Your Lab
J. Back, N. Lane and J. Lannigan. Wayne State Univ.,
U.S.A., Univ. of Cambridge, U.K. and Sch. of Med.,
Univ. of Virginia, U.S.A.
Scholars &
Emerging
Leaders
1440 73
Novel Insights Into Human SLE Pathogenesis
V. Pascual. Baylor Res. Inst., Dallas, U.S.A.
WORKSHOP 13
Poster
Session
Chair: Andreas Radbruch
Cochair: Joseph Tario
1415 72
High-Dimensional Modeling of B and T Cells
C.B. Bagwell. Verity Software House Inc., Topsham,
ME, U.S.A.
Wednesday
15 June
Tuesday
14 June
WORKSHOP 12
Monday
13 June
Featured Companies:
Index
47
Speaker/Author
Congress
Overview
P
OSTER SESSION 1
Saturday
11 June
Special
Lectures
Authors of ODD Numbered Boards Present
HAPPY HOUR
EXHIBITOR SHOWCASE
Sunday
12 June
SHARED RESOURCE LAB FORUM –
NETWORKING: CREATING LASTING
CONNECTIONS
Index
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Scholars &
Emerging
Leaders
Poster
Session
Wednesday
15 June
Tuesday
14 June
Monday
13 June
Chairs: Joanne Lannigan and Rachael Walker
Shared Resource Laboratory (SRL) staff are often faced
with a broad range of applications and technological
implementations while servicing a varied user base on
a daily basis. The 2016 CYTO Shared Resource Lab
Forum offers a unique opportunity to network with
SRL staff and experts in cytometric technology. The
forum is being hosted by the ISAC SRL Emerging
Leaders and particular focus will be placed on
providing networking opportunities for SRL managers
and staff. The goal is to develop strong and lasting
connections between SRL members across ISAC.
Enjoy an evening dedicated to Shared Resource
Laboratory staff and get to know fellow SRL staff/
managers and others through a series of formal and
informal networking activities. Advance registration is
required. Dinner and one complimentary drink ticket
will be provided for pre-registered participants.
TUESDAY, JUNE 14, 2016
POSTER VIEWING
FRONTIERS SESSION 2: THE NEXT FRONTIERS IN
DEEP PROFILING
Chair: Paul Smith
Cochair: Attila Tarnok
945
82
Learning and Communicating Cell Identity in the
Context of Cancer Microenvironments
J. Irish. Vanderbilt Univ., U.S.A.
COFFEE BREAK
COMMERCIAL EXHIBITS
PARALLEL 11: EXTRACELLULAR VESICLES &
BIOMARKERS
Chair: Jonni Moore
Cochair: Nancy Fisher
1100 83
Flow Cytometric Quantification of White Blood Cell
ȕ-Adrenergic Receptor Density and Urinary
Endothelial-Derived Microparticles as Biomarkers of
Right Ventricle Failure in Pulmonary Arterial
Hypertension
K. Asosingh, J. Rose, N. Wanner, K. Queisser, P.
Barret, M. Park, C. Hite, S.V. Naga Prasad and S.
Erzurum. Cleveland Clin., U.S.A.
1120 84
A Functional Role for Heart-Derived Extracellular
Vesicles in Heart Failure-Associated Electrical
Remodeling
K. Danielson, J. Tigges, V. Camacho, V. Toxavidis and
S. Das. Beth Israel Deaconess Med. Ctr., Boston,
U.S.A.
1140 85
Circulating Microparticles as a Novel Biomarker for
Early Prediction of Preeclampsia and Intrauterine
Growth Restriction
A. Jadli, K. Ghosh and S. Shetty. Natl. Inst. of
Immunohaematol, Mumbai, India
1200 86
Label-Free Detection of Extracellular Vesicles in
Human Breast Milk Compared to Infant Formula
L. de Rond, E. van der Pol, A. Sturk, R. Nieuwland,
F.A. Coumans and T.G. van Leeuwen. Acad. Med.
Ctr., Amsterdam, Netherlands
900
81
The Next Dimension in Single Cell Cytometry
P. Chattopadhyay, S. Perfetto, J. Hill and M. Roederer.
NIAID, NIH, U.S.A.
48
PARALLEL 12: NOVEL FLOW & IMAGE
CYTOMETRY APPLICATIONS
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
PARALLEL 15: FLOW CYTOMETRY
INSTRUMENTATION
Chair: Jessica Houston
Cochair: Micheal Zordan
1100 99
Imaging Flow Cytometer and Cell Sorter Using
Spatial-Temporal Transformation
Y. Han, S. Cho, Y. Gu, A. Zhang, and Y. Lo. Univ. of
California, San Diego, and Nanocellect Biomedical
Inc.,U.S.A.
Oral Session
Abstracts
Poster Session
Abstracts
Index
49
Speaker/Author
1140 93
Biomarkers for Predicting Tolerance in Living Donor
Liver Transplantation
K. Jain, S. Choudhury, N. Mehta, S. Nundy and M.
Gupta. NIMS Univ. and Sir Ganga Ram Hosp., New
Delhi, India
Poster
Session
1120 92
Modulation of HLA-DR on Monocyte Subpopulation
in ICU Patients
A. Cattaneo, E. Trombetta, C. Frugoni, P. Cetrangolo,
A. Protti, J. Colombo and L. Porretti. Fndn. IRCCS Ca'
Granda Policlin., Milan, Italy
1140 97
A Statistically Robust Automated Method for
Leukemia Detection Using Multi-parametric Analysis
on Imaging Flow Cytometry Data
V. Venkatachalam, Y. Konishi, H. Pugsley, P.
Morrissey, W. Ortyn, E. Peerschke, P. Maslak, T. Tsuji
and S. Tabata. Amnis, part of MilliporeSigma, Seattle,
U.S.A., Sysmex Corp., Kobe, Japan and Mem. SloanKettering Cancer Ctr., New York, U.S.A.
1200 98
Deep Phenotypic Profiling of Small Clinical Samples
through Multispectral Imaging Cytometry
T. Gierahn, D. Loginov and J.C. Love. MIT, U.S.A.
Wednesday
15 June
Chair: Jack Dunne
Cochair: Frank Schildberg
1100 91
High Level of Activation and Altered Mitochondrial
Functionality in T Cells from Patients with
Progressive Forms of Multiple Sclerosis
S. De Biasi, E. Bianchini, M. Nasi, L. Gibellini, S.
Pecorini, A.M. Simone, D. Ferraro, F. Vitetta, P. Sola,
A. Cossarizza and M. Pinti. Univ. of Modena and
Reggio Emilia, Modena, Italy
1120 96
Direct Learning of a Generative Model for
Endoplasmic Reticulum Distribution from
Fluorescence Microscopy Images
K. Liu-Huang, T. Lee, G. Rohde and R. Murphy.
Carnegie Mellon Univ., U.S.A.
Tuesday
14 June
PARALLEL 13: IMMUNE MONITORING
Chair: Bartek Rajwa
Cochair: Greg Finak
1100 95
Cell Painting Parametisation of Cellular Phenotypes
within Heterogeneous Populations
R.A. Howard-Jones, K. Tuvstog, M. Wiltshire, V.
Griesdoorn, M.R. Brown, A.J. Sloan and R.J. Errington.
Sch. of Med. and Sch. of Dent., Cardiff Univ. and
Swansea Univ. Col. of Engin., U.K.
Monday
13 June
PARALLEL 14: AUTOMATED IMAGE ANALYSIS
Sunday
12 June
1200 90
Inertial Focusing and Imaging Flow Cytometry of
Euglena gracilis for Biodiesel Applications
M. Li, H.E. Enrique Munoz, J. Lin, K. Owsley, K.
Goda, E. Diebold and D. Di Carlo. UCLA, Omega
Biosystems Inc., U.S.A. and Univ. of Tokyo, Japan
Saturday
11 June
1140 89
Tracking Ancient Global Change Using Flow and
Imaging Cytometry
R. Tennant, A. Power, R. Lee, R. Jones and J. Love.
Univ. of Exeter, U.K.
Special
Lectures
Chair: Gergely Toldi
Cochair: Gelo dela Cruz
1100 87
High-Resolution Flow Cytometry of the Intestinal
Microbiota in Inflammatory Bowel Disease
J. Zimmermann, T. Hübschmann, F. Schattenberg, J.
Schumann, P. Durek, M. Friedrich, R. Glauben, B.
Siegmund, A. Radbruch, S. Müller and H-D. Chang.
DRFZ, Berlin, Helmholtz Ctr. for Envrn. Res., Leipzig
and Charité Univ. Med., Berlin, Germany
1120 88
Integration of Acoustic and Light Sensing for Marine
Bio-mining
G. Wiegand. Med. Univ. of South Carolina, U.S.A.
1200 94
Parallel Monitoring of Human Rhinovirus- and
Allergen-Specific CD4+ T Cells during Experimental
Infection in Allergic Asthmatics
L. Muehling, P. Heymann, P. Wright, D. Mai, W.
Kwok and J. Woodfolk. Univ. of Virginia and
Benaroya Res. Inst., Seattle, U.S.A.
Congress
Overview
Poster
Session
Wednesday
15 June
Tuesday
14 June
Monday
13 June
Sunday
12 June
Saturday
11 June
Special
Lectures
Congress
Overview
1120
100
Time Domain Analysis by Scanning Small Laser Beam
Cytometry
M. Yamamoto, K. Hernandez and J.P. Robinson.
Miftek Corp., West Lafayaette, IN and Purdue Univ.,
U.S.A.
1505 113
A Glorious Path of Plant Flow Cytometry
J. Dolezel. Inst. of Exptl. Botany, Olomouc, Czech
Republic
1140 101
A Frequency-Multiplexed Parallel Inertial Flow
Cytometer for High-Throughput Screening and Drug
Discovery
J. Lin, K. Owsley, E. Diebold and D. Di Carlo. UCLA
and Omega Biosysts. Inc., U.S.A.
COFFEE BREAK
1200 102
Flow Cytometry Adds Imaging: Introducing a New
High Throughput Imaging Flow Cytometer from
Omega Biosystems
E. Diebold, J. Lin, K. Owsley, M. Bahr and D. Borlaug.
Omega Biosysts Inc., Los Angeles, U.S.A.
EXCEPTIONAL STUDENT AWARD
PRESENTATIONS
1245 – 1345, TCC
Featured Companies:
ACEA Biosciences Inc. – Tahoma 5, TCC Level 3
BD Biosciences – Tahoma 1, TCC Level 3
Beckman Coulter – Skagit 4, TCC Lower Level
Fluidigm Corporation – Tahoma 3, TCC Level 3
MilliporeSigma – Tahoma 4, TCC Level 3
Thermo Fisher Scientific – Yakima 1, TCC Level 1
Scholars &
Emerging
Leaders
Chair: Mario Roederer
Cochair: Tomáš Kalina
1415 103
Phytoplankton Analysis with Flow Cytometry
G. van den Engh. Crr for Marine Cytometry, Concrete,
WA, U.S.A.
1440 104
Sending Sharks to School: Novel Methods for
Quantifying Cognitive Ability in Fishes
K. Yopak. Univ. of Western Australia
WORKSHOP 16
1600 105
Biological Reference Materials for Enabling
Quantitative Flow Cytometry Measurements in
Antibodies Bound Per Cell
L. Wang, R.A. Hoffman and J.P. Nolan. NIST,
Gaithersburg, MD and Scintillon Inst., San Diego,
U.S.A.
WORKSHOP 17
1600 106
Best Practices for Providing Cell Sorting Services in a
SRL
M. Gregory and M. DeLay. NYU Med. Ctr. and
Cincinnati Children’s Hosp. Med. Ctr., U.S.A.
WORKSHOP 18
1600 107
Workshop on Perivascular Cell Subsets
A. Donneberg, V.S. Donnenberg, J. Phillippi, A.
Brooks and M. Corselli. Univ of Pittsburgh Sch. of
Med., U.S.A., Univ. of Auckland, Australia and BD
Biosciences, San Jose, U.S.A.
WORKSHOP 19
1600 108
Managing Operations in Multi-user, Multi-site and
Consolidated SRLs
C. Groves, R. Walker, C. Dumrese and D. Davies.
MedImmune, Gaithersburg, MD, U.S.A., Babraham
Inst., Cambridge, U.K., Univ. of Zurich, Switzerland
and The Francis Crick Inst., London, U.K.
Index
Abstracts
Oral Session
Abstracts
PLENARY SESSION 2: BEYOND THE
MAINSTREAM
Commercial
Tutorials &
Exhibits
50
WORKSHOP 20
Authors of EVEN Numbered Boards Present
HAPPY HOUR
EXHIBITOR SHOWCASE
POSTER VIEWING
ISAC BUSINESS MEETING
Chair: Joanne Lannigan
Cochair: Lola Martinez
1100 114
Deep Profiling of the Murine Myelopoietic System:
Signaling and Cell Cycle Responses to Neurotropic
Viral Infection Profiled by Flow and Mass Cytometry
(CyTOF)
T. Ashhurst, P. Niewold, D. Cox, A. Smith and N.
King. Univ. of Sydney, Centenary Inst. and
Ramaciotti Facility for Human Systs. Biol.,
Camperdown, Australia
1120 115
Dynamics of T-Lymphocyte Differentiation Revealed
by Tracing Single Cell Proliferative History
Z. Good, N. Vivanco-González, N. Samusik, L.
Borges, G. Nolan and S. Bendall. Stanford Univ.,
U.S.A.
1140 116
Deep Immuno-phenotyping and Functional
Assessment of Leukocyte Responses to M.
tuberculosis Using Cytometry by Time-of-Flight
V. Rozot, S. Gupta, M. van Royeen, M. Hatherill, H.
Maecker and T.J. Scriba. Univ. of Cape Town, South
Africa and Stanford Univ., U.S.A.
1200 117
Complex Modulation of the Immune Response to
Surgery by Immune Enhancing Nutrients
B. Gaudilliere, N. Aghaeepour, M. Angst and G.
Nolan. Stanford Univ., U.S.A.
Poster Session
Abstracts
Chair: Rachel Errington
Cochair: János Szöllõsi
900
111
Light Sheet-Based Illumination Provides for Highly
Corrected, Sensor-Based and Fully Automated
Microscopy
E. Stelzer. Goethe Univ. Frankfurt am Main, Germany
PARALLEL 16: MASS CYTOMETRY
Oral Session
Abstracts
FRONTIERS SESSION 3: HIGH CONTENT
MICROSCOPY
Commercial
Tutorials &
Exhibits
POSTER HIGHLIGHTS TOUR
Scholars &
Emerging
Leaders
Poster
Session
WEDNESDAY, JUNE 15, 2016
Wednesday
15 June
COMMERCIAL EXHIBITS
Tuesday
14 June
POSTER SESSION 2
Monday
13 June
Chair: J. Paul Robinson
1730 110
The Hidden Layer of Regulatory RNA in Human
Development and Cognition
J. Mattick. Garvan Inst. of Med. Res., Sydney,
Australia
COFFEE BREAK
Sunday
12 June
KEYNOTE LECTURE
Saturday
11 June
Special
Lectures
1600 109
Organizing Local Events and Groups; Common Issues
to Overcome
G. Dela Cruz, J. Sederstrom, K. Clise-Dwyer and B.
Daniel. Danish Stem Cell Ctr., Copenhagen,
Denmark, Baylor Col. of Med., M.D. Anderson
Cancer Ctr., Houston and Univ of Texas Hlth. Sci.
Ctr., San Antonio, U.S.A.
930
112
Rapid, High Dimensional Immunohistochemistry
with Next Generation Instrumentation for
Multiplexed Ion Beam Imaging
M. Angelo. Stanford Univ., U.S.A
Congress
Overview
Index
51
Speaker/Author
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Index
Abstracts
P
ARALLEL 17: IMMUNE CELLS, STEM CELLS AND
CANCER BIOLOGY
Chair: Yolanda Mahnke
Cochair: Rachael Walker
1100 118
Immune and Mesenchymal Cells in the CD45+
Compartment of the Adipose Stromal Vascular
Fraction
A. Donneberg, V.S. Donnenberg, J.P. Rubin, E.M.
Meyer, M. Corselli and A. Guar. Univ of Pittsburgh,
Univ. of Pittsburgh Sch. of Med. and BD Biosciences,
San Jose, U.S.A.
1120 119
Combining Imaging and Flow Cytometry to Delineate
Erythropoietic Intermediates
K. McGrath, S. Catherman, K. Fegan and J. Palis.
Univ. of Rochester, U.S.A.
1140 120
CD Maps – Antigen Density Measurements of CD1CD100 on Human Lymphocytes
T. Kalina, D. Kužílková, M. Cuenca, E. Blanco
Álvarez, S.J.W. J. W. Bartol, M. Andres-Perez, M. van
Zelm and P. Engel. Charles Univ. in Prague, Czech
Republic, Univ. of Barcelona, Spain, Univ. de
Salamanca, Spain and Erasmus MC, Rotterdam,
Netherlands
1200 121
Genetically Encoded Fluorescent Proteins for
Tracking Circulating Tumor Cells with Flow
Cytometry
E. Galanzha, D. Nedosekin, M. Juratli, V. Verkhusha
and V. Zharov. Univ. of Arkansas for Med. Sci.,
U.S.A., Frankfurt Univ. Hosps., Germany and Albert
Einstein Col. of Med., U.S.A.
PARALLEL 18: ADVANCES IN
INSTRUMENTATION
Chair: Rui Gardner
Cochair: Yiqing Lu
1100 122
Concentration Dependent Acoustic Positioning of
Nanoparticles in Flow
F. Fencl, J. Lopez, S. Graves and M. Piyasena. Univ.
of New Mexico, Colorado Sch. of Mines and New
Mexico Inst. of Mining and Technol., U.S.A.
1120 123
Acoustophoretic Separation of Mononuclear Cells
from Red Blood Cells in Peripheral Blood
A. Urbansky, A. Lenshof, P. Ohlsson, S. Scheding and
T. Laurell. Lund Univ. and Univ. Hosp. Skåne,
Sweden
52
1140 124
12-Channel Fluorescence Lifetime Flow Cytometry
for Investigation of Protein-Protein Interactions in
Intact Cells
J. Nedbal, I. Rahman, A. Ivetic and S. Ameer-Beg.
King's Col. London, U.K.
1200 125
Droplet-Based Microfluidics for Single-Cell Analysis
D. Eastburn, M. Pellegrino, W. Hyun, A. Sciambi and
J. Yates. Mission Bio, Inc., San Francisco and UCSF,
U.S.A.
PARALLEL 19: STANDARDS AND CALIBRATION
Chair: David Haviland
Cochair: Michael Thomson
1100 126
quantiflash-Based Method for Calibration,
Standardization and Optimization of High Parameter
Fluorescence Flow Cytometers
S. Perfetto, J.C.S. Wood, P. Chattopadhyay, J. Hill, R.
Nguyen, D. Ambrozak and M. Roederer. NIAID, NIH,
Bethesda and Wake Forest Univ. , U.S.A.
1120 127
Synthetic, Cell-Like Particles with Optical,
Fluorescent, and Biochemical Multiplexing
B. Miller, W. Hyun, O. Liu and J. Kim. Slingshot
Biosciences, Everyville, CA and UCSF, U.S.A.
1140 128
Multiplexed Cell-Based Sensors for Assessing the
Impact of Engineered Systems on Cell Health
S. Varma, A. Box and J. Voldman. MIT and Stowers
Inst. for Med. Res., Kansas City, MO, U.S.A.
1200 129
CLEAN Corrects Variations in CyTOF Sample
Preparation
J. Hokanson, R. McCarthy, C. Benton, A. AlRawi and
M. Andreeff. MD Anderson Cancer Ctr., Houston,
U.S.A.
PARALLEL 20: CYTOMETRY DATA ANALYSIS
Chair: Ryan Brinkman
Cochair: Josef Spidlen
1100 130
Multiplexed Imaging Cytometry Analysis Toolbox
Coupled to Imaging Mass Cytometry Reveals Patterns
of Cell Interactions amongst the Heterogeneity of
Breast Cancer
D. Schapiro, H. Jackson, S. Raghuraman, V. Zanotelli,
R. Catena and B. Bodenmiller. Univ. of Zurich and
Life Science Zurich Grad. Sch., Switzerland
ICCS/ESCCA GUEST SYMPOSIUM A
Scholars &
Emerging
Leaders
AWARDS CEREMONY
Master of Ceremonies: John Nolan, Awards
Committee Chair and Past President
Commercial
Tutorials &
Exhibits
Recognition of New ISAC Marylou Ingram Scholars
Poster Session
Abstracts
Sara De Biasi, Postdoc, University of Modena and
Roggio Emilia, Italy
Matthew Linden, Associate Professor of Haematology,
The University of Western Australia, Australia
Jakub Nedbal, Postdoc, King’s College London, UK
Matthew Saunders, Research Scientist, Glycosensors
and Diagnostics, LLC, USA
Yvan Saeys, Principal Investigator, Ghent University,
Belgium
Oral Session
Abstracts
Index
53
Speaker/Author
Chair: Frederic Preffer
Cochair: Jeannine Holden
1530 136
Exosomes Present in Human Tumor
Microenvironments Arrest T- Cell Activation,
Suppress Proliferation, and Block Cytokine
Production
R. Bankert, R. Kelleher Jr., S. Balu-Iyer, J. Loyall, G.
Shenoy, C. Berenson, P. Wallace, J. Tario, Jr. and K.
Odunsi. SUNY at Buffalo and Roswell Park Cancer
Inst., U.S.A.
1555 137
Mass Cytometric Functional Profiling of Acute
Myeloid Leukemia Defines Cell-Cycle and
Immunophenotypic Properties That Correlate with
Known Responses to Therapy
G. Behbehani, N. Samusik, Z. Bjornson, W. Fantl, B.
Medeiros and G.P. Nolan. The Ohio State Univ and
Stanford Univ., U.S.A.
Poster
Session
ICCS and ESCCA are ISAC associated societies that
foster the education, know-how, dissemination,
standardization of flow-cytometry and its
implementation in pre-clinical and clinical cell
analysis. ICCS founded in 1984 to address the
growing need for an exchange of diagnostic and
monitoring information between clinical flow
cytometry laboratories. Today besides its Annual
Meeting and Clinical Cytometry B Journal, has very
active advocacy, standardization and educational
programs. ESCCA represent more than 45 European
countries and in association with numerous European
national societies, ESCCA organizes international
conferences and training courses and national
education program in local languages. Both ICCS and
ESCCA are involved with ISAC in the ICCE
certification program.
ICCS/ESCCA GUEST SYMPOSIUM B
Wednesday
15 June
Chair: Frederic Preffer
Cochair: Hervé Luche
The ICCS/ESCCA symposium hosted by ISAC features
some of the cutting edge technologies from these two
Societies. The talks were selected by both Presidents,
Fiona Craig (ICCS) and Katherina Psarra (ESCCA) and
the Clinical Cytometry Editor Fred Preffer. The Session
highlights recent developments in translational and
clinical cytometry with featured talks by Brent Wood,
Ruth de Tute, Richard Bankert and Greg Behbehani.
They will speak about evolving applications for
multiple myeloma minimal residual disease testing,
the immuno-regulatory impact of tumor derived
exosomes and the application of mass cytometry to
leukemia testing.
Tuesday
14 June
COFFEE BREAK
Monday
13 June
Beckman Coulter Life Sciences – Skagit 4, TCC Lower
Level
Sunday
12 June
Featured Company:
Saturday
11 June
COMMERCIAL TUTORIAL
Special
Lectures
1245 – 1345, TCC
1415 134
Minimal Residual Disease Detection in Acute
Leukemia
B. Wood. Univ. of Washington, U.S.A.
1440 135
Consensus in Minimal Residual Disease Monitoring in
Multiple Myeloma: Development of PlatformIndependent Assays
R. de Tute. Leeds Teaching Hosps. NHS Trust, U.K.
Congress
Overview
1120 131
ggcyto: A New Flow Cytometry Data Visualization
Framework for Bioconductor Based on ggplot
G. Finak, W. Jiang and R. Gottardo. Fred Hutchinson
Cancer Res. Ctr., Seattle, WA, U.S.A.
1140 132
Improving Marker Gene Discovery from HighDimensional Single-Cell Snapshot Data
R. Cannoodt, W. Saelens, K. De Preter and Y. Saeys.
Ghent Univ. and Cancer Res. Inst. Ghent, Belgium
1200 133
Reproducible Reduction: Deterministic tSNE Using
Regression Trees Enables Intra-sample Comparison
M. Stadnisky, S. Siddiq, J. Almarode, J. Quinn and A.
Hart. Flowjo LLC, Ashland, OR, U.S.A.
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Recognition
of New ISAC Shared Resource Lab (SRL)
Emerging Leaders
Suat Dervish, Flow Cytometry Sorter Operator,
Westmead Institute of Medical Research, Australia
Christian Kukat, Head of FACS & Imaging Core
Facility, Max Planck Institute for Biology of Ageing,
Germany
Radhika Rayanki, Research & Development Associate,
MedImmune, USA
Erica Smit, Flow Cytometry Core Manager (Faculty of
Health Sciences, IDM), Senior Scientific Officer
(SATVI), South African TB Vaccine Initiative, Faculty
of Health Sciences, University of Cape Town, South
Africa
Gert Van Isterdael, Flow Core Facility Manager, Staff
employee, Inflammation Research Center, Ghent
University, VIB (Flanders Institute for Biotechnology),
Belgium
To Be Announced:
Cytometry Part A: 2015 Best Paper Award
Distinguished Service Award
Exceptional Student Award Finalists and Winner
Membership Award
Outstanding Poster Awards
President’s Award for Excellence Finalists and
Winner
The Fulwyler Award for Innovation Excellence
CLOSING RECEPTION
Transportation will not be provided.
Ticket required for adminttance. See page 23 for full
details.
Index
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Scholars &
Emerging
Leaders
Poster
Session
1900 – 2300, Seattle Aquarium
54
Monday
13 June
One of the ways this grant will be used to support and
strengthen the ISAC Marylou Ingram Scholars program is by
paying for one day of activities for ISAC Marylou Ingram
Scholars before the start of CYTO, ISAC’s annual Congress.
The first ISAC Marylou Ingram Scholars day will be on
Friday, June 10, 2016.
Tuesday
14 June
Marylou Ingram’s contribution to the field of cytometry
spanned nearly 70 years. Dr. Ingram’s distinguished career
in academic medicine and research included time as faculty
Wednesday
15 June
ISAC Marylou Ingram Scholars 2012-2016
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Enrico Lugli
Principal Investigator, Laboratory of
Translational Immunology
Head, Humanitas Flow Cytometry Core
Humanitas Clinical and Research
Center
Rozzano, Italy
Education
Ph.D., University of Modena and
Reggio Emilia, Italy (supervisor: Prof. Andrea Cossarizza)
Post-doctoral fellow, ImmunoTechnology Section, Vaccine
Research Center, NIAID, NIH, Bethesda, MD USA
(supervisor: Dr. Mario Roederer)
Scholars &
Emerging
Leaders
Participation/Support of ISAC
Within ISAC, I have been involved in a number of activities,
including developing tutorials and day-long pre-congress
courses on mass cytometry, data analysis, and
polychromatic cytometry. I am currently a member of the
Education Committee, and I lead the Electronic Learning (elearning) Task Force. As part of this group, I played a key
role in the early development of our on-line education
portal (CYTO U), and I currently manage the electronic
delivery/production of new CYTO U courses. As an ISAC
Scholar, my colleague Nima Aghaeepour and I applied for
(and won) funding for the first Scholar Collaboration
project, and have developed a proposal for more highprofile presentations of Scholar work at the CYTO meeting.
Poster
Session
Pratip K. Chattopadhyay
Staff Scientist, ImmunoTechnology
Section, Vaccine Research Center,
NIH, Bethesda, Maryland, USA
Education
Bachelors of Arts, Biology: University
of Virginia
Ph.D., Molecular Microbiology and
Immunology: Johns Hopkins
University School of Public Health
Postdoctoral Training: National Institutes of Health
Scientific Interest and Avenues for Collaboration
My work aims to identify the characteristics of T- and Bcells that predict successful immune responses. For this
work, I develop and optimize multi-parametric proteomic
and transcriptomic technologies, collaborating closely with
engineers and bioinformaticians. I then apply these new
technologies to better understand basic T-cell immunology
and the immune response to vaccines or natural disease,
using large cohorts of individuals with well-defined clinical
outcomes (particularly in HIV and EBV-associated diseases).
I have extensive experience collaborating with groups 1)
developing new hardware platforms or reagents, 2)
searching for settings to test their data analysis tools, and 3)
clinicians or epidemiologists interested in comprehensive
characterization of immune responses. I am always eager
to work with new collaborators in these areas.
Sunday
12 June
ISAC recently received a grant from the Wallace H. Coulter
Foundation to support and strengthen the ISAC Scholar
Program. As of receipt of the grant and going forward, the
ISAC Scholar program shall be known as the ISAC Marylou
Ingram Scholars Program, and current and future Scholars
should refer to themselves as ISAC Marylou Ingram
Scholars.
Saturday
11 June
at the University of Rochester, Caltech and the University of
Miami; at Los Alamos National Laboratory; and as a
consultant to the National Cancer Institute, the FDA,
Brookhaven National Laboratory, NASA and other
organizations. She was a pioneer in automated cell analysis,
played a key role in developing automated cell analysis
systems, and was founding director of the Institute for Cell
Analysis at the University of Miami. Dr. Ingram spent two
of her sabbaticals working directly with Wallace Coulter in
the very early days of automated cell analysis, and she
regularly interacted with Wallace Coulter as he developed
new technologies for analysis. From 1982 to 2013, she was
a Senior Research Scientist at the Huntington Medical
Research Institute in Pasadena, California. She headed the
Tissue Engineering & In Vitro Systems program and led a
research program on tumor spheroids with the aim of
creating better models for studying tumor growth and drug
responsiveness.
Special
Lectures
The ISAC Marylou Ingram Scholars program is designed to
enhance the scientific and leadership experiences of
emerging leaders in the field of cytometry. It is a career
development program for those under the age of 40 on the
date of the application. The program provides opportunities
for leadership training, an opportunity to apply for a mentor
training opportunity with another Society member,
presentation opportunities, financial support for
membership in ISAC, as well as other valuable mentoring
activities. In addition, ISAC Marylou Ingram Scholars are
given the opportunity to be an integral part of the Society
through committee memberships, advisory boards, the
opportunity to review manuscripts submitted to Cytometry,
Part A (the official journal of ISAC) and creating educational
material to further support the goals of ISAC. Being chosen
as an ISAC Marylou Ingram Scholar is an indication that you
are recognized for your leadership potential, scientific skills
and ability to achieve your career goals.
Congress
Overview
ISAC Marylou Ingram Scholars
Index
55
Speaker/Author
Index
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Scholars &
Emerging
Leaders
Poster
Session
Wednesday
15 June
Tuesday
14 June
Monday
13 June
Sunday
12 June
Saturday
11 June
Special
Lectures
Congress
Overview
Scientific
Interest and Avenues for Collaboration
x
Mechanisms of memory T cell differentiation and
maintenance in humans
x
Cytokine-based cancer immunotherapy
x
Immune reconstitution in acquired
immunodeficiencies (bone marrow
transplantation, HIV infection)
Organization committee of CYTO 2014
Attended CYTO 2007, 2012, 2013, 2014
ISAC-sponsored flow cytometry workshop @ ESID
(Prague, Nov. 1-2, 2014)
Cytometry Part A Editorial Board
Member of the Scientific Communications
Committee
Gergely Toldi
clinical fellow, Birmingham Women's
Hospital, Birmingham, UK
assistant professor, First Dept. of
Obstetrics and Gynecology, Semmelweis
University, Budapest
Education
MD (2011), PhD (2012), Board
Certification in Pediatrics (2015),
Specialty Training in Neonatology (2015-present)
T lymphocyte activation, kinetic flow cytometry,
autoimmunity, immunology of pregnancy and neonates
CYTO 2013, 2014, 2015 & 2016: Program Committee
Member, Session Chair
Outstanding Poster Award, CYTO 2012
Reviewer for Cytometry A
Michael Zordan
Staff Engineer, Sony Biotechnology,
Inc.
San Jose, CA
Education
Duke University B.S.E. Biomedical
Engineering
Purdue University. Ph.D. Biomedical
Engineering
Post-Doctoral Fellow, Purdue
University, Laboratory of Prof. J. Paul Robinson
My research interests are in the design and development of
novel cell analysis instruments and technology. I would be
interested in collaborating with anyone interested in
developing their technology or with researchers to design
technology to perform novel measurements they cannot
currently perform.
Member of ISAC Data Standards Task Force
Co-Chaired Parallel Sessions at CYTO 2013 and CYTO
2014
Invited Speaker, GLIIFCA 2013
Invited Speaker, Metroflow 2014
56
Qianjun Zhang
General Manager, Emerald Biotech,
located in Hangzhou, China
Education
M.S., University of Colorado Health
Sciences Center
Scientific Interest and Avenues for
Collaboration
I am enthusiastic about the new
technology within flow cytometry and
also dedicated to enhancing the training and knowledge of
flow techniques within resource limited areas such as South
East Asia and China. I have helped organize a few
international flow cytometry workshops and served as
faculty teaching at those venues. I would like to continue
contributing and help the educational committee with my
resource and expertise.
ISAC Live Education Delivery Task Force member (2013 –
present)
Actively involved in CYTO 2012, 2014
Co-Chaired Parallel Sessions at CYTO 2015
Serve as abstract reviewer for CYTO 2016
Website
www.linkedin.com/in/qianjunzhang/
Nima Aghaeepour
Postdoctoral Fellow, Stanford
University
Education Ph.D., Bioinformatics,
University of British Columbia, 20082012 B.Sc., Computer Science,
University of Tehran, 2003-2008
Collaboration: Nima Aghaeepour is
currently a postdoctoral fellow in Garry
Nolan’s laboratory at Stanford University, a Fellow of the
Canadian Institute of Health Research, and an Ann
Schreiber Mentored Investigator of the Ovarian Cancer
Research Fund. His research is focused on computational
analysis of single cell proteomics and genomics data to
enable a systems level understanding of complex cancers as
well as hematologic and immune system malignancies.
He currently contributes to ISAC as a member of the finance
and CYTO innovation committees and was a former of the
scientific communications committee and the CYTO
conference’s program committee. He has also chaired
several CYTO parallel sessions, has co-organized the
FlowCAP parallel sessions, workshops, and tutorials, and
was an invited faculty in ISAC’s pre-congress advanced data
analysis course.
Website: http://linkedin.com/in/naghaeep
Anis Larbi
Principal Investigator, Immunity and
Aging Program, Singapore Immunology
Network (SIgN) Director, A*STAR Flow
Cytometry Platform Associate
Professor, Department of Geriatrics,
Faculty of Medicine, University of
Sherbrooke, Canada
Education
2005 - PhD Immunology: Research
Center on Aging, Laboratory of immunology and signaling,
Sherbrooke, Faculty of Medicine, University of Sherbrooke,
Canada
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Index
57
Speaker/Author
Saturday
11 June
Katarzyna Piwocka
Associate Professor, Nencki Institute of
Experimental Biology
Head of the Laboratory of Cytometry
Head of the Department of
Biochemistry
Education
2013 Habilitation in Biological
Sciences, Nencki Institute, Warsaw,
Poland
Bruno Diaz Paredes
Researcher I
Hospital São Rafael – Center of
Biotechnology and Cell Therapy
(CBTC)
Salvador City, Bahia - Brazil
Education
Graduate of Biology Sciences –
Biomedicine Module in Federal
University of Rio de Janeiro (UFRJ)
Master’s degree in Physiology and Doctor’s degree in
Sciences at Carlos Chagas Filho Biophysics Institute (UFRJ)
Research Technologist in Flow Cytometry sector of National
Center of Bioimage (CENABIO)
Assistant Professor in Department of Gastroenterology and
Hepatology of Yamaguchi University
Stem cells (adult, embryonic, iPS) for liver, neural and
cardiac diseases; Immunology of stem cells; Innovation on
Cell-based therapies; Cell sorting.
Attended 4 CYTO meetings (2011-2014)
Participation as poster’s referee on CYTO meetings
Co-coordinator of 1st ISAC Scholars and SRL Emerging
Leaders Business Meeting
Website
http://lattes.cnpq.br/0347294341083336
Special
Lectures
Sung Hwan Cho
CTO, NanoCellect Biomedical Inc.,
located in San Diego, CA, USA
Education
Ph.D., University of California San
Diego (UCSD), USA
Research Engineer at West Wireless
Health Institute, San Diego, CA, USA
CTO, Principal Investigator at
NanoCellect Biomedical Inc.
As an inventor of the core technology of the company
(NanoCellect), I am interested in integrated microfluidic
systems where fluidics, optics, electronics and acoustics all
work together synergistically to develop next generation
flow cytometer and medical devices.
ISAC Membership Committee member (2013 – present)
Organization committee of CYTO 2013, 2014, 2015
Attended CYTO 2012, 2013, 2014
Website
http://www.linkedin.com/pub/sung-hwan-cho/6/140/108
2001 PhD with honours in Biochemistry, Nencki
Institute, Warsaw, Poland
1994 MSc in microbiology, Biology Department, Warsaw
University, Poland.
Post-graduate training:
2003-2004 Cell Development and Disease Laboratory,
BioSciences Institute, University College Cork, Ireland
My scientific interest concerns the investigation of
prosurvival mechanisms promoting leukemia development
and resistance to therapy as well as looking for novel potent
therapeutic targets to treat leukemia. A major focus of my
research is related to signaling pathways regulating cellular
stress response, chromosomal instability and aneuploidy as
well as the cross-talk of leukemia cells with the
microenvironment. These aims are realized using in vivo
and in vitro models, including co-culture studies. We use
flow cytometry mostly to study apoptosis/viability, cell cycle
and proliferation, signal transduction, cell tracking, RNA
detection, immune phenotyping and many others. We are
looking for collaboration in the field of cancer biology,
signal transduction and leukemia microenvironment.
Member of the Program Committee - CYTO 2014,
CYTO 2015, CYTO 2016
Meeting attendance: CYTO 2010 (oral
presentation), CYTO 2011, CYTO 2012, CYTO
2013, CYTO 2014, CYTO 2015 (oral presentation),
CYTO 2016 (oral presentation)
CYTO 2014, CYTO 2015, CYTO 2016 - Co-Chair
of the Parallel Sessions
CYTO 2014 - Organization of the ISAC Marylou
Ingram Session
2007
ISAC membership for prospective young
researchers
Website
http://cytometry.nencki.gov.pl/
Congress
Overview
2000 - BSc Biology, Claude Bernard University, IUT-A,
Lyon 1, France
Scientific Interest and Avenues for Collaboration I am
interested in human aging and the major component we are
focusing on is immunology. We are developing an immune
mapping during human aging. This involves studying
circulating as well as resident immune cells. The associated
functionality of cells is used as a surrogate marker for onset
and/or progression of age-associated diseases. Our interest
goes from phenotyping immune cells, understanding subset
functionality, identifying biomarkers in health and diseases
which can be found in >100 of my publications. My
laboratory is a key player in the field of biology of aging in
Singapore and we intend to compete at the international
level. For this, we have developed world-class cohort
studies on aging, unique technological platforms, one of
them being the flow cytometry, and gathered collaborations
with key scientists elsewhere. We are opened to
collaboration in immunology, immunosenescence,
immunophenotyping, and cohort studies.
I have been involved in the development of modules of the
CYTO U. I organized ISAC Scholar's event at CYTO2015
and currently involved in disseminating flow cytometry in
Asia. I co-Chaired Parallel Sessions at several CYTO
conferences and organizing a workshop this year on “Flow
Cytometry for cohort, longitudinal and multi-center
studies”. With my fellows in Singapore we hope to offer
soon a better visibility to ISAC in Asia by offering CYTO-like
events.
Website
http://www.a-star.edu.sg/sign/Research/PrincipalInvestigators/tid/11/LARBI-Anis.aspx
Index
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Scholars &
Emerging
Leaders
Poster
Session
Wednesday
15 June
Tuesday
14 June
Monday
13 June
Sunday
12 June
Saturday
11 June
Special
Lectures
Congress
Overview
Er Liu
Senior Scientist, Roche Tissue
Diagnostics, Tucson, Arizona
Education
B.S. Chemical Engineering, Wuhan
University of Technology, 2001
M.S. Biomedical Engineering, Wuhan
University of Technology, 2004
Ph.D. Biomedical Engineering, Rutgers
University, 2011
Interested in bioanalytical instrumentation development
(e.g. portable fluorescence imager, next generation
flow/image cytometer, nano-particle analyzer and
sequencing instrumentation). Developing multiplex-able
screening/single cell analysis platform for cancer profiling
and drug screening.
2013
CYTO 2013 conference President’s Award of
Excellence
2013
Program committee, CYTO 2013: ISAC’s XXVIII
International Congress.
2014
Program committee, CYTO 2014: ISAC’s XXIX
2015
Program committee, CYTO 2015: ISAC’s 30th
Frank Schildberg
Investigator – Harvard Medical School,
Boston, MA, USA
Education
Ph.D. (Immunology), University of
Bonn, Germany
Postdoctoral Fellow, Dana-Farber
Cancer Institute, Boston, MA, USA
Collaboration
A liver immunologist by training, I work on different cellular
and molecular aspects of how our immune system is
regulated and how this knowledge could be translated into
novel therapies for infectious diseases, autoimmune
diseases, cancer immunotherapy, transplantation and
fibrosis. A major focus of my research is related to stromal
cell biology, myeloid derived suppressor cells, coinhibitory
molecules and microvesicles, and how these entities shape
our immune responses. My research involves advanced
genetic in vivo mouse models, functional assays with
human tissue samples, multiplex biomarker assays and
several cutting-edge cytometric analyses. I am always eager
to collaborate with new partners from industry and
academia in these areas.
Member of Parallel Session Committee (2016)
Member of Cyto A-ISAC Scholar Mentorship Program (2015
– present)
Reviewer for Cytometry Part A (2015 – present)
Parallel Session Co-chair (2015 – present)
Member of Membership Services Committee (2014 –
present)
Member of Program Committee (2014 – present)
CYTO attendee (2012 – present)
58
Website
https://www.linkedin.com/in/frankschildberg
https://www.researchgate.net/profile/Frank_Schildberg
Joseph D. Tario, Jr.
Senior Flow Cytometry Specialist,
Department of Flow and Image
Cytometry
Education
PhD (Pathology)
BS (Biology)
Collaboration
I have employed flow cytometry to make contributions in
the areas of dendritic cell biology, cell tracking &
proliferation, cancer immunotherapy, pathology,
transplantation, extracellular vesicles, and molecular
cytometry. I am also interested in the use of flow cytometry
as a clinical tool for the diagnosis of hematologic
malignancy.
Participation / Support of ISAC
ISAC / CYTO U eLearning Delivery Task Force, Member
ISAC / CYTO U Image Cytometry Content Task Force,
eLearning Liaison
CYTO Program Committee, Member
CYTO Organizing Committee, Member
CYTO Workshop Committee, Member
CYTO Parallel Session Committee, Chairperson
Plenary Session Co-chair, CYTO 2016
Parallel Session Co-chair, CYTO 2015
Invited Speaker, CYTO 2014
Invited Speaker, CYTO 2013
Attendance: CYTO 2010, CYTO 2011, CYTO 2013, CYTO
2014, CYTO 2015, CYTO 2016
Michael Halter
Scientist, National Institute of
Standards and Technology
Education
Ph.D. in Bioengineering from the
University of Washington at Seattle in
2004
B.S. in Materials Science and
Engineering from Purdue University at
West Lafayette in 1998
-Live cell imaging of stem cell populations
-Engineering fluorescent protein gene reporter stem cell
lines
-Modeling gene expression dynamics in pluripotent stem
cells
-Quality control for quantitative optical microscopy
CYTO U Course on Quality Control for Optical Microscopy
Attendance at CYTO conferences since 2008
Website
http://www.nist.gov/mml/bbd/cell_systems/michael_halter.cf
m
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Index
59
Speaker/Author
Hervé Luche
R&D Manager at Center for
Immunophenomics (CIPHE), FRANCE
Education
Ph.D. in Immunology, Medical School
of Ulm, GERMANY
Post-doctoral fellow on T cell and
dendritic cell development in the
laboratory of B&M MALISSEN,
FRANCE
Scientist at CIPHE, US-012, INSERM
I am interested in trying to understand how immune cells
integrate signals and communicate with their environment
to actually change their transcriptional programs, fate or
acquire a new function. I use a combination of mouse
genetics with high content multi-parameter flow and mass
cytometry as well as transcriptomic analysis. I am looking
for collaborations in the field of meta-analysis and data
integration of multiple datasets.
Attended CYTO 2012, 2014, 2015, 2016
Parallel session chair (2015-2016), workshop evaluator
(2016), and poster judge (2014)
Volunteered for French translation of CYTO-U tutorial
material
Website
https://www.linkedin.com/profile/view?id=30427463&trk=n
av_responsive_tab_profile
http://ciphe.marseille.inserm.fr/
Tuesday
14 June
Education
2004 Ph.D., University of Peking, Beijing, P.R.China
2004-2008 Postdoctoral Researcher at MRC-Protein
Phosphrylation Unit with Prof. Dario Alessi, Dundee, U.K.
2008-2014 Senior Research Associate at CRUK Manchester
Institute with Dr. Tim Somervaille, Manchester, U.K.
My lab has recently identified and validated few potential
candidates as therapeutic treatment targets in Acute
Myeloid Leukaemia (AML). (Huang et al. Leukaemia 2014,
Harris and Huang et al. Cancer Cell 2013). The future
research will focus on epigenetic regulation in leukaemia
cancer stem cells, and continue to identify novel pathways
and targets in leukaemia by using combinations of different
systems biology approaches and by exploring novel
polyomics methods in the study of cancer stem cells.
Attended CYTO 2014
Presented short talk at scholar evening session at CYTO
2014
Website
http://www.gla.ac.uk/researchinstitutes/cancersciences/resea
rch/units/paulogormanleukaemiaresearchcentre/xhuang
Monday
13 June
Xu Huang
Lecturer/Principal Investigator of
Haemato-Oncology/Systems Medicine
Group
Paul O’Gorman Leukaemia Research
Centre
Institute of Cancer Sciences, MVLS
University of Glasgow
Glasgow, Scotland
U.K.
Sunday
12 June
Jessica P. Houston
Associate Professor
Department of Chemical & Materials
Engineering
New Mexico State University, Las
Cruces, NM
Education
B.S., Chemical Engineering, New
Mexico State University, 2000
M.S. Chemical Engineering Texas
A&M University, 2002
Ph.D. in Chemical Engineering, Texas A&M University,
2005
Director’s Postdoctoral Fellow, Los Alamos National
Laboratory, 2006
Flow cytometry instrumentation development; fluorescence
lifetime measurements; fluorescence decay kinetics; phasesensitive flow cytometry
Chair, Scientific Communications Committee
ISAC Councilor Term 2014-2018
Program committee member, session chair, workshop
evaluator, presenter, and poster judge: CYTO 2016
Website
http://dept-wp.nmsu.edu/flowcytometry/
Saturday
11 June
Special
Lectures
applications. I am particularly interested in high-throughput
techniques for rare-event detection and multiplexed analysis
using tunable photoluminescence. Currently, I am
investigating new nanophotonic approaches for early-stage
diagnosis of cancers and infectious diseases.
ISAC membership since 2011
Member of the E-Learning Delivery Task Force
Program Committee of CYTO 2014, 2015 and 2016
Website
http://web.science.mq.edu.au/directory/listing/person.htm?i
d=ylu
Congress
Overview
Yiqing Lu
Macquarie University Research Fellow
ARC Centre of Excellence for
Nanoscale BioPhotonics (CNBP)
Macquarie University, Australia
Education
I completed my B.Eng. degree in
Electronic Engineering from Tsinghua
University, and PhD degree in the
cross-disciplinary area of bio-nanophotonics with a joint program between Tsinghua
University and Macquarie University.
My research is centred on the development of analytical
methods and instrumentation for biomedical and photonic
Yolanda Mahnke
Flow Cytometry Consultant
Education
BSc in Biology; Imperial College of
Science, Technology and Medicine,
University of London, London, UK
PhD in Immunology with Prof. V
Schirrmacher; The Open University,
London, UK; and Dept. of Cellular
Tumour Immunology, German Cancer Research Center,
Heidelberg, Germany
PostDoc with Prof. V Schirrmacher, Dept. of Cellular
Tumour Immunology, German Cancer Research Center,
Heidelberg, Germany
PostDoc with Prof. P Romero, Div. of Clinical OncoImmunology, Ludwig Institute for Cancer Research,
Lausanne Branch, Lausanne, Switzerland
PostDoc with M Roederer, ImmunoTechnology Section,
Vaccine Research Centre, National Institutes of Allergies
and Infectious Diseases, National Institutes of Health,
Bethesda, MD, USA
Senior Research Investigator, Translational and Correlative
Studies Laboratory, University of Pennsylvania,
Philadelphia, PA, USA
I have a long-standing interest in multi-parameter flow
cytometry, and used this tool to study the evolution of T-cell
responses as well as other immune or tumour cell types in
the context of tumours, infections, vaccinations, or cellular
therapies. I now assist and train others in building multicolour reagent panels, as well as in data analysis and
interpretation.
CYTO meeting attendance 2011, 2014, 2015
Developed a novel publication platform in
Cytometry A for the publication of optimized
multicolor immunofluorescence panels (OMIP),
together with Mario Roederer and Pratip
Chattopadhyay
Ad hoc reviewer for Cytometry A
Taught at pre-CYTO Workshop 2011
Lectured at CYTO 2011
Judged posters at CYTO 2014, 2015
Scholars &
Emerging
Leaders
Poster
Session
Wednesday
15 June
Tuesday
14 June
Monday
13 June
Sunday
12 June
Saturday
11 June
Special
Lectures
Congress
Overview
Co-chairing a parallel session at CYTO 2015, 2016
Workshop evaluation CYTO 2016
Carolina Wählby
Professor in Quantitative Microscopy,
Centre for Image Analysis
Dept. of Information Technology and
Science for Life Laboratory
Uppsala University, Uppsala, Sweden
Education
MSc in Molecular Biotechnology,
Uppsala University, 1998, PhD in Digital
Image Analysis with thesis on ’Algorithms for Applied
Digital Image Cytometry’ at the Dept. of Information
Technology, Uppsala University, 2003. PostDoc at the
Dept. of Genetics and Pathology, Uppsala University
Associate Professor in Digital Image Processing 2009.
Principal Investigator, Imaging Platform of the Broad
Institute of Harvard and MIT
Cambridge, MA, USA 2009-2015.
My research is focused on development of algorithms for
image analysis of microscopy data with applications in
biomedical research. This includes contributing to Broad’s
free and open-source CellProfiler project, and development
of advanced methods to quantify and mine information in
microscopy images, primarily from high-throughput
experiments and tissue samples. I’m interested in
collaborations in the exciting intersection between
computer science and biomedicine as microscopy is
becoming a quantitative measurement tool, and not only a
means of visual observation. I now lead a research group at
Uppsala University, with support from SciLifeLab Sweden,
the Swedish research council, and the ERC.
I’m interested in having a more active part in the ISAC
community by attending meetings and networking activities.
I was a member of the CYTO 2016 Program Committee.
Website
http://www.cb.uu.se/~carolina/
Greg Finak
Vaccine Immunology Statistical Center
Methods Development Lead
Senior Staff Scientist
Department of Biostatistics,
Bioinformatics and Epidemiology
Vaccine and Infectious Disease
Division
Fred Hutchinson Cancer Research
Center
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Index
Abstracts
Seattle, WA
Education
MSc in Biochemistry, NMR protein structure, 2002, McGill
University, Montreal, Canada.
PhD in Bioinformatics, Thesis on functional genomics of
breast cancer tumor-stroma interactions. 2008, McGill
University, Montreal, Canada.
Post-doctoral researcher at the IRCM (Institut de Recherches
Cliniques de Montreal) 2008-2010 and at the Fred
Hutchinson Cancer Research Center (2010-2012), focused
on computational flow cytometry including automated
60
gating, quality control, and infrastructure for data sharing
and reproducibility.
I develop computational software and statistical methods for
analysis of high throughput immunological assay data (e.g.
single-cell RNASeq, flow cytometry, Fluidigm Biomark,
CyTOF etc.) applied to immune correlates discovery in
vaccine trials. Interesting study designs, novel assays, and
interesting data sets that pose analytic, computational and
statistical challenges are of interest.
Website
https://www.fredhutch.org/en/labs/profiles/finak-greg.html
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Index
61
Speaker/Author
Saturday
11 June
Raluca Aura Niesner
Group leader, Biophysical Analytics
German Arthritis Research Center,
Berlin (DRFZ, Berlin), Germany
Education
2001 – 2005
PhD thesis in
Biophysical Chemistry at TU,
Braunschweig
May
Ma
ay 2001
20
00
01
1
Diploma in Chemistry at TU
Braunschweig
Scientific Interests and Avenue for Collaboration
The development of new techniques which better meet the
general requirement in biosciences and biomedicine to
monitor vital processes at high spatial and temporal
resolution, on a molecular basis, in their genuine
environment is of central relevance for biophysics in
general, and for microscopy in particular. In this frame, we
focus on the improvement and extension of multi-photon
laser-scanning microscopy (MPLSM) with application to
intravital microscopy (imaging in living rodents) to better
understand immunological processes in different disease
contexts. Thereby, we focus on the improvement of optical
performance, e.g. spatial resolution, imaging depth,
photobleaching/ phototoxicity, imaging time window, and
of molecular selectivity to monitor cell and tissue functions
by time-resolved fluorescence techniques.
Josef Spidlen
Staff Scientist, BC Cancer Agency
Vancouver, BC, Canada
Education
Postdoctorate Fellow, 2005 - 2008, BC
Cancer Agency, BC, Canada
Ph.D. in Biomedical Informatics, 20022005, Charles University in Prague,
First Faculty of Medicine, Czech
Republic
M.Sc. in Computer Science, 1996-2002, Charles University
in Prague, Faculty of Mathematics and Physics, Czech
Republic
My scientific interest and research are focused on the
development and application of software tools for
automated high throughput cytometry data analysis. My
formal training is in computer science and software
engineering, which I have been applying in the fields of
medical informatics and computational biology. I am the
author and maintainer of several R packages, and the author
of a number of flow cytometry data analysis tools, including
most of the modules in the GenePattern Flow Cytometry
Suite, and the FlowClean FlowJo plugin. I am also the main
author of most recent ISAC data standards, a.k.a. file
formats, such as FCS, Gating-ML, CLR, ICEFormat. Finally, I
am the chief developer of ISAC-supported flow cytometry
data repository - FlowRepository.org. I am always excited to
engage in collaborations allowing me to apply my
computational background to address data analysis
challenges and to gain novel biological insights.
ISAC Data Standards Task Force: first author of most ISAC
data standards
FlowRepository Steering Committee: Chief Developer of
FlowRepository
Member of the CYTO 2016 Parallel Session Committee
Attended several past CYTO conferences, taught at
workshops, helped by chairing sessions and judging posters
Website
http://spidlen.ca
Special
Lectures
Elisa Nemes
Senior Scientist
South African Tuberculosis Vaccine
Initiative
Cape Town, South Africa
Education
MSc in Medical Biotechnology and
PhD (topic: Immunological effects of
antiretroviral therapy interruption in
HIV infection) from University of
Modena and Reggio Emilia, Italy.
x Clinical trials of novel tuberculosis vaccines
x Prospective immunological biomarkers of risk of
tuberculosis disease and immune reconstitution
inflammatory syndrome
x Paediatric immunology
x Development and standardization of novel assays to
measure immune cell function and phenotype
Attendance at CYTO 2014 (oral presentation), 2015 (session
co-chair), 2016 (abstract and workshop review, poster
presentations).
Website
http://www.satvi.uct.ac.za/
Invited talk: CYTO2011, CYTO2014
Talk: CYTO2013
Chair: CYTO2012, CYTO2014, CYTO2015
Reviewer: CYTO2012, CYTO2014, CYTO2015, CYTO2016
Organizing committee: CYTO2011, CYTO2014
Congress
Overview
Pia Kvistborg
Faculty member at the Netherlands
Cancer Institute
Education
PhD in Tumor immunology from
University of Copenhagen
Collaboration
Main interest is to dissect the
(dys)functional state of tumor-specific T cells in cancer and
how anti-cancer therapies may influence this state using
RNA sequencing and high-dimensional flow cytometry. I
primarily assess this using patient material obtained during
clinical trials.
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Index
Abstracts
Sara De Biasi
Post Doc, Department of Surgery,
Medicine, Odontoiatrics and
Morphological Sciences
University of Modena and Reggio
Emilia, Modena, Italy
Education
Ph.D., University of Modena and
Reggio Emilia, Italy (supervisor: Prof.
Special
Lectures
Congress
Overview
Andrea Cossarizza)
Post-doctoral fellow, Department of Surgery, Medicine,
Odontoiatrics and Morphological Sciences (supervisor: Prof.
Andrea Cossarizza)
I am interested in the dynamics of T cell homeostasis and
functions in conditions of acquired immunodeficiencies,
with a special focus on HIV infection and solid organ
transplantation and related therapies. More recently, my
work focused on multiple sclerosis as a model to study
autoimmunity. In particular, I am investigating the role of
innate-like T cells (iNKT cells), rare cells populations among
peripheral blood mononuclear cells. In addition, I took
advantage of the experience acquired in the field of rare
events detection such as iNKT cells to study circulating
endothelial cells (CEC), their precursor (EPC) in different
type of cancers.
Attended:
CYTO 2012: Evidences for Autophagy in SW872
Adipocytic Cells Treated with Atazanavir (Poster
presentation).
CYTO 2013: Complex changes in invariant Natural
Killer T (iNKT) cells in patients with different clinical
forms and treatments of Multiple Sclerosis (Oral
presentation).
CYTO 2014: Fine tuning of Treg and iNKT cells after
treatment with Fingolimod in Multiple Sclerosis
patients (Oral presentation). Principal Component
Analysis allows to cluster patients with Multiple
Sclerosis on the basis of different subsets of CD8+ and
iNKT cells (Oral presentation).
CYTO 2015: Polyfunctional Response of Invariant
Natural Killer T Cells in Patients Affected by Multiple
Sclerosis Displays Th-1 and Th-17 Profiles (Outstanding
poster Award). Inhibition of Lon protease by
triterpenoids alters mitochondria and is associated to
cell death in human cancer cells (Poster prensentation).
iNKT Cells and Their Subpopulations Are Not Restored
in HIV+ Patients with Low CD4/CD8 ratio after
Prolonged Effective Therapy, and Display a
Pronounced Th-1 and Th-17 Pro-inflammatory Profile
(Poster prensentation).
Attendance at CYTO 2016: High Level of Activation and
Altered Mitochondrial Functionality in T Cells from Patients
with Progressive Forms of Multiple Sclerosis (Oral
presentation)
62
Matthew Linden
Associate Professor of Haematology
School of Pathology and Laboratory
Medicine
University of Western Australia
Nedlands, WA, Australia
Education
BSc Hons PhD W.Aust
I earned my PhD in haematology from The University of
Western Australia in 2003 and joined the Centre for Platelet
Function Studies at The University of Massachusetts
Medical School (now the Centre for Platelet Research and
Harvard Medical School) initially as a postdoctoral fellow
and then research instructor. In 2007 I returned to Australia
as an academic at RMIT University in Melbourne, where I
launched the RMIT Flow Cytometry core facility in 2009. In
2012 I was recruited to the University of Western Australia
as academic head of the cytometry core at the Centre for
Microscopy, Charactisation and Analysis (a node of the
National Microscopy and Microanalysis Research Facility).
In 2015 I took up an associate professorship of haematology
at the same institution in order to develop postgraduate
haematology coursework and progress research on platelets
systems biology.
I am a highly motivated and well-rounded academic with a
passion for cytometry. I hold significant roles spanning
teaching, research and leadership of cytometry in Australia.
Working at the interface of the shared resource laboratory,
discovery and translational research, I have developed
novel cytometry techniques for the measurement of blood
platelets and employed these in the development of new
antiplatelet therapies. I am committed to advancing
cytometry through strong, sustainable shared resource
laboratories and cutting edge research. Through
participation in the ISAC Scholar program, I seek to expand
across research, education and leadership of cytometry at
an international level.
My research focuses on two main fields; advancing
understanding of blood platelets in health and disease, and
advancement and development of cytometry tools for cell
biology. I have developed widely adopted flow cytometric
methods for the laboratory monitoring of antiplatelet
therapy, applied these to develop novel approaches to
inhibition of platelet function, and extended this research
and developed cytometric methods for measuring the
interaction of platelets with immune cells. I also develop
novel flow, imaging and mass cytometry approaches which
broadly underpin research in other areas of biological and
medical sciences.
I am also involved in cytometry education at both
undergraduate and postgraduate levels. Through an
innovative blend of theoretical and practical learning, I seek
to motivate, inspire and influence student learning with a
practically focused curriculum. I provide a strong
foundation in the diagnosis of haematological malignancies
by standard 6 – 8 colour flow cytometry, but also introduce
emerging developments in cytometry, so that my graduates
(as the next generation of clinical cytometrists) might
champion and lead the translation of these novel research
tools to the clinical laboratory.
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Yvan Saeys
Associate Professor, Ghent University
Group leader, Data Mining and
Modeling for Biomedicine group at the
VIB Inflammation Research Center
Ghent, Belgium
Education
I obtained my PhD in computer science
from Ghent University, and after
spending time abroad at the University
of the Basque Country and the Université Claude Bernard
(Lyon 1, France) I obtained an FWO postdoctoral fellowship
at Ghent University. Since 2015 I am an Associate
Professor at the faculty of medicine at Ghent University and
a Principal Investigator at the VIB Inflammation Research
Center. I am heading the Data Mining and Modeling group,
an interdisciplinary research team of 15 people, consisting
of mathematicians, computer scientists, engineers and
bioinformaticians.
The main research theme of my group is the design and
application of data mining and machine learning techniques
for high-throughput single-cell technologies. These include
flow and mass cytometry based techniques but also singlecell transcriptomics platforms. Our group developed the
FlowSOM and FloReMi R packages for flow cytometry
visualization and biomarker discovery, and obtained the
best results at the FlowCAP IV challenge on predicting HIV
to AIDS progression. We are actively welcoming
collaborations with wet-lab groups that would like to
explore computational flow cytometry, as well as
experimentalists that design new technologies and willing to
explore the potential of machine learning techniques for
data analysis and interpretation.
I participated in Cyto 2014, where I enjoyed the data
analysis sessions and FlowCAP discussions. At Cyto 2016
my group has both an oral and a poster presentation. I
would also like to take this ISAC scholarship as an
opportunity to get more actively involved in the society. In
particular I would like to actively promote computational
flow cytometry and assist in training the next generation of
scientists to get acquainted with the novel tools that
computational flow cytometry offers.
Website
http://www.dambi.ugent.be
Saturday
11 June
Poster Session
Abstracts
Index
63
Speaker/Author
Special
Lectures
Jakub Nedbal
Post Doc, Division of Cancer Studies
and Randall Division of Cell and
Molecular Biophysics, King's College
London
Education
2012
King's College London, UK,
PhD Molecular Immunology
2007
King's College London, UK,
MSc Immunology
2006
Masaryk University Brno, Czech Republic, MSc
Biophysics
Development of prototype flow and image cytometry
instruments and their applications in novel biological
experiments are my research interests. Specifically, I
develop new tools enabling fluorescence lifetime and
anisotropy spectroscopic readouts of photophysical states of
fluorophores inside cells. These techniques find most
applications in Förster resonant energy transfer (FRET)
measurements probing biomolecular interactions inside
intact cells, such as protein di-/oligomerization, signalling
pathway activity or receptor-ligand and drug-target
interactions. My current focus is on three novel techniques,
listed by their increasing complexity:
Compact open-source 3D printed fluorescence lifetime flow
cytometer.
3-laser, 12-channel fluorescence lifetimes flow cytometer.
Fluorescence lifetime imaging and sorting platform for
patient-derived circulating tumor cells.
This effort can only be justified if the developed techniques
can prove themselves by new biomedical discoveries made
in collaboration with researchers experiencing unmet
methodological needs. Collaborators must be brave enough
to accept the risks and teething problems associated with
pre-commercial techniques, but hopefully will be rewarded
with novel discoveries and the opportunity to shape future
of a segment of flow cytometry.
To maximize chance of success, my work in the field of
fluorescence lifetime flow cytometry draws inspiration, tools
and experiences from the mature field of fluorescence
lifetime imaging. Furthermore, I design my instruments to be
efficiently replicable, amendable and transferable to give a
potential collaboration a head start.
If you have questions or ideas regarding FRET, fluorescence
spectroscopy in cells, fluorescence lifetime imaging or flow
cytometry, please find me at CYTO or drop me a line.
CYTO 2014 Poster presentation
CYTO 2015 Oral presentation (President's Award for
Excellence)
CYTO 2016 Oral presentation (12-Channel Fluorescence
Lifetime Flow Cytometry for Investigation of Protein-Protein
Interactions in Intact Cells)
Congress
Overview
I was chair the 38th Annual Scientific Meeting of the
Australasian Cytometry Society (ACS) (the Australian affiliate
of ISAC), which I hosted in Perth in 2015
(http://cytometry2015.org.au/) . This four day conference,
opened by Nobel Laureate Professor Barry Marshall and
featuring keynote presentations by Dr Mario Roederer, Prof J
Paul Robinson and Dr Jonathan Irish (and many others) was
amongst the largest and most successful cytometry meetings
in the Southern Hemisphere.
I continue to be an active member of ISAC, having attended
both the 2014 and 2015 conferences. I look forward to
expanding my involvement in ISAC through the Scholar’s
program, and contributing to the working groups and
conference planning. In 2016 I have submitted 2 abstracts
for consideration at CYTO, one outlining the first method
for analysis of platelet function by mass cytometry, while
the second outlines a method for multiparameter imaging of
surface and intracellular molecules by secondary ion mass
spectrometry (SIMS).
Website
http://www.web.uwa.edu.au/people/matthew.linden
Matthew Saunders
Research Scientist
Glycosensors and Diagnostics, LLC
Athens, GA/San Diego, CA
Education
B.S. Biochemistry, University of
Oregon, 2002
Ph.D. Biomedical Science,
University of New Mexico, 2009.
Graduate research support from Los Alamos National
Laboratory, National Flow Cytometry Resource (NFCR).
Postdoctoral Researcher. Carnegie Mellon University,
Molecular Biosensor and Imaging Center,
2010-2014
Flow cytometry based assay development and optimization.
Multiplex flow cytometry. Directed evolution and protein
engineering. Glycan-specific detection and measurement.
Product development and commercialization.
Oral Presentation CYTO 2008 (Budapest, Hungary)
Oral Presentation CYTO 2010 (Seattle, WA)
Index
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Scholars &
Emerging
Leaders
Poster
Session
Wednesday
15 June
Tuesday
14 June
Monday
13 June
Sunday
12 June
Saturday
11 June
Special
Lectures
Congress
Overview
64
The ISAC SRL Emerging Leaders program is one of a number
of programs and services ISAC recently launched to better
serve its members who are Shared Resource Lab managers
and staff. You can read more about these services online at:
http://isac-net.org/News/Society/ISAC-Expands-MemberServices-to-Shared-Resource-La.aspx
Sunday
12 June
ISAC SRL Emerging Leaders 2014-2018
Michael Gregory
Laboratory Manager, NYU Langone
Medical Center, New York, NY
Education
Masters, Biology (in progress), New York
University
B.A., Biology (2005), New York
UniversityScientific Interest and Avenues
Monday
13 June
Tuesday
14 June
for Collaboration
T-Cell Immunology, Centrifugal Counter-Flow Elutriation
CYTO U E-Learning Delivery Task Force Member
CYTO 2014 attendance
Website
http://ocs.med.nyu.edu/cytometry-and-cell-sorting-core
Wednesday
15 June
Commercial
Tutorials &
Exhibits
Prostate Cancer)
Dip Eng (Electrical)
My interests lie in the accurate application of technology to
develop deeper understanding of science, particularly the
biological sciences. I believe that strong, sustainable shared
resource laboratories, which not only provide access to
instrumentation but also expertise, have the capacity to
enhance scientific understanding and to participate in the
discovery of cures for many diseases.
I have a particular interest in the application of Cellular and
Single Cell Genomic approaches to understanding the
underling processes involved in real disease states. I would
welcome collaboration in the areas of single cell genomics,
microfluidics in biology and novel applications involving
cell sorting and cytometric analysis.
Scholars &
Emerging
Leaders
Robert (Rob) Salomon
Flow Cytometry Manager/ Senior Flow
Cytometry Scientist, Garvan Institute of
Medical Research, Sydney, NSW,
Australia
Education
BSc (Anatomy and Physiology)
Honors 1st class (Animal models of
Prostate Cancer)
MSc (Molecular Profiling of Human
Poster
Session
Oral Session
Abstracts
Poster Session
Abstracts
Andrew Filby
Flow Cytometry Core Facility Manager
and co-manager of the Newcastle
University Single Cell Unit (NUSCU),
Faculty of Medical Sciences, Newcastle
upon Tyne, UK
Education
B.Sc. Hons. Biochemistry (University of
Huddersfield, UK)
PhD Molecular and Cellular Immunology (National Institute
for Medical Research, NIMR, UK; affiliated with University
College London).
Post-doctoral training also at the NIMR
I specialise in imaging (flow) cytometry, conventional flow
cytometry (sorting and analysis) as well as molecular and
cellular immunology. I am very interested in the multipronged application of different cytometry technologies to
address key questions in human health and disease. I have
a keen interest in the use of machine learning approaches to
analyse image cytometry data. I am also very involved in
the educational and training initiatives within the
UK/European and international cytometry communities. I
am also an active voice for the shared resource laboratory
best practices. As a Cytometry Part A journal scholar I am
also very interested in how science is communicated
effectively and accurately.
I serve on the image cytometry content and e-learning
delivery task forces. I have attended five out of the last six
Cyto conferences organizing a total of six workshop
sessions at these meetings as well as submitting scientific
abstracts for talks and posters.
Websites
http://scholar.google.co.uk/citations?user=TpilJ8YAAAAJ&hl
=en
http://www.researchgate.net/profile/Andrew_Filby
http://www.ncl.ac.uk/fccf/
Saturday
11 June
This five-year program provides ISAC SRL Emerging Leaders
with a complimentary membership in the Society,
subscription to Cytometry Part A, and complimentary
registration and partial travel funds to attend the annual
society CYTO conference as well as eligibility for one-time
funding for projects that benefit the society and cytometry.
In addition, ISAC SRL Emerging Leaders are given the
opportunity to be an integral part of the Society through
committee memberships, advisory boards, and creating
educational material to further support the goals of ISAC.
Being chosen an ISAC SRL Emerging Leader is an indication
that you are recognized for your leadership potential,
technical expertise and ability to achieve your career goals.
Special
Lectures
The ISAC SRL Leadership Development program is designed
to enhance and develop the next generation of emerging
leaders in SRL (Shared Resource Lab) operations who will
also help further the field of cytometry. New SRL managers
with less than three years of experience as a manager and
deputy or assistant SRL managers with at least 3 years of
experience are eligible to apply.
Congress
Overview
ISAC SRL Emerging Leaders
Index
65
Speaker/Author
Participation/Support
of ISAC
ISAC 2016 Workshop Presenter – “Systems for Simplifying
Core Management”, Sunday, June 12, 2016, from 15:45 17:00.
CYTO U E-Learning Delivery Task Force Member
CYTO 2014 and 2015 Outstanding Poster Award Judge and
Abstract review
Shared Resource Lab Task Force Member
Australasian Cytometry Society Website Administrator
www.cytometry.org.au
Australasian Cytometry Society Treasurer 2013-15
Australasian Cytometry Society Conference Chair 2016
www.acs2016.org.au
Websites
www.flow.garvan.org.au
https://www.linkedin.com/pub/robert-salomon/13/775/26
http://www.slideshare.net/Robert_Salomon
Tuesday
14 June
Monday
13 June
Sunday
12 June
Saturday
11 June
Special
Lectures
Congress
Overview
Rachael Walker
Head of Flow Cytometry
Babraham Institute, Cambridge, UK
Education
PhD in Tissue Engineering, Department
of Clinical Engineering, University of
Liverpool – 2005
BMedSc (Honours) – Biomedical
Materials Science, University of
Birmingham – 2001
I head a very busy and fast growing flow cytometry core
facility, working closely with both academic researchers
and local biotech companies. I have recently been
awarded a government grant for writing and hosting a Flow
Cytometry training course for Industry, the first course to be
held, Oct 2014.
Co-chair of Membership Services Committee
Member of SRL task force
Secretary of RMS Cytometry Committee and Secretary of
FlowcytometryUK (ISAC affiliated societies)
Lecturer in ISAC Pre-congress courses 2010-11
Attended CYTO regularly since 2006
Member of program committee for CYTO since 2010
ISAC Scholar, 2012 - 2014
Website
http://www.babraham.ac.uk/science-services/flowcytometry/rachael-walker
Index
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Scholars &
Emerging
Leaders
Poster
Session
Wednesday
15 June
Jessica B. Back
Associate Director, Microscopy,
Imaging, and Cytometry Resources
(MICR) Core
Wayne State University and Karmanos
Cancer Institute
Detroit, MI
Education
B.S., Biochemistry – Ohio Northern
University, Ada, OH
Ph.D., Biochemistry – Wayne State University, Detroit, MI
Post-Doctoral Fellow, Tumor Immunology -- Karmanos
Cancer Institute, Wayne State University, Detroit, MI
My goal is to ensure the success of the researchers using
MICR by providing expertise in flow cytometry techniques,
individualized assay design consultation, and data analysis
support. I also strive to push my users out of their comfort
zones, expand their use and understanding of flow
cytometry, and make their data more functional and
reproducible. My personal research interests are in
carcinogenesis and tumor immunology.
Meeting Attendance: CYTO 2013, CYTO 2014, CYTO
2015, GLIIFCA 2013, GLIIFCA 2015
CYTO 2015 Outstanding Poster Judge
CYTO 2016 Workshop Organizer
GLIIFCA Steering Committee
GLIIFCA 2015 Roundtable Topic Leader
GLIIFCA 2016 Poster Organizer
Website
http://micr.med.wayne.edu
66
Anna Belkina
Senior Research Specialist
Flow Cytometry Core Facility
Education
MD (Russian State Medical University)
1997-2003
Ph.D. (Boston University School of
Medicine) 2007-2012
Postdoctoral Fellow, Department of
Microbiology 2012-now
As a Research Specialist and acting manager of a larger
institution Core Facility, I am focused on engaging our
research community in practices matching the highest
standards in the field. My current research efforts include a
number of collaborative projects focusing on characterizing
T cell immunity in chronic inflammatory diseases (Type 1
and Type 2 diabetes, periodontal disease, scleroderma) and
cancer. Specifically, I work on immune exhaustion
pathways and inhibitory receptor phenotyping using highdimensional flow cytometry. We employ both automated
and expert-guided data analysis to identify novel
populations of exhausted immune cells and characterize
their function.
ISAC E-Learning Delivery Task Force/CytoU
Attended CYTO 2014, 2015, 2016
Outstanding Poster Award Judge at CYTO2015, 2016
Co-chair of CYTO 2016 Parallel Session 3: Immunology
CYTO 2016 SRL Forum Organizing Committee member
Websites:
http://www.bu.edu/flow-cytometry
https://www.researchgate.net/profile/Anna_Belkina/
Saturday
11 June
Collaboration
My interests involve providing a high standard of education
to enable researchers utilising the core facility‘s Flow and
Imaging Cytometry capabilities to expand their scope for
scientific discovery. I have assisted groups with a research
focus in type 1 and 2 diabetes immunology, cancer,
virology, allergy and hematology; thus have an interest in
developing and providing appropriate biosafety practices.
Cyto 2015 Session Chair & Poster Judge
Cyto 2016 Session Chair
Cyto 2016 Workshop Facilitator
Member of organising committee for ACS (ISAC affiliated
society) Melbourne Meeting
Website
https://www.svi.edu.au/research_themes/facs
Sunday
12 June
Monday
13 June
Tuesday
14 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Christian Kukat
Head of FACS & Imaging Core Facility
Max Planck Institute for Biology of
Ageing, Cologne, Germany
Education
Postdoctoral Fellow, Max Planck
Institute for Biology of Ageing, Cologne
– 2013
Postdoctoral Fellow, Karolinska
Institute, Stockholm – 2009
PhD, Center of Biotechnology and Biomedicine, University
of Leipzig – 2008
Diploma in Biology, University of Würzburg – 2004
As the head of our institute’s combined cytometry,
microscopy, and histology core facility, I am interested in
helping users synthesize these complementary technologies
to enable better experimental design. My personal scientific
interest is mitochondria, as these organelles accompanied
me for a long time and are wonderful objects for flow
cytometry and microscopy analyses.
First time attendee at CYTO 2015 in Glasgow
Founder of the Cytometry Club Cologne
Website
http://www.age.mpg.de/science/core-facilities/facs-imaging/
Wednesday
15 June
Suat Dervish
Flow Cytometry Sorter Operator
Westmead Institute of Medical Research /
Sydney University, Sydney, NSW,
Australia
Education
BMedSc (Hons) – Immunology,
Biochemistry - Sydney University
GradCertInn&Ent - Sydney University
I have always been fascinated at the complexity and depth
of interactions within biological systems. I enjoy applying
cytometry to better understand and describe these processes
using novel methods. Designing and building both software
and hardware solutions to better serve this purpose is an
area I am actively interested in.
Cyto2013 – ISAC
Australian Cytometry Society
SYDFlow
Australian Society of Immunology
Special
Lectures
University
I am interested in novel and cell sorting technologies. Also,
I am interested in how cell sorting affects the survival,
viability and functionality of different stem cell lines,
progenitors and differentiating cells.
Certification Advisory Committee member 2015-present
Meeting Attendance: CYTO 2011, CYTO 2012, CYTO
2013, CYTO 2014, CYTO 2015
Outstanding Poster Judge CYTO 2015
Programming Committee CYTO 2016
Workshop Organizer CYTO 2016
Danish Society for Flow Cytometry Board Member 2016
CPH FLOW (Copenhagen-area flow cytometry user group)
founder
Website
http://danstem.ku.dk/research1/core_facilities/flowcytometry-core-facility/
Michael Thomson
Flow Cytometry Coordinator, St
Vincent’s Institute of Medical Research
Melbourne, Australia
Education
M.Bus-Biotech – RMIT University
B.AppSc (Biochemistry/Chemistry) –
Swinburne University
Congress
Overview
Gelo Victoriano B. Dela Cruz
Flow Cytometry Core Facility Manager,
The Danish Stem Cell Center
Copenhagen, Denmark
Education
BSc Molecular Biology and
Biotechnology, University of the
Philippines - Diliman
MS Applied Recombinant DNA
Technology credits, New York
Index
67
Speaker/Author
Radhika Rayanki
Research & Development Associate
Flow Cytometry Core Facility
Medimmune/Astrazeneca
Gaithersburg, MD
Education
BSc (Chemistry, Botany and Zoology)
MSc (Biochemistry)
Certificate in Biotechnology
I am interested in cross-instrument cell sorting capabilities,
improving the longitudinal performance of the analyzers by
identifying more thorough and simpler QA/QC methods and
implement /optimize SOPs of all manner of FACS Core lab
operations
My personal research interests are in the exploration of
Cancer Biology and autoimmune disease; comparative
studies of granulocyte subsets between cancer blood and an
autoimmune disease
First time attendee at CYTO
Attend local Flow Cytometry US meetings (Flow Cytometry
Interest Group meeting – NIH, Annual Flow Cytometry
Conferences)
Participate in organizing Chesapeake Cytometry Consortium
(ISAC afflicated society)
Index
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Scholars &
Emerging
Leaders
Poster
Session
Wednesday
15 June
Tuesday
14 June
Monday
13 June
Sunday
12 June
Saturday
11 June
Special
Lectures
Congress
Overview
Erica Smit
Senior Scientific Officer/ Flow Cytometry
Core Manager
South African Tuberculosis Vaccine
Initiative (SATVI), University of Cape Town,
Institute of Infectious Disease and Molecular
Medicine (IDM)
Cape Town, South Africa
Gert Van Isterdael
Flow Cytometry Core Facility Manager
Inflammation Research Center
VIB (Flanders Institute for Biotechnology)
Ghent University, Belgium
Education
BSc Biomedical Laboratory Techniques
(Pharmacy and Biotechnology)
KAHO Sint-Lieven, Technology campus Ghent
My primary role is to provide flow cytometry expertise to
the users of the core facility. This is achieved through
providing technical help to the scientists, maintaining the
machines, implicating high quality SOPs as well as
providing both experimental setup and data analysis
consultations. My scientific interest lies in the development
of novel automated analysis techniques for multi-parameter
flow cytometry data to remove user bias. To achieve this we
are closely collaborating with our on-site Bioinformatics
team headed by Prof. Yvan Saeys. In addition to this, as I
help scientists at the core on a daily basis, I am also actively
involved in several research projects ranging from basic
science topics, such as understanding the role of specific
transcription factors in immune cell development and
homeostasis, to more applied research projects, including
those investigating mechanisms of allergy, asthma and
cancer.
Attended CYTO 2014, Fort Lauderdale and CYTO2015,
Glasgow
Website
http://www.irc.ugent.be
http://orcid.org/0000-0001-6626-1316
https://www.researchgate.net/profile/Gert_Van_Isterdael
Twitter: @IRCFlowCore
Education
National Diploma (ND) Biomedical Technology – Cape
Peninsula University of Technology
Baccalaureus Technologiae (B Tech) – Biomedical
Technology - Cape Peninsula University of Technology
I work as a Senior Scientific Officer at a TB Vaccine Trial
Laboratory within a larger Research Institute. My research
interests include Immunology and Vaccinology. My
personal interests in flow cytometry involve troubleshooting
and correcting technical problems on the flow cytometers
as well as implementation of latest calibration and
optimization procedures. My goal is to establish a high
standard foundation for the research community at the
Institute to engage, educate and train students and staff the
basic principles of and the latest advances and applications
in multiparameter flow cytometry.
CYTO conference attendance: CYTO 2013
CYTO 2016: Poster title: Qualification and implementation
of an inter-laboratory quality assurance program for a whole
blood intracellular cytokine staining assay.
CYTO 2016: Attending SRL Forum and related workshops
Website
http://www.satvi.uct.ac.za/
68
Multimedia and Poster Sessions
Author presentation and discussion times:
Monday, June 13
800 – 1930
1800 – 1900
Tuesday, June 14
800 – 2000
1830 – 1930
Wednesday, June 15
800 – 1415
1100 – 1200
Index
69
Speaker/Author
B8
145
Interest of Raman Microspectroscopy in the
Diagnosis and Prognosis of B-Chronic Lymphocytic
Leukemia
N. Bailly, D. Gheldof, S. Walbrecq, L. Liu, M. Fere, C.
Gobinet, J. Clossat, E. Cornet, X. Troussard, F. Mullier
and B. Chatelain. Catholic Univ. of Louvain, Belgium,
Univ. of Reims Champagne-Ardenne, TRIBVN,
Chatillon and CHU Caen, France
Poster Session
Abstracts
B312 449
In Memorium: Dick Sweet and His Contributions to
Flow Cytometry and Cell Sorting
M. Bigos, D. Houck. Stanford Univ. and BD
Biosciences (retired), U.S.A.
AUTOMATED MICROSCOPY
Oral Session
Abstracts
FLOW CYTOMETRY INSTRUMENTATION
B6
143
A Fluorescent Cell Barcoding Method to Measure
Human CTL Responses: Development of a "CytotoxFlow" Approach for Small Samples of Living Cells
F-E. L'Faqihi, M. Farcé, S. Mueller, D. Lestrade and S.
Valitutti. INSERM U1043, Toulouse, France
B7
144
Recombinant BCG Over Expressing Ag85C Generates
Durable Central Memory T Cells and Showed
Enhanced Anti-mycobacterial Activity in Cattle
A. Kumar and H. Krishna Prasad. All India Inst. of
Med. Sci., New Delhi, India
Commercial
Tutorials &
Exhibits
B1
138
Pre-enrichment of ILC2 from Whole Blood Reduces
Sort Time and Results in Improved Purity
J. Wilshire, Y. Valdez, S. Kyei, G. Poon, F. Takei, C.
Peters, S. Woodside, A. Eaves and T. Thomas.
STEMCELL Technols. and Terry Fox Lab., Vancouver,
Canada
ANTIGEN-SPECIFIC IMMUNE RESPONSES
Scholars &
Emerging
Leaders
CELL SORTING AND SELECTION
POSTER PRESENTATIONS
Poster
Session
MULTIMEDIA PRESENTATIONS
B5
142
Flow and Image Cytometry Educational Outreach
Opportunities: Roswell Park Cancer
Institute Experience
A. Conway, A. Kisailus, O. Maguire, J.D. Tario, E.
Podniesinski, H. Minderman and P.K. Wallace.
Roswell Park Cancer Inst., Buffalo, U.S.A.
Wednesday
15 June
The number listed following the letter “B”
signifies the poster board number/location in
the Exhibit Hall 4EF on WSCC Level 4. The
number next to the board number signifies the
program number which can be referenced to
the abstract located in the back of the
program book.
OTHER BIOLOGICAL APPLICATIONS
Tuesday
14 June
1430
Poster Viewing
Poster Session III (Consists of Poster
Highlights Tour)
All poster material must be removed
from the boards
B4
141
CyTOF Studio: Automated Classificationand Analysis
of High-Dimensional Mass Cytometry Data
S. Davis, M. Nolan and J. Hill. GlaxoSmithKline,
U.S.A.
Monday
13 June
Poster Viewing
Poster Session II: Authors of EVEN
numbered poster boards present
NEW SOFTWARE DEVELOPMENT
Sunday
12 June
Poster Viewing
Poster Session I: Authors of ODD
numbered poster boards present
Saturday
11 June
Sunday, June 12
1600 – 2000
Authors must set up posters on
assigned board
B2
139
Automated Identification of Novel Cell Types
Basedupon Signaling Characteristics in Chronic
Myeloid Leukemia
J. De and P. Kane. Deepath Med. Diagnostics, Palo
Alto, U.S.A.
B3
140
Survival Stratification in Acute Myeloid Leukemia by
Single Cell Signal Profiling
J. Skavland, S-E. Gullaksen, S. Gavasso, H. Reikvam,
Ø. Bruserud and B.T. Gjertsen. Univ. of Bergen and
Haukeland Univ. Hosp., Norway
Special
Lectures
Congress
Overview
HEMATOLOGICAL DISORDERS
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Index
Abstracts
AUTOMATED SAMPLE PREPARATION
B9
146
‘Car Wash’ an Integrated Continuous-Flow
Microfluidic Device to Prepare Leukocytes for Flow
Cytometry by Deterministic Lateral Displacement
Y. Chen, J.L. D’Silva, R.H. Austin, J.C. Sturm, X. Chen,
K. Reeder, C. Civin, T. Ward, A. Skelley, K. Gandhi, L.
Aurich, Z.P. Lee and C. Dosier. Princeton Univ.,
Univ. of Maryland Sch. of Med. and GPB Scientific,
Richmond, VA, U.S.A.
B10 147
Automated Processing of Human Blood Leukocytes
by a Plastic, Disposable Microfluidic Deterministic
Lateral Displacement Device
A. Skelley, T. Ward, K. Gandhi, Z.P. Lee, C. Dosier,
J.L. D’Silva, Y. Chen, M. Kim, X. Chen, L. Aurich, D.
Recktenwald, R.H. Austin, M. Grisham, C. Civin and
J.C. Sturm. GPB Scientific, Richmond, VA, Princeton
Univ. and Univ. of Maryland Sch. of Med., U.S.A.
B11 148
Preparation of Polychromatic Antibody Cocktails for
Immunophenotypic Whole Blood Assays Using a
Semi-automated Approach
I. Gonzalez, T. Meeuwsen, W. Miller, D. Haley, V.
Conrad, T. Poplonski, A. Tanibata-Branham, K. Bahjat
and Y. Koguchi. Earle A. Chiles Res. Inst., Portland,
OR, Sony Biotechnol., San Jose, Beckman Coulter,
Inc. Life Sci., Brea, CA, Bristol-Myers Squibb,
Redwood City, CA, U.S.A.
BIOMARKERS
B12 149
Automated Analyses of Mass Cytometry Data Enables
Identification of Biomarkers That Associate with
Manufacturing Success for CAR-T Cell Therapies
I. Pruteanu-Malinici, P. Pierog, A. Colantonio, A.
Veloso and S. Kassim. Novartis, Cambridge, MA,
U.S.A.
B13 150
Gene Expression Profiling and Molecular
Characterization of Miltefosine Resistance in L.
donovani.
P. Tiwary, D. Kumar and S. Sundar. Banaras Hindu
Univ., India
B14 151
Optimal Detection of Urinary Extracellular Vesicles
in Evaluating Chronic Kidney Disease
M. Mizus, E. Mohler, III, R.D. Schretzenmair, L.
Zhang, W.T. Rogers, L.B. Holzman, R.R. Townsend
and J.S. Moore. Perelman Sch. of Med., Univ. of
Pennsylvania, U.S.A.
B15 152
Evaluation of the Oxidant Status of Patients with
Rheumatoid Arthritis as a Potential Biomarker for
Disease Progression
M. Chatterjee and S. De. Inst. of Postgrad. Med.
Educ., Kolkata, India
70
B16 153
Issues with Mesenchymal Stem Cell Markers in
Amniotic Cells
W. Weston, R. Frisch and S. Namin. UMTB/Vivex
Biomed., Miami, U.S.A.
B17 154
Mass Cytometry: TNF Signaling in Patients with
Rheumatoid Arthriti
L. Bader, S-E. Gullaksen, J. Skavland, C. Vedeler, C.
Gram Gjesdal and S. Gavasso. Haukeland Univ.
Hosp. and Univ. of Bergen, Norway
B18 155
NIST Measurement Service for ERF Value
Assignments of Calibration Microspheres in Flow
Cytometry
P. DeRose, A. Gaigalas and L. Wang. NIST,
Gaithersburg, MD, U.S.A.
B19 156
Development, Validation, and Outsourcing of a Flow
Cytometry Assay to Measure Pharmacodynamic
Response in Patients with B-Cell Malignancies
Treated with BTCT4465A, an Anti-CD20/CD3 T-Cell
Dependent Bispecific Therapy
K. Dalpozzo, C. Green, G. Hernandez, C. Marathe
and J. Wallin. Genentech, South San Francisco,
U.S.A.
B20 157
Detection of PDL1 and PDL2 on Circulating Tumor
Cells Using Microfluidic Based Chipcytometry
J. Teo, M. Tan, A. Mirenska, J. Oh, L. Hong, A.
Bhagat, R. Wnek, J. Detmers, C-L. Chin and D.
Skibinski. MSD Intl. Singapore, ClearBridge
Biomedics, Singapore, Zellkraftwerk GmbH,
Hannover, Germany, Merck & Co. Inc., Rahway,
U.S.A and Zellkraftwerk GmbH, Leipzig, Germany
B21 158
Performing a ‘Fit for Purpose’ Validation Exercise of
Flow Cytometry Assays for Lymphocyte Populations
Assessed as Pharmacodynamic Biomarkers during
Drug Development Clinical Trials
D. Lanham and M. Patel. Eurofins Pharma Bioanalysis
UK Ltd, Abingdon, U.K.
B22 159
Development and Validation of a Clinical Flow
Cytometry Assay to Measure Intracellular Ki67
Expression in Memory T Subsets, NK and Treg Cells
in Human Whole Blood
Y. Sun, T. Bridal, K. Yang, L. Patti-Diaz, M.N.
Hedrick, M. Cleary and A.G. Ehrhardt. Bristol-Myers
Squibb, U.S.A.
B23 160
Detection of Durvalumab-Induced Changes in
Activated and Proliferating T Cells in Melanoma
Patients Using Flow Cytometry-Based
Immunophenotyping Assays
N. Standifer, J. Pearson, D. Sinibaldi and M. Liang.
MedImmune, CA and MD, U.S.A.
Poster
Session
B38 175
Innovations in Fluorescence Activated Cell Sorting
for Genomic Studies of Single Cells
M. Crow, T. Petersen, M. Rohani, D. Horner, A. Tam
and J.C. Mason. Becton, Dickinson & Co, Seattle and
San Jose, U.S.A.
Scholars &
Emerging
Leaders
Oral Session
Abstracts
Poster Session
Abstracts
B39 176
Clonogenic Potential of a Rare Marrow-Derived after
Purification with Droplet versus Microchip Sorting
L. O'Flynn, E. Horan, E. Rodriguez-Mesa, J. Dunne
and S. Elliman. Orbsen Therapeut., Galway and
Miltenyi Biotec, Santa Barbara and Livermore, CA,
U.S.A.
B40 177
Cell Differentiation Approaches Using 5-Way Cell
Sorting: Facts and Artifacts
A.L. Bertho and R. Ferraz. Oswaldo Cruz Inst.,
FIOCRUZ, Rio de Janeiro, Brazil
Commercial
Tutorials &
Exhibits
B31 168
Comparison of Label-Free Cell Cytotoxicity Image
Cytometric Detection Method to Cell Titer-Glo
R. Patel, L. Chan, O. Dery and G. Wei. Ignyta, San
Diego and Nexcelom Bioscience, Lawrence, MA,
U.S.A.
B36 173
Two Photon Flow Cytometry and Sorting on a
Beckman Coulter MoFlo High Speed Sorter: A
Provisional Study
A. Riddell. Univ. of Cambridge, U.K.
B37 174
A Size Restricted Cell Sorting Strategy for
Applications in Modeling Assisted Evolution in the
Algae, Chlamydomonas reinhardtii
I. Larma-Cornwall, C.E. Seed and J.E. Tomkins. Univ.
of Western Australia
Wednesday
15 June
B30 167
Using Image-Based Flow Cytometry to Monitor and
Track Satellite Cells In Vitro
H-Y. Luk, D. Levitt, B. Mcfarlin and J. Vingren. Univ.
of North Texas, U.S.A.
B35 172
A Novel Method for Single Cell Deposition Setup:
Target Practice
S. Monard. Walter and Eliza Hall Inst., Parkville,
Australia
Tuesday
14 June
B29 166
Development of a Multiplexing Imaging Flow
Cytometry Assay for Apoptosis Evaluation
D. Bonilla, K. Acklin, K. Ruissard, M. Pichler, R.
Jones, A. Sergueeva, D. Dwyer, R. Jewell, V. Papanna,
S. Vaidyanathan, K. Ramirez and K. Clise-Dwyer. MD
Anderson Cancer Ctr., Houston and EMD Millipore,
Seattle, U.S.A.
B34 171
CD81 T-Catch Cell Isolation Approach as an Ideal
Tool for an Advanced Cell Analysis on Mass
Cytometer
O. Pelak, D. Kuzilkova, J. Stuchly, M-L. Kiene, K.
Stanar, M. Vaskova, H. Stadler and T. Kalina. 2nd Fac.
of Med., Charles Univ. in Prague, Czech Republic and
IBA GmbH, Goettingen, Germany
Monday
13 June
CELL PROLIFERATION AND DEATH
CELL SORTING AND SELECTION
Sunday
12 June
B27 164
Uber Your Analysis: Creating and Sharing a Custom
Analysis Platform Integrating Single Cell Data
A. Panopoulos, S. Siddiq, J. Almarode and M.
Stadnisky. FlowJo LLC, Ashland, OR, U.S.A.
B28 165
Leveraging Mass Cytometry for Deep Phenotyping
and Quantification of Target Proteins in a Macaca
fascicularis Model
C. Stevens, K. Atkuri and H. Neubert. Pfizer Inc.,
Andover, MA, U.S.A.
B33 170
Flow Cytometric Analysis of Intracellular ROS and
NS Production and Curcumin Inhibition
Z. Luo, Q. Zhao, J. Liu, R. Peng, J. Liao and Z. Diwu.
AAT Bioquest Inc., U.S.A.
Saturday
11 June
BIOPHARMACEUTICAL APPLICATIONS
Special
Lectures
B25 162
Development of a Dual mRNA (ISH) and Protein
(IHC) Biomarker Assay in Psoriasis Skin Biopsies
D. Krull. GlaxoSmithKline, U.S.A.
B26 163
Multidrug Resistance Protein Activity of T
Lymphocytes Assessed by Flow Cytometry Is a
Predictor of Biological Treatment Response in
Rheumatoid Arthritis
G. Toldi, P. Szerémy, A. Apjok, H. Bhattoa, J.
Kappelmayer, Z. Szekanecz, S. Szántó, G. Szûcs, S.
Szamosi, Á. Horváth and A. Domján. MDQuest Ltd.,
Szeged and Univ. of Debrecen, Hungary
B32
169
Real-Time Caspase 3/7 Measurement of Suspension
and Adherent Cells Using Celigo Image Cytometry
S. Bonasu, S. Kessel, L. Chan, J. Qiu and D. Kuksin.
Nexcelom Bioscience, U.S.A.
Congress
Overview
B24 161
Development and Validation of Immunophenotyping
Assays for Use on Cryopreserved Peripheral Blood
Mononuclear Cells Isolated in Trials of ImmuneMediated Therapies for Cancer Programs
J. Pearson, M. Liang and N. Standifer. MedImmune,
Mountain View, U.S.A.
Index
71
Speaker/Author
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
B43 180
Evaluating the Effects of Cell Sorting on Gene
Expression
M. DeLay, A. Bergeron, A. Box, K. Brundage, M.
Cochran, R. del Rio-Guerra, M.A. Handley, P. Lopez,
E.M.M. Meyer and A. Saluk. Cincinnati Children's
Hosp., Dartmouth Col., Stowers Inst. for Med. Res.,
Kansas City, West Virginia Univ. Hlth. Sci. Ctr., Univ
of Rochester, Univ. of Vermont, Massachusetts Gen.
Hosp., NYU, Univ of Pittsburgh and The Scripps Res.
Inst., U.S.A.
B44 181
Enrichment of CRISPR-Mediated HomologousDirected Repair in Mammalian Cells by Cell CycleBased Flow Sorting
M. DeLay, H. Xie, Y. Chen, S. Thornton and Y-C. Hu.
Cincinnati Children's Hosp. and Med. Ctr., U.S.A.
B45 182
Dilute Sample and High Flow Rate Maximize Sort
Recovery
G. Buruzula, A. Wood and T. Serwold. Joslin Diabetes
Ctr., Boston, U.S.A.
B46 183
Development of a Sensitive Flow Cytometry-Based
Platform for Isolation and Molecular
Characterization of Circulating Tumor Single Cells
and Clusters
C. Pletcher, N. Bhagwat, L. Wang, S. Yee, L. Yu, J.
Moore, B. Stanger, E. Dixon and E. Carpenter. Univ.
of Pennsylvania, BD Technologies, Durham, NC and
BD Biosciences, San Jose, U.S.A.
B47 184
New Sorting and Genomics Technologies to
Facilitate High-Throughput, High-Precision, Single
Cell Gene Expression Analysis of Breast Cancer Cells
in Bone Marrow Samples
L. Yu, R. Johnson, M. Bigos, C. Lomas and A. Wang.
BD Biosciences, San Jose and Stanford Univ., U.S.A.
Index
Abstracts
Oral Session
Abstracts
B41
178
Nodexus' Label-Free Cell Screening and Isolation
Platform Allows for Unprecedented Clinical Utility
K. Balakrishnan. Nodexus Inc., Berkeley, U.S.A.
B42 179
Multi-parametric Microfluidic Flow Cytometer for
Directed Evolution and Characterization of
Fluorescent Protein Libraries
P. Manna, F. Vietmeyer, A. Palmer and R. Jimenez.
Univ. of Colorado Boulder and JILA, NIST Univ. of
Colorado Boulder, U.S.A.
B48
185
BD Influx 6-Way 13 PopulationSorting. Vaccine
Signature Deconvolutionon Leukocyte Subsets.
®
Tracking Immune Response to Heplisav
J-P. Blanck, E. Gatewood, J. Cardozo, N. Raman, E.
Ruchaud, R. Coffman, G. Obermoser and K.
Kayembe. Baylor Inst. for Immunol. Res., Dallas,
Dynavax Technols. Corp., Berkeley and Stanford
Univ., U.S.A.
B49 186
A Novel Method for Characterising Cell Properties
Based on Pulse Shapes
K. Feher, K. von Volkmann, J. Kirsch, R. Addo, A.
Radbruch, J. Popien and T. Kaiser. German
Rheumatism Res. Ctr. (DRFZ) and APE Angewandte
Physik & Elektronik GmbH, Berlin, Germany
B50 187
Translation of the Human Immunophenotyping
Panels from a FACS Aria Fusion to an Influx Platform:
Evolving Polychromatic Cell Sorting Services in RL
E. Perez, L. Bergamachi, C. Bowman, N. Savinykh, L.
Turner and A. Petrunkina. Univ. of Cambridge, U.K.
B51 188
Evaluation of Methods to Verify Aerosol
Containment in Cell Sorters: Collection of Aerosols
with a Cyclone Air Sampler
K. Holmes, J. Baijer, D. Ambrozak, R. Nguyen and S.
Perfetto. NIAID, NIH, U.S.A. and CEA-DSV, France
B52 189
Beyond the Norm with the Beckman Coulter
AstriosEQ: Sorting with 50 and 200 Micron Tips
A. Dean, C. Ross, R. Morris and A. Kachynski.
Beckman Coulter Inc., Fort Collins, U.S.A.
B53 190
Fast and Easy Immunomagnetic Positive Selection of
PE- or Biotin-Conjugated Antibody Labelled Cells
with Releaseable RapidSpheres Results in
Functional, Pure, and Particle-Free Cells
C. Ewen, A.I. Kokaji, S. Clarke, D. Kellerman, M.
Chambers, M. Chan and S. Woodside. StemCell
Technols Inc., Vancouver, Canada
B54 191
Microfluidic Platform for On-Chip Single Cell
Analysis
K. Lee, A. Truetsky, J. Song, R.J. Giedt, E. Kim, A.E.
Kovach, E.P. Hochberg, C.M. Castro, R. Weissleder
and H. Lee. Massachusetts Gen. Hosp./Harvard Med.
Sch., U.S.A.
CELL-DERIVED MICROVESICLES
B55 192
A Methodological Approach for Characterization of
Extracellular Vesicles: Small-Particle Flow Cytometry
J. Tigges, V. Camacho and V. Toxavidis. Beth Israel
Deaconess Med. Ctr., Boston, U.S.A.
72
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
B70 207
Automated Cell Type Annotation
S. Van Gassen, M.J. Van Helden, M. Guilliams, T.
Dhaene and Y. Saeys. Ghent Univ., iMinds, Ghent
and VIB Inflammation Res. Ctr., Ghent, Belgium
Tuesday
14 June
COMPUTATION AND INFORMATICS
Monday
13 June
B71 208
ZellGate: Automated Quality Control and Gating of
High-Dimensional Cytometry Data in Clinical
Validation Settings
A. Mirenska and C. Hennig. Zellkraftwerk GmbH,
Hannover, Germany
Poster Session
Abstracts
B62 199
A Multiplex Bead Platform for Protein Profiling of
Exosomes by Flow Cytometry
S. Wild, C. Dose, N. Koliha, Y. Wiencek, U. Heider
and A. Bosio. Miltenyi Biotec GmbH, Germany
B63 200
Submicron Particle Analysis and Counting Is Highly
Favored by the Use of Side Scatter from the Violet
Laser on the CytoFLEX Flow Cytometer
P. Poncelet, T. Bouriche, S. Robert, C. Franco and R.
Lacroix. Biocytex, Aix-Marseille Univ., INSERM Fac.
of Pharm. and CHU La Conception, APHM, Marseille,
France
B66 203
Cell-Based Biomarkers in Clinical Research:
Developing, Validating and Implementing Sample
Preparation, Biobanking and Image Cytometry Assays
to Support Drug Development
J. Detmers, A. Mirenska and C. Hennig. Zellkraftwerk
GmbH, Leipzig and Hannover Med. Sch., Germany
B67 204
Isolation of Antigen Specific CD8+ T Cells Using the
MACSQuant Tyto: A Closed, Sterile, Single-Use
Microchip-Based Cell Sorter
M. Ragland, S. Walter, I. Lai and C. Yee. Owl
Biomed., Goleta, CA, Immatics US, Inc., Houston and
Univ. of Texas MD Anderson Cancer Ctr., U.S.A.
B68 205
Stability Assessment of Various Flow Cytometry
Markers in Stabilized Whole Blood
D. Gagnon, P. Pouliot, B. Yassine-Diab, R-M. Brito, V.
Hebert, A. Gagnon, J-F. Poulin and Y. Peretz. Caprion
Biosciences Inc., Montreal, Canada
B69 206
Oral Supplementation with Baker’s Yeast b Glucan Is
Associated with Alterations in Granulocyte Function
following Strenuous Exercise in a General Population
Cohort
E. Bowman, A. Henning, A. Venable and B. McFarlin.
Univ. of North Texas, U.S.A.
Sunday
12 June
B61 198
Tumor-Derived Exosomes Regulate Expression of
Immune Function-Related Genes in Human T Cell
Subsets
P. Simms, M. Laurent and T. Whiteside. Loyola Univ.
Chicago, Maywood, U.S.A., Univ. Hosp., Basel,
Switzerland and Univ. of Pittsburgh, U.S.A.
B65 202
Utilizing Data Integration Software to Create an
Analysis Platform for Immunotherapy Clinical Trials
H. Smithers, O. Finney, S. Rawlings-Rhea, R. Hicks,
B. Futrell, D. Doolittle, L. Ton, J. Park, R. Gardner and
M. Jensen. Seattle Children's Res. Inst., Seattle
Children's Hosp. and Univ. of Washington, U.S.A.
Saturday
11 June
B60 197
Exosomes Alter mRNA Gene Profile in T
Lymphocytes
P. Simms, L. Muller, T. Whitesides and P. Le. Loyola
Univ. Chicago, Maywood, U.S.A., Univ. Hosp., Basel,
Switzerland and Univ. of Pittsburgh Sch. of Med.,
U.S.A.
CLINICAL TRIALS
Special
Lectures
B59 196
Novel Extracellular Vesicle Assay Combined with
Mie Scatter Allows Differentiation Between
Apoptotic Vesicles, Microvesicles and Residual
Platelet
J. Welsh, P. Horak, D. Smith, J. Wilkinson, J.
Holloway and N. Englyst. Univ. of Southampton, U.K.
B64
201
Differences in Light Scatter Characteristics of
Phenotypic Subsets of Extracellular Vesicles
W.R. Overton, M. McGrane, M. Wesberry-Schmierer,
T. Arango and T. Johnson. CytoVas LLC, Philadelphia,
U.S.A.
Congress
Overview
B56
193
Capabilities and Limitations of Microvesicle Analysis
with Light-Scattering Flow Cytometry
A. Konokhova, M. Yurkin, D. Korneev and V. Maltsev.
Voevodsky Inst. of Chem. Kinet. and Combustion SB
RAS, Novosibirsk, Novosibirsk State Univ. and SRC
VB Koltsovo, Novosibirsk region, Russia
B57 194
Identifying Exosome Binding and Internalization in
Blood Cell Subsets by Imaging Flow Cytometry
H. Pugsley, S. Friend, C. Probst and P. Morrissey.
Amnis part of MilliporeSigma, Seattle, U.S.A.
B58 195
Surface Plasmon Resonance Imaging Detects Lower
Antigen Densities Than Flow Cytometry
F. Coumans, E. Gool, I. Stojanovic, A. Böing, R.
Schasfoort, E. van der Pol, G. Sturk, T. van Leeuwen
and R. Nieuwland. Acad. Med. Ctr., Amsterdam and
Univ. of Twente, Netherlands
Index
73
Speaker/Author
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Index
Abstracts
B72
209
Characterizing Cell Subsets in Heterogeneous Tissues
Using Marker Enrichment Modeling
K. Diggins and J.M. Irish. Vanderbilt Univ., Nashville,
U.S.A.
B73 210
High Dimensional Cytometry Data Visualization
Using Parallel Coordinates
J. Hokanson, C. Wakefield, J. Weinstein, M. Andreeff
and J. Burks. MD Anderson Cancer Ctr., Houston,
U.S.A.
B74 211
A New Statistical Agglomerative Clustering
Algorithm for High Dimensional Single-Cell Data
A. Terekidi, C. Loh, O. Ornatsky and V. Baranov.
Fluidigm Canada Inc., Markham, ON
B75 212
Data to Discovery: A ‘Nuts-And-Bolts’ Level
Discussion on Linking Flow Analysis to a Database
J. Quinn, J. Almarode and M. Stadnisky. Flowjo LLC,
Ashland, OR, U.S.A.
B76 213
Verification and Validation of tSNE Results
S. Siddiq, J. Almarode, M. Golden and M. Stadnisky.
FlowJo LLC, Ashland, OR, U.S.A.
B77 214
Fully Automated Fluorescence Intensity
Normalization (Cluster Registration) for Flow
Cytometry Data to Minimize Batch Effects and
Optionally Remove Variation in Individual Cluster
Locations
J. Rebhahn, S. Quataert, G. Sharma and T. Mosmann.
Univ. of Rochester, U.S.A.
B78 215
CyteGeist: An Open-Source Project for Integration,
Visualization, and Collaborative Analysis of Diverse
Biomedical Data
A. Treister, A. Mirenska and C. Hennig. CyteGeist,
Ashland, OR, U.S.A. and Zellkraftwerk GmbH,
Hannover
B79 216
Automated Comparison of 200 Surface Marker
Stainings to Identify Markers Specific for Memory B
Lymphocytes by Flow Cytometric Screening Assay
H-D. Chang, R. Riedel, S. Kröger, U. Stervbo and A.
Radbruch. DRFZ Berlin
B80 217
The Characterization of Receptor Density on Human
Blood Cells
R. Balderas. BD Biosciences, San Jose, U.S.A.
74
B81
218
High-Dimensional Modeling of Peripheral Blood
Mononuclear Cells from a Helios Instrument
C.B. Bagwell, H. Maecker, M. Leipold and G. Stelzer.
Verity Software House Inc., Topsham, ME, Stanford
Univ. Sch. of Med. and Fluidigm, South San
Francisco, U.S.A.
B82 219
Sometimes Simpler Is Better: An Easy-to-Implement
Log-Like Transform for Cytometry
C.B. Bagwell. Verity Software House Inc., Topsham,
ME, U.S.A.
B83 220
Statistical Tests of Unimodality Supporting Difficult
Gating Decisions
K. Johnsson and M. Fontes. Lund Univ., Sweden and
Inst. Pasteur, Paris
CYTOMETRY IN RESOURCE POOR SETTINGS
B84 221
Teaching Advanced Flow Cytometry in Africa:
Lessons Learned Over the Last 10 Years
E. Nemes, W. Burgers, C. Riou, E. Andersen-Nissen,
G. Ferrari, C. Gray and T. Scriba. Univ. of Cape
Town, Hutchinson Ctr. Res. Inst. of South Africa,
Cape Town and Duke Univ., U.S.A.,
DIAGNOSTICS
B85 222
Macrophages Gold Nanoparticle Uptake Analysis by
Image and Flow Cytometry
A. Tarnok, S. Melzer, R. Ankri and D. Fixler. Heart
Center Leipzig, Germany and Bar Ilan Univ. Israel
B86 223
Epithelial and Mesenchymal Circulating Tumor Cells
Isolated from Breast Cancer Patients and Injected at
High Cell Dose Are Not Tumorigenic in NOD-scid
Il2rȜ null Mice
A. Donnenberg, P. Basse and V.S. Donnenberg. Univ
of Pittsburgh Sch. of Med., McGowan Inst. for Regen.
Med. and Cancer Inst., U.S.A.
B87 224
Utilization of Imaging Flow Cytometry for the
Identification of Rare Spermatozoa in Patients with
NonoObstructive Azoospermia
M. Filice, A. Gauthier-Fisher, K. Hahn, S. Swanson, K.
Lo, S. Moskovtsev and C. Librach. Create Fertility Ctr.
and Mount Sinai Hosp., Toronto and Univ. of
Toronto, Canada
B88 225
Flow Cytometry Knowledge Transfer: From the
Research Laboratory to the Yeast Production Facility
A. Mercier and F. D'Aoust. Lallemand, Montreal,
Canada
B102 239
Education Overhaul: Active Learning Approaches to
User Education in a Core Facility Setting
T. Baumgarther and J. Wilshire. Mem. Sloan-Kettering
Cancer Ctr., U.S.A. and STEMCELL Technols. Inc.,
Vancouver, Canada
Oral Session
Abstracts
B104 241
Expanding the MoFlo Legacy Capabilities with Easy
Laser Swapping
J. Dow, S. Coquery and N. Fisher. Univ of North
Carolina at Chapel Hill, U.S.A.
B105 242
Accellix Automated Flow Cytometry
J. Meissonnier, H. Kasdan, M. Rosen, E. Carlebach
and A. Reiter. LeukoDx / Accellix, Jerusalem, Israel
Commercial
Tutorials &
Exhibits
FLOW CYTOMETRY INSTRUMENTATION
Scholars &
Emerging
Leaders
B103 240
Evaluating Cell Sorter Cleaning Procedures across
ABRF-Flow Cytometry Research Group Institutions
by Testing for Common Contaminants
R. del Rio, K. Brundage, A. Bergeron, A. Box, M.
Cochran, M. DeLay, M.A. Handley, E.M.M. Meyer, A.
Saluk and P. Lopez. Univ. of Vermont, West Virginia
Univ., Dartmouth Col., Stowers Inst. for Med. Res.,
Univ. of Rochester, Cincinnati Children’s Hosp. Med.
Ctr., Massachusetts Gen. Hosp., Univ of Pittsburgh,
The Scripps Res. Inst. and NYU, U.S.A.
Poster
Session
Poster Session
Abstracts
B95 232
Automation of Cytokinesis-Block Micronucleus Assay
Using Imaging Flow Cytometry
C. Probst, M. Rodrigues, L. Beaton-Green, B. Kutzner
and R. Wilkins. MilliporeSigma, Seattle, U.S.A. and
Health Canada, Ottawa
B101 238
Standardization in a Multi-core Platform
K. Leone. Dana-Farber Cancer Inst., U.S.A.
Wednesday
15 June
DNA DAMAGE AND REPAIR
Tuesday
14 June
B93 230
Revealing the Treatment Response in Patients with
CTLa-4 Immunodeficiency with Single-Cell PhosphoFlow and Hyperspectral Flow Cytometry
V. Kanderova, M. Novakova, E. Fronkova, M. Svaton,
J. Kayserova, P. Smisek, O. Hrusak and T. Kalina. 2nd
Fac. of Med., Charles Univ. in Prague and Univ.
Hosp. Motol, Czech Republic
B94 231
Flow Cytometric and PCR-Based Characterization of
Immune Dysregulation in Shingles Patients
B. Crucian, S. Mehta, S. Tyring, H. Kunz, D. Chew, H.
Quiriarte, A. Renner and D. Pierson. NASA, JES Tech,
Univ. of Texas and Wyle Labs., Houston, U.S.A.
Monday
13 June
B91 228
Transcriptomic and Polychromatic Analysis of New
Subtype of B Cell Acute Lymphoblastic Leukemia
with a Switch to Monocytic Lineage
A. Dobiášová, M. Nováková, T. Kalina, M. Vášková,
E. Froòková, L. Slámová, M. Kubrièanová Žaliová, O.
Hrušák, K. Fišer and E. Mejstøíková. Charles Univ. in
Prague and Univ. Hosp. Motol, Czech Republic
B92 229
CD4+ Naive T Cells from HIV+ Patients with
Suppressed Viremia Contain Fewer Copies of Proviral
DNA and Greater Numbers of sjTRECs Compared to
Central or Effector Memory Lymphocytes
A. Cossarizza, S. De Biasi, E. Bianchini, M.
Digaetano, S. Pecorini, M. Pinti, L. Gibellini, V.
Borghi, C. Mussini and M. Nasi. Univ. of Modena and
Reggio Emilia and Univ. Hosp., Modena, Italy
B98 235
Purdue Cytometry Email Discussion–27 Years and
Growing
J.P. Robinson and B. Rajwa. Purdue Univ., U.S.A.
B99 236
Mathematical Model for Optimising the Operational
Performance of Shared Laboratory Resources
A M. Petrunkina. Univ. of Cambridge, U.K.
B100 237
Withdrawn
Sunday
12 June
DISEASE PROGRESSION MONITORING
B97 234
The Need to Know: Cytometry Education for
Biotechs
R. Walker and D.C. Davies. Babraham Inst.,
Cambridge, U.K. and The Francis Crick Inst., London,
U.K.
Saturday
11 June
®
Touch-Screen Based Muse AutoCD4/CD4% System
J. Clor, P. De Borja, D. Perry, C. Karaman and K.
Tyagarajan. MilliporeSigma, Hayward, CA, U.S.A.
B96 233
Merging Two Large Flow Cores: Expectations and
Challenges
D. Davies and G. Preece. The Francis Crick Inst.,
London, UK
Special
Lectures
B90 227
Simplified and Affordable CD4 Monitoring Using the
FACILITY MANAGEMENT
Congress
Overview
B89
226
+
CD34 Stem Cell Enumeration with Supplemental T
Cell and NK Cell Evaluation: A Comparison between
Manual and Automated Analysis Methods
J. Tario, Jr., J. Greenway, P. Wallace, B. Hill, C.B.
Bagwell and D. Herbert. Roswell Park Cancer Inst.,
Buffalo and Verity Software House Inc., Topsham,
ME, U.S.A.
Index
75
Speaker/Author
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Index
Abstracts
B106 243
Fifty Four Parameters of Red Blood Cells Population
Obtained by Scanning Flow Cytometer
E. Chernyshova, A. Chernyshev, K. Gilev, D.
Strokotov and V. Maltsev. Voevodsky Inst. of Chem.
Kinet. and Combustion SB RAS and Novosibirsk State
Univ., Russia
B107 244
Utilization of 3D Printing Technology in a Flow
Cytometry Shared Resource Facility
A. Saluk and M. Haynes. The Scripps Res. Inst., U.S.A.
B108 245
Comparing In Vitro Lung Adenocarcinoma Cell
Growth Using 2D and 3D Coculture Tissue Models
J. De Lora, J. Velasquez, C. Sher, J. Freyer and A.
Shreve. Univ. of New Mexico, U.S.A.
B109 246
Evaluation of Single Cell Plate Sorting in a
Multiinstrument Core Facility
M. Buonopane. Dana Farber Cancer Inst., U.S.A.
B110 247
Novel Ultrasensitive Blood Tests Using In Vivo Flow
Cytometry Platform
V. Zharov. Univ. of Arkansas for Med. Sci., U.S.A.
B111 248
Improving Flow Cytometer Performance by a Novel
Strategy for Optimizing Bandwidth Filters
K. Witte. Univ. Hosp. Tuebingen, Germany
B112 249
Automated Analysis and Sorting of Human iPS Cell
Clusters by Large Particle Flow Cytometry
R. Bongaarts. Union Biometrica Inc., Geel, Belgium
B113 250
Analytical Performance of the BD FACSVia™ System
in the Enumeration of Residual Leucocytes from
Leucoreduced Blood Products
Y. Zeng, M. Saleminik, F. Mosqueda, J. Nguyen, L.
Shatsman, A.D. Chau, R. Hershock-Quintana, M.
Dabay, S. Seetharaman, A. Lin and K. Judge. BD
Biosciences, San Jose and American Red Cross
Biomed. Svcs., Rockville, MD, U.S.A.
B114 251
Small Particle Flow Cytometry: Light Scatter
Performance for Extracellular Vesicle Studies
O. Kenyon. Apogee Flow Systems (UK), Castelldefels,
Spain
B115 252
42CH Hyperspectral Detection System for Flow
Cytometry
K. Hernandez, M. Yamamoto and J.P. Robinson.
Miftek Corp.,West Lafayette, IN and Purdue Univ.,
U.S.A.
76
B116 253
Standardization and Monitoring of Multiple Flow
Cytometers for Global Flow Cytometric Testing
B. Chitteti, C. Gonneau, N. Anfossi and V. Litwin.
Covance Inc., Indianapolis, U.S.A. and Geneva,
Switzerland
B117 254
Custom Integrated Water-Cooled Sort Collection
Tube Holders for BD FACSAria
S. Mazel, B. Mazel, M. Mazel and J.M. Petrillo.
Rockefeller Univ. and Columbia Univ., U.S.A.
B118 255
A Semi-automated Approach to Tracking and
Maintaining Instrument Performance on a BD Influx
Flow Cytometer
G. Osborne, F. Battye and S. Dervish. Queensland
Brain Inst., Brisbane, Flow Cytometry Consulting,
Viewbank, Australia and Univ. of Sydney
B119 256
Recent Advances in Instrumentation in Cambridge
Biomedical Research Centre Cell Phenotyping Hub
V. Radjabova, C. Bowman, N. Savinykh and A.
Petrunkina. Univ. of Cambridge, U.K.
B120 257
Acoustic Focusing for Single Cell Impedance
Spectroscopy
D. Kalb, J. Coons, C.K. Sanders and B. Marrone. Univ.
of New Mexico, CDC, Los Alamos and Los Alamos
Natl. Lab., U.S.A.
B121 258
New Capabilities for Characterization of Expanded
Fluorescent Protein Reporter Cell Lines Using
Microcapillary Cytometry
K. Gillis, M. Farbarik, P. de Borja, V. Nguyen, S.
Ahuja and K. Tyagarajan. EMD Millipore, Hayward,
U.S.A.
B122 259
Optimizing a Custom Benchtop Flow Cytometer for
the Detection of Extracellular Vesicles, with
Subsequent Application of Enhancements to a
Commercial Instrument
E. Podniesinski, J.D. Tario, Jr., P.K. Wallace, K.A.
Muirhead and R. Bankert. Roswell Park Cancer Inst.,
Buffalo, SciGro Inc-MidWest Ofc., WI and Univ. at
Buffalo SUNY, U.S.A.
B123 260
High Parameter Flow Cytometry: Going Beyond 18
Colors
A.J. Tyznik, S. Widmann, A. Nguyen, J. Rabenstein
and A. Stall. BD Biosciences, San Diego, U.S.A.
B124 261
Cytometry Systems That Collect Fluorescence
Lifetimes of Photoswitchable, Near-Infrared, and
Other Unique Fluorescent Protein Variants
J. Houston. New Mexico State Univ., U.S.A.
B129 266
Single Tube Flow Cytometry Quantification of ZAP70 Using Antibody Bound Per Cell Value of CD4 as a
Reference Standard
H. Degheidy, L. Wang, F. Abbasi, H. Mostowski and
S. Bauer. FDA, Silver Spring, MD and NIST,
Gaithersburg, MD, U.S.A.
B137 274
Flow Cytometry Multiplexing Used to Analyze
Metabolic Activity and Assess Pharmaceutical
Compound Toxicity as a High Throughput Screening
Tool
V. Calderon, L. Montoya, M. Yan, D. Beacham, A.
Anderson, C. DeMarco, K. Kihn, M. Wickett and M.
O'Grady. Thermo Fisher Scientific, Eugene, OR,
U.S.A.
Scholars &
Emerging
Leaders
B138 275
The Emulsion Sorting Technology Platform for HighThroughput Screening Assays in Various Fields of
Biology
Y. Bansho, J. Akagi, T. Ino, K. Osawa, T. Tanaka, M.
Ishige, Y. Fujimura and K. Takeda. On-chip
Biotechnologies Co. Ltd., Tokyo, Japan
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
B139 276
Application of Functional Genomics Methods to
Luminex-Based Assay Data
B. Capaldo, L. Muehling, J. Wisniewski, J. Woodfolk
and J. Lannigan. Univ. of Virginia Sch. of Med.,
U.S.A.
Poster Session
Abstracts
Index
77
Speaker/Author
Poster
Session
B132 269
Quantitative Label-Free Chemical Imaging of Cell
Compartments Using Hyperspectral CARS
Microscopy
R. Errington, A. Karuna, F. Masia, M. Wiltshire, P.
Borri and W. Langbein. Sch. of Med., Sch. of Phys.
and Astron., Sch. of Biosci., Cardiff Univ., U.K.
B136 273
Proteomic Changes Associated with Neoplastic
Transformation, EMT and Invasion in Metastatic
Breast Cancer
V. Donnenberg, J-J. Zhang, M. Corselli, E.M.M.
Meyer, C.T. Carson and A.D. Donnenberg. Univ. of
Pittsburgh Sch. of Med. and BD Biosciences, La Jolla,
CA, U.S.A.
Wednesday
15 June
HIGH CONTENT ANALYSIS
HIGH THROUGHPUT SCREENING
Tuesday
14 June
B131 268
Single Cell Analysis of Human Monocyte Subset
Heterogeneity Using Flow Cytometry Index Sorting
and High-Content mRNA Sequencing
M. Tycon, T. Le, C. Lomas, K. Dembski, J. Goglio, C.
Bush-Donovan and X. Wang. BD Life Sciences, San
Jose, U.S.A.
B135 272
An Objective Test to Qualify Plate Samplers for
Complex Flow Cytometric Assays
E. Meyer and A. Donnenberg. Univ of Pittsburgh
Cancer Inst. and Sch. of Med., Univ. of Pittsburgh,
U.S.A.
Monday
13 June
B130 267
Platelet Function Testing by Mass Cytometry
M. Linden. Univ. of Western Australia Sch. of Pathol.
and Lab. Med., Australia
B134 271
A High-Throughput 3D Tumor Spheroid Screening
Method for Drug Discovery Using Imaging
Cytometry
S. Kessel, S. Cribbes, O. Dery and L. Chan. Nexcelom
Bioscience, Lawrence, MA, U.S.A.
Sunday
12 June
B133 270
Profiling of ROS Sensitivity from Whole Blood
Samples on the iQue Screener PLUS: Featuring the
Automation of Acquisition/Analysis of Large Data Set
from Complex Biology
Z. Liu and K. Luu. IntelliCyt Corp., Albuquerque,
U.S.A.
Saturday
11 June
B125 262
Application of Flow Cytometry in Guiding the
Molecular Diagnosis of Severe Combined
Immunodeficiency Disorder
J. Aluri, M. Gupta, M. Kulkarni, A. Dalvi, P. Taur, M.
Desai and M. Madkaikar. Natl. Inst. of
ImmunoHaematol. and Bai Jerbai Wadia Children's
Hosp., Mumbai, India
B126 263
Evaluation of Pediatric B- and T-ALL Minimal
Residual Disease Levels in Turkey by Flow Cytometry
G. Deniz, A. Yilmaz, M.Y. Yilmaz, I. Tahrali, G. Ozcit
and S. Cinar. Istanbul Univ. and DETAE, Istanbul,
Turkey
B127 264
Imaging Flow Cytometry: A Potentially Powerful Tool
in the Assessment of Haematological Malignancies
H. Hui, K. Fuller, L. Grimwade, S. Bennett, A.
Chakera and W. Erber. Univ. of Western Australia,
Perth and Cambridge Univ. Hosps. NHS Fndn., U.K.
B128 265
Analysis of the Tumor Imprint on the Residual
Normal B Cell Compartment of CLL Patients by Mass
Cytometry
T. Andrieu, A. Michallet, P. Mondiere, F. Davi, S.
Dussurgey and T. Defrance. SFR Biosci., Lyon,
INSERM U1111, Hospices Civils de Lyon and Pitie
Salpetriere Hosp., Paris, France
Special
Lectures
HIGH THROUGHPUT INSTRUMENTATION
Congress
Overview
HEMATOLOGICAL DISORDERS
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Index
Abstracts
IMAGE PROCESSING AND ANALYSIS
B140 277
Towards Quantitative Cellular Astronomy
G. Gonzalez, E. Pardo, J. Tucker-Schwartz, A. de Lara
Rubio, S. Borromeo, N. Malpica and S. Dave. MIT,
U.S.A. and Univ. Rey Juan Carlos, Spain
IMMUNE MONITORING
B141 278
Leukocyte Surface Marker Expression Correlates with
Gender, Age and Lifestyle Factors: Cytomics within
the Life Study
S. Melzer, J. Bocsi, S. Zachariae, C. Engel, M. Löffler,
I. Dähnert and A. Tarnok. HELIOS Heart Ctr. Leipzig
and LIFE – Leipzig Re. Ctr. for Civilization Dis.,
Germany
B142 279
Autoimmune Signature in Narcolepsy Revealed by
High-Dimensional Mass Cytometry
F. Hartmann, R. Bernard-Valnet, D. Mrdjen, E. Galli,
C. Krieg, L. Weber, M. Robinson, R. Liblau and B.
Becher. Univ. of Zurich, Switzerland and INSERM
U1043, Toulouse, France
B143 280
Automatic Measurement of the T Cell Activation via
Flow Cytometry FRET
K. von Kolontaj, G. Horvath, E. Latz and M. Büscher.
R&D Engineering, Bergisch Gladbach and Univ. of
Bonn, Germany
B144 281
Comparison of CyTOF Assays across Sites: Results of
a Six-Center Pilot Study
M. Leipold, G. Obermoser and H. Maecker. Stanford
Univ., U.S.A.
B145 282
Standardization and Quality Control Using Control
Samples Minimize Batch Effects and Improve
Reproducibility of Mass Cytometry Assays in Human
Immunology Studies
K. Kleinsteuber, B. Corleis, N. Rashidi, N. Nchinda, A.
Lisanti, D. Kwon and B. Walker. Ragon Inst. of MGH,
MIT and Harvard, Cambridge, U.S.A., Heinrich Pette
Inst. for Exptl. Virol., Hamburg, Germany and HHMI,
Chevy Chase, MD, U.S.A.
B146 283
Qualification and Implementation of an Interlaboratory Quality Assurance Program for a Whole
Blood Intracellular Cytokine Staining Assay
E. Smit, N. Bilek, B.M. Kagina, A. Penn-Nicholson, S.
Suliman, V. Rozot, H. Mearns, N. du Plessis, G.
Walzl, W.A. Hanekom, E. Nemes and T.J. Scriba.
Univ. of Cape Town and Univ. of Stellenbosch, South
Africa
78
B147 284
Using Intracellular Amplified Nucleic Acid Detection
Technique to Measure Telomere Length in Individual
T-Cell Subsets
A. Henning, D. Levitt, J. Vingren and B. McFarlin.
Univ. of North Texas, U.S.A.
B148 285
Towards Implementation of High-Dimensional
CyTOF Immunophenotyping in Large-Scale Human
Immune Monitoring Studies.
A. Rahman, X. Guo and O. Mayovska. Icahn Sch. of
Med. at Mount Sinai, U.S.A.
B149 286
HIV Induced CD4+ T Cell Dysregulation: A Case for
Supplementary Immune Modulation
S. Salwe, A.K. Singh, V. Padwal, S. Velhal, A.
Bandivdekar and V. Patel. Natl. Inst. for Res. In
Reprod. Hlth., Mumbai, India
B150 287
Characterization of Adaptive Immune Responses
across Species and Tissues
S. Guenounou, C. Joly, C. Cassan, R. Le Grand, A.
Cosma and V. Contreras. CEA, Fontenay-aux-Rosas,
France
B151 288
Adaptation of the Truculture® System for Flow
Cytometry Analysis of Leukocyte Activation Markers
L. Green, W. White and M. Roberts. Covance Inc.,
Greenfield, IN and MedImmune LLC, Gaithersburg,
MD, U.S.A.
B152 289
Swaying Ȝį T Cell Subset Phenotypes Influence HLA
B27 Responsive Auto Immunity
K. Thyagarajan and B.N. Narayanan. Beckman
Coulter India Pvt. Ltd., Bangalore, India
B153 290
Highly Reliable Quality Assurance Procedure for
Whole Blood Immunophenotyping Assays Using
Stabilized Whole Blood Process Controls
T. Poplonski, I. Gonzalez, T. Meuwsen, W. Miller, V.
Conrad, A. Howells-Ferreira, K. Bahjat and Y.
Koguchi. Earle A. Chiles Res. Inst., OR and BristolMyers Squibb, CA, U.S.A.
B154 291
Assessment of Specific T Cell Cytokine Responses
Using a Standardized Approach Devoid of Reagent
Pipetting
N. Dupas, F. Malergue, A. Blum, K. Thyagarajan, M.
Kapinsky and T. Miloud. Beckman Coulter, Marseille,
France, Charité Virchow Clin., Berlin, Gemany and
Beckman Coulter, Bangalore, India
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
B169 306
A High-Throughput Image Cytometry-Based
Screening Method for the Detection of IL2-Induced
Peripheral Blood Mononuclear Cell-Mediated
Cytotoxicity
L. Chan, K. McCulley and P. Banerjee. Nexcelom
Bioscience, Lawrence, MA and Baylor Col. of Med.,
U.S.A.
B170 307
A Novel Image Cytometric Analysis Method for T
Cell-Mediated Cytotoxicity of 3D Tumor Spheroids
L. Chan, L. Humbert and S. Cribbes. Nexcelom
Bioscience, U.S.A. and Immunocore Ltd., Abingdon,
U.K.
Scholars &
Emerging
Leaders
Poster Session
Abstracts
Index
79
Speaker/Author
B168 305
Centenarians Show a Better Response to Induced
Oxidative Stress Compared with Offspring and Their
Age-Matched Controls
F. Sizzano, S. Collino, O. Cominetti, D. Monti, P.
Garagnani, R. Ostan, C. Pirazzini, M.G. Bacalini, C.
Franceschi and A. Palini. Nestlé Inst. of Hlth. Sci.,
Lausanne, Switzerland and Univ. of Florence, Univ. of
Bologna and IRCCS Inst. of Neurol. Sci. of Bologna,
Italy
Poster
Session
B162 299
Using Flow Cytometry to Assess Monocyte to
Macrophage Differentiation, in an In Vitro Model of
Human Non-infectious Posterior Uveitis
J. Washington, Y. Ma, C. Macardle, S. Bailey and J.
Smith. Flinders Univ. and SA Pathology, Bedford Park,
Australia
B167 304
CD107a Marker and Cytokine Detection for Study
the Role of Distinct Cytotoxic Cell Population in
Cutaneous Leishmaniasis
R. Ferraz, C.F. Cunha, M.I.F. Pimentel, M.R. Lyra,
A.O. Schubach, A.M. Da-Cruz and A.L. Bertho.
Oswaldo Cruz Fndn., Rio de Janeiro, Brazil
Wednesday
15 June
B161 298
Study of New Specific Populations from HIV Infected
Patients by Mass Cytometry
A. Leite Pereira, J. Elhmouzi-Younes, C. Tardiveau, O.
Lambotte and R. Le Grand. CEA, Fontenay-aux-Roses
and Bicêtre Hosp., Le Kremlin-Bicêtre, France
Tuesday
14 June
B160 297
CASKCyto to Accelerate Mass Cytometry Analysis in
IRM Discovery
S. Wang, J. Ma, Y. Qu and A. Chi. Merck, Rahway, NJ
and Boston, MA, U.S.A.
Monday
13 June
B159 296
Characterizing the Role of Broadly Neutralizing
Antibodies in HIV Immune Response
P. Van, C. Murie, R. Gottardo and F. Porichis. Fred
Hutchinson Cancer Res. Ctr., Seattle and and Ragon
Inst., Cambridge, MA, U.S.A
B165 302
Comparison of Electronic Cigarette Vaping and
Smoking in a Mouse Model of COPD
B. Saxton, M. Madison, C. Landers, H-Y. Tung, B-H.
Gu, Y. Qian, F. Kheradmand, D. Corry, J. Sederstrom
and P. Porter. Baylor Col. of Med., U.S.A.
B166 303
Flow Cytometry Approach to Understand the
Mechanisms Involved in the Immunopathogenesis of
Human Cutaneous Leishmaniasis
C. Cunha, R. Ferraz, M. Pimentel, M. Lyra, A.
Schubach, A. DA-Cruz and A.L. Bertho. Oswald Cruz
Inst., Rio de Janeiro, Brazil
Sunday
12 June
IMMUNOLOGY
B164 301
OptiSort: Towards Optimal Neutrophil Purification
A. Bartholomäus, M. Mengel, D. Grummit, C. Peth
and M. Büscher. Miltenyi Biotec GmbH, Bergisch
Gladbach, Germany and Owl Biomed., Santa
Barbara, CA, U.S.A.
Saturday
11 June
B158 295
Breaking the Barrier for Polychromatic Flow Cell
Sorting
J.F. Whitesides, A-L. Savoye, J. Van Dyke, B.F. Haynes
and M.A. Moody. Duke Univ. sch. of Med. and Univ.
of California-Davis, Sacramento, U.S.A.
Special
Lectures
B157 294
Toward the Quantification of Integrin Conformations
in Human Lymphoblastic Cells Using HighThroughput, Multifrequency Flow Cytometry
J. Sambrano, Jr., A. Chigaev, L.A. Sklar and J.P.
Houston. New Mexico State Univ. and Univ. of New
Mexico Sch. of Med., U.S.A.
B163 300
Modulation of T Cell Immune Response by
Differential Ration of Fish Oil and Corn Oil in
Experimentally Induced Colon Carcinoma
R. Malik, N. Agnihotri and A. Bhatnagar. Panjab
Univ., India
Congress
Overview
B155 292
TREM-1 and HLA-DR Expression in Human
Monocytes from Septic Patients Is Not Related with
Pro-inflammatory Cytokine Milieu
L. Arriaga-Pizano, L. Cabrera-Rivera, L-A. FloresMejia, J. Prieto-Chavez, A. Cérbulo-Vázquez, C.
López-Macias, E. Ferat-Osorio and A. Isibasi. IMSS
Hosp. de Espec. "CMN Siglo XXI", ENCB IPN and
UNAM Fac. of Med., Mexico City, Mexico
B156 293
Immunophenotypic Lymphocyte Characterization in
Colostrum, Early and Mature Human Milk
A. Cérbulo-Vázquez, O. Pralta-Méndez, I. MancillaHerrera and L. Arriaga-Pizano. Hosp. de la Mujer,
Natl. Inst. of Perinatol. and IMSS, Mexico City,
Mexico
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Index
Abstracts
B171 308
Identification and Characterization of Circulating,
Tumor-Associated Double Positive T-Cells in the
Melanoma Pig Model by Flow Cytometry
Z. Sinkorova, J. Sinkora, M. Sinkora, M. Cervinkova, J.
Cizkova and P. Kucerova. Univ. of Defence, Hradec
Kralove, BD Biosciences, Prague, Inst. of Microbiol.
and Inst. of Animal Physiol. and Genet., AS CR,
Liebechov, Czech Republic
B172 309
Chronic In Vitro Alcohol Increases Mitochondrial
DNA Depletion in T Cells and Promotes
Immunosenesence
C. Porretta, D. Welsh and R. Siggins. LSU Hlth. Sci.
Ctr., New Orleans, U.S.A.
B173 310
Multicolor Phenotypic Characterization of Human
Antibody Secreting Cells: Tissue Differences and
Validation of the ASC Immunoglobulin Secretion and
Gene Expression Profile
J. Carrell, B. Rajan, R. Rayanki, R. Grady, R. Kolbeck
and C. Groves. MedImmune LLC, Gaithersburg, MD,
U.S.A.
B174 311
Polychromatic Flow Cytometry and Single Cell
Sorting Reveal Transcriptional Heterogeneity of
Natural Killer Cells
D. Kim, M. Costanzo, M. Creegan, K. Lal, J. Ake, M.
Robb, N. Michael, D. Bolton, N. Steers and M. Eller.
Walter Reed Army Inst. of Res., Silver Spring, MD,
Henry M. Jackson Fndn., Bethesda and Columbia
Univ., U.S.A.
B175 312
Quantifying Neutrophil Extracellular Trap Formation
Using Flow Cytometry
C. Durandt, J-G. Nel, A. Theron, R. Pool, G.R.
Tintinger and R. Aderson. Univ. of Pretoria, South
Africa
INFECTIOUS DISEASES
B176 313
Association of Interleukin-18 Gene Polymorphism
with Susceptibility to Visceral Leishmaniasis in
Endemic Area of Bihar, an Indian Population
A.
Kumar, P. Tiwary, J. Chakravorty and S.
Sundar. Banaras Hindu Univ., India
B177 314
High-Dimensional Mapping of B Cell Subsets in HIV1 Infection Using Multiparametric Flow Cytometry
Reveals Perturbations in Naive and Memory B Cell
T. Liechti, D. Braun, H. Kuster, J. Böni, H. Günthard
and A. Trkola. Univ. of Zürich and Univ. Hosp.
Zürich, Switzerland
80
B178 315
Memory and Activation Profiles of Mycobacterium
tuberculosis-Specific CD4 T Cells in Acute and
Established Infection
E. Nemes, C. Mpande, O. Dintwe, H. Mahomed, W.
Hanekom, M. Hatherill and T. Scriba. Univ. of Cape
Town, South Africa
B179 316
Magneto-optical Diagnosis of Malaria in Patient
Samples
B. Grimberg, V. Byrd, D. Blankenship, R. Bihary and
R. Deissler. Case Western Reserve Univ., U.S.A.
LIVE CELL IMAGING/TRACKING
B180 317
Novel Reporter Cell Lines Developed by Endogenous
Gene Tagging
D. Malkov, A. Klarich, A. Samsonov, N. Zenser, F.
Zhang and H. Zhang. MilliporeSigma, St. Louis,
U.S.A.
B181 318
Selective Analysis of RNA in Live and Fixed Cells
with Strandbrite RNA Green
Z. Luo, Q. Zhao, J. Liu, R. Peng, J. Liao and Z. Diwu.
AAT Bioquest Inc., U.S.A.
B182 319
The Role of Monocytes in Foam Cell Formation:
Leveraging Microperfusion Cell Culture and Live Cell
mRNA Detection to Elucidate Mechanisms of Action
B. McFarlin, A. Henning and A. Venable. Univ. of
North Texas and MilliporeSigma, Denton, U.S.A.
MICROBIOLOGY AND AQUATIC SCIENCES
B183 320
Effect of Proanthocyanidins on the Biofilm of
Streptococuus Mutans in Vitro
M-Y. Li. Shanghai Jiao Tong Univ., China
B184 321
Quantification of Symbionts in the Protist
Paramecium Bursaria Using the CyoFLEX™ Flow
Cytometer
K. Hogg, E. Minter, G. Park, M. Brockhurst and P.
O'Toole. Univ. of York, U.K.
B185 322
Identification and Enumeration of Marine Organisms
in Water Using Imaging Flow Cytometer
S. Vaidyanathan, B. Davidson, S. Friend and P.
Morrissey. Amnis part of MilliporeSigma, Seattle,
U.S.A.
B186 323
FACS Sorting Is an Effective Way to Purify Live
Microorganisms from Seawater
F. Chen, X. Wu, X. Yang and L. Fang. Becton
Dickinson Med. Devices (Shanghai) Co. Ltd., China
Oral Session
Abstracts
B202 339
Near Ultraviolet Laser Diode Excitation of Late
Generation Brilliant Ultraviolet Fluorochromes
W. Telford, N. Hawk and V. Kapoor. NCI, NIH,
Bethesda, U.S.A.
Commercial
Tutorials &
Exhibits
Poster Session
Abstracts
Index
B203 340
Chlorins: A Novel Family of Violet Laser-Excitable
Red to Far-Red Fluorophores for Polychromatic Flow
Cytometry
J.B. Pitner, R.B. Evans-Storms, D.A. Olsen, M.
Taniguchi and J.S. Lindsey. NIRvana Sciences,
Research Triangle Park, NC and North Carolina State
Univ., U.S.A.
81
Speaker/Author
B200 337
Point-Of-Care Diagnosis for Iron Deficiency Anemia
A. Velasquez, J. Tigges, J. Felton, S. Lu, V. Camacho,
V. Toxavidis and I. Ghiran. Beth Israel Deaconess
Med. Ctr., U.S.A.
B201 338
Live Cell Flow Cytometric Assay to Assess Mu Opioid
Receptor Internalization Using a Fluorogen
Activating Proten-Based Biosensor
A. Baumann, D. Gebhard, L. Pustilnik, S. Humphreys,
D. Cawkill and R. Doyonnas. Pfizer, Groton, CT,
U.S.A. and Pfizer, Cambridge, U.K.
Scholars &
Emerging
Leaders
B195 332
The Relative Quantification of Antigen Density: A
Key Step Towards the Rationalization of Panel
Design in Multi Parametric Flowcytometry
H. Luche, C. Santa Maria, P. Grenot, L. Hadjem and
M. Malissen. CIPHE, Marseille, France
NEW PROBES AND ASSAYS
Poster
Session
B192 329
Sentinel Gating: A Case Study
H. Sobon. Dana-Farber Cancer Inst., U.S.A.
B193 330
Cross-Platform Comparison of 18-ParameterMeasurements Using Spectral Flow Cytometry,
Conventional Flow Cytometry and Mass Cytometry
F. Mair and B. Becher. Univ. of Zurich, Switzerland
B194 331
RNA Detection of CD163 in Conjunction with
Multiple Cell Surface Markers to Identify
Macrophage Populations Using Flow Cytometry
S. Thornton, A.A. Sproles, T. Do, J. Schick, R. Tan, M.
DeLay and G. Schulert. Cincinnati Children’s Hosp.
Med. Ctr., U.S.A.
B199 336
Multiparametric Cell Surface and Intracellular
Imaging of Cells by Nanosims
M. Linden, H. Jiang, I. Larma, A. Buckley and M.
Kilburn. Univ of Western Australia, Australia
Wednesday
15 June
B191 328
New Cytometry Principles, Spectral and Mass
Cytometry: Comparison of Methods in Diagnostic
and Research Laboratory
M. Novakova, O. Pelak, D. Kuzilkova, D. Thurner, P.
Semerak, E. Mejstrikova, O. Hrusak, M. Vaskova and
T. Kalina. 2nd Fac. of Med., Charles Univ. in Prague
and Univ. Hosp. Motol, Czech Republic
Tuesday
14 June
MULTI-DIMENSIONAL FLOW CYTOMETRY
B198 335
Single Cell Analysis of Human Islets in Type I
Diabetes Using Highly Multiplexed Imaging
S. Engler, P. Herrera, M.A. Atkinson and B.
Bodenmiller. Univ. of Zurich and Univ. of Geneva,
Switzerland and Univ. of Florida, U.S.A.
Monday
13 June
B190 327
Acoustic Trapping Enables Novel Means to Access
Submicrometer Particles and Microvesicles in
Biobank Sample Volumes
T. Laurell, O. Gidlöf, D. Erlinge, A. Ku, K. Petersson,
H. Lilja, P. Ohlsson, Y. Ceder and M. Evander. Lund
Univ., Sweden
Sunday
12 June
B189 326
Critical Components of Design of Portable
Microfluidic Cytometry Devices for Diagnostics
J. Leary. Aurora Life Technols. LLC, Santa Fe, U.S.A.
B197 334
CNS and Systemic “Oxidative Stress Memory” in
Chronic Neuroinflammation Revealed by
Simultaneous Intravital Marker-Free NAD(P)H-FLIM
and Neuronal Calcium Imaging
R. Niesner, D. Bremer, R. Mothes, A. Mossakowski, F.
Paul, A. Hauser and H. Radbruch. DRFZ and Charité Univ. of Med., Berlin, Germany
Saturday
11 June
MICROELECTRO-MECHANICAL SYSTEMS (MEMS)
AND MICROFLUIDICS
MULTI-DIMENSIONAL IMAGE CYTOMETRY
Special
Lectures
B188 325
Determining Phytoplankton Community Composition
and Biomass from Different Biogeochemical Regimes
in the N. Atlantic Gyre Using Flow and Imaging
Cytometry
N. Poulton and L. Lubelczyk. Bigelow Lab. for Ocean
Sciences, E. Boothbay, ME, U.S.A.
B196 333
Single-Cell Mass Cytometry Reveals Phenotypic and
Functional Heterogeneity in Acute Myeloid Leukemia
at Diagnosis, Remission and Relapse
L. Han, P. Qiu, J.L. Jorgensen, D.H. Mak, J.K. Burks,
T.J. McQueen, F. Ravandi, S.O. Ciurea, G. AI-Atrash,
H.M. Kantarjian, S.M. Kornblau, M. Andreeff, M.L.
Guzman and M. Konopleva. MD Anderson Cancer
Ctr., Georgia Tech, Emory Univ. and Weill Med. Col.
of Cornell Univ., U.S.A.
Congress
Overview
B187 324
Recruitment of an Effective Efflux System Is Crucial
for 2-Hydroxybiphenyl Metabolism in Pseudomonas
azelaica HBP1
K. Czechowska, C. Reimmann and J. van der Meer.
Lausanne Univ., Switzerland
Congress
Overview
Special
Lectures
B205 342
Assessment of SiR-Hoechst by Flow Cytometry: A
Potential Far-Red DNA Stain for Live Cell
V. Valerie, G. Grazvydas, L. Loic, A. André and M.
Miguel. Ecole Polytech. Fed. de Lausanne,
Switzerland
Poster
Session
Wednesday
15 June
Tuesday
14 June
Monday
13 June
Sunday
12 June
Saturday
11 June
B204 341
Cytometry of HbA1c
A. Van Agthoven, F. Malergue, C. Godefroy and E.M.
Rabellino. Beckman Coulter Immunotech, Marseille,
France and Beckman Coulter Inc., Miami, U.S.A.
B206 343
Dual Fluorescence/Mass-Tag Antibody Conjugates
for Pre-selection of Cells by FACS for Subsequent
Mass Cytometry
S. Baumgart, A. Peddinghaus, A. Schulz, S.
Krauthäuser, C. Dose, A. Radbruch, H. Mei and A.
Grützkau. German Rheumatism Res. Ctr. (DRFZ),
Berlin and Miltenyi Biotec GmbH, Bergisch
Gladbach, Germany
B207 344
A Novel Non-cytotoxic Fluorescent Dye for Cell
Proliferation Analysis
P. Melquist. Thermo Fisher Scientific, U.S.A.
B208 345
Monitoring Proteolyic Cleavage at the Cell Surface
through a Novel Cell-Based Platform
C. Smurthwaite, A. Dharmawan, D. Abbadessa, C.
Reed and R. Wolkowicz. San Diego State Univ.,
U.S.A.
B209 346
Horizon Brilliant UV Dyes - Testing New Colors Out
L. Martinez, T. Lopez Briones, U.P. Cronin, E. Garrido
and M.A. Sanchez Luengo. CNIO-Spanish Natl
Cancer Res. Ctr., Madrid
Index
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Scholars &
Emerging
Leaders
NEW SOFTWARE DEVELOPMENT
B210 347
Automated Data Analysis and Interpretation in Flow
Cytometry: Application for the Unsupervised
Diagnosis of Lymphoproliferative Neoplasms
E. Papadopoulou, K. Kotta, P. Moschonas, V. Douka,
A. Anagnostopoulos, K. Stamatopoulos and D.
Tzovaras. Ctr. for Res. & Technol. and G.
Papanikolaou Hosp., Thessaloniki, Greece
B211 348
Crowdsource Your Controls: Promoting and
Validating Dynamic FMO-Based Analysis
T. Crawford, J. Panopoulos, J. Almarode, M. Simm, C.
Simmons, M. Swindle and M. Stadnisky. FlowJo LLC,
Ashland, OR, U.S.A.
B212 349
onTrack 2.0: A New Automated Approach to QC on
the BD Influx
J. Van Dyke and B.E. McLaughlin. Univ. of California,
Davis and UC Davis, Sacramento, U.S.A.
82
OTHER BIOLOGICAL APPLICATIONS
B214 351
Laser-Induced Breakdown Spectroscopy Using
Lanthanide Tagged Bio-molecules
H. Kim, I-J. Doh, E. Biela, C. Gondhalekar, B. Rajwa,
V. Patsekin, P. Diwakar, E. Bae, L. Stanker and J.P.
Robinson. Purdue Univ. and USDA, West Lafayette,
IN, U.S.A.
B215 352
A Portable Atomic Spectrometer Using Electrical
Spark and Metal-Conjugated Biomolecules
H. Kim, I-J. Doh, C. Gondhalekar, E. Biela, B. Rajwa,
V. Patsekin, E. Bae, P. Kulkarni and J.P. Robinson.
Purdue Univ. and Ctr. for Dis. Control, Cincinnati,
U.S.A.
B216 353
The Effect of Laser Power on Data Resolution in Flow
Cytometry
C. Hall, J. Graham, B.L. Ng and C. Kirton. Wellcome
Trust Sanger Inst., U.K.
B217 354
Streamlined Workflow for Sterile Cell Sorting Using
Microfluidics Chip Based Sorter
E. Ikeda, R. Serita and Y. Sakai. Sony Corp., Tokyo,
Japan
B218 355
Flow Cytometry as a Tool to Quantify the Cellular
Immune Response in Biomphalaria glabrata
Haemocytes: Actions of Nanoparticles, Plant
Products and Synthetic Pesticides
R. Ferraz, C.L. Coelho da Silva, M.J. Faro, D.
Fernandes Baptista and A.L. Bertho. Oswaldo Cruz
Inst., FIOCRUZ, Rio de Janeiro, Brazil
B219 356
Yeast Count and Viability Measurements Using the
Simplified, Touchscreen-Based Muse Cell Analyzer
K. Tran, L. Rollins and K. Tyagarajan. EMD Millipore,
Hayward, CA, U.S.A.
OTHER CLINICAL APPLICATIONS
B220 357
Withdrawn
B221 358
Application of Circulating Platelet Activation and
Platelet-Leukocyte Adhesion Using Flow Cytometry
W. Cui, S. Liu, L. Dong, L. Cai, Y. Li and J. Du.
Beijing Anzhen Hosp., China
B222 359
Prevalence of Regulatory T Cell Subtypes in
Preeclampsia
C. Orban, Z.E. Vásárhelyi, A. Bajnok, A. Molvarec, J.
Rigó Jr and G. Toldi. Semmelweis Univ., Hungary
B223 360
Barth Syndrome: Cardiolipin Alterations Linked to
Tafazzin Mutations Lead to Apoptosis and Mitophagy
Alterations
P.X. Patrice X.. Univ. Paris-Sorbonne
Monday
13 June
Tuesday
14 June
PROTEIN ENGINEERING
B239 376
Quantitation of Sub-visible Particles in Therapeutic
Protein Formulations Using Imaging Flow Cytometry
C. Probst, Y. Zeng and R-R. Zhu. MilliporeSigma,
Seattle and Bedford, MA, U.S.A.
Wednesday
15 June
RARE EVENT DETECTION
Poster
Session
B240 377
Microvesicles in Amiotrophic Lateral Sclerosis:
Potential Biomarkers for Disease Propagation and
Therapeutic Targets
S. La Salvia, F. Colombo, D. Sproviero, L. Diamanti,
O. Pansarasa, S. Gagliardi, B. Paola, M. Ceroni, L.
Porretti and C. Cereda. "C. Mondino" Natl. Neurol.
Inst., Univ. of Pavia, Fndn. IRCCS Ca' Granda
Ospedale Maggiore Policlin., Milan, Res. Inst.
Humanitas, Rozzano, IRCCS Natl. Inst. of Neurol.
Fndn., Pavia, Italy
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
B241 378
Single-Particle Characterization of Vaccinia Virus by
Flow Virometry
V. Tang, T. Renner, O. Varette, F. Le Boeuf, J. Wang,
J-S. Diallo, J. Bell and M-A. Langlois. Univ. of Ottawa
and Ottawa Hosp. Res. Inst., Canada
Oral Session
Abstracts
Poster Session
Abstracts
B242 379
High Throughput Detection of Rare T Cell
Populations Enhanced by Acoustic Focusing
M. Brudner, K. Hopsen, L. Kien, D. Smith, E. Flano
and L. McNeil. Genocea Biosciences, Cambridge,
U.S.A.
Index
83
Speaker/Author
Sunday
12 June
B233 370
Concurrent Labeling of Fluorescent Tagged
Antibodies with Metal Tags for Flow and
MassCytometry
J. Lannigan, C. Chew, M. Solga and B. Capaldo. Sch.
of Med., Univ of Virginia, U.S.A.
B236 373
Application of Isotype Control in Mass Cytometry
(CyTOF)
L. Lai, J. Li, C.T. Ng, R. Ong, V. Chew, J.Y. Leong and
S. Albani. Singapore Hlth. Svcs. Pte Ltd., Singapore
Gen. Hosp. and Duke-NUS Med. Sch., Singapore
B237 374
Light Scattering Properties of Nanomaterials
A. Rosner, A. Morales Kastresana, W. Telford, J.
Berzofsky and J.C. Jones. NCI, NIH, U.S.A.
B238 375
Methods to Simplify the Design of Multicolor Panels
S. Widmann, X. Wang, M. Corselli, J. Rabenstein and
A. Stall. BD Biosciences, San Diego, U.S.A.
Saturday
11 June
B227 364
Discovery of New Gene Targets for Synergistic
Cystic Fibrosis Therapies
L. Perkins, G. Fisher, M. Naganbabu, B. Schmidt, F.
Mun and M. Bruchez. Carnegie Mellon Univ., U.S.A.
B228 365
A Budget Version of Mass-Cell-Barcoding Used for
Mass Cytometry Antibody Validation on Multiple
Stimulation Conditions
C. Schwärzler, T. Andrieu and M. Garcia. EPFL,
Lausanne, Switzerland and INSERM, Lyon, France
B229 366
Industry 4.0 Reaching Flow Cores: Comprehensive
Robotisation of a Cytometry Workflow
C. Hennig, A. Mirenska and J. Detmers. Zellkraftwerk
GmbH, Hannover, Germany
B230 367
Novel Method for the Analysis and Interpretation of
Extracellular Vesicles in an Exercise Model
V. Camacho, J. Tigges, V. Toxavidis, S. Das and I.
Ghiran. Beth Israel Deaconess Med. Ctr., Boston,
U.S.A.
B231 368
Fluorophore Selection for Extracellular Vesicle Labels
with High Resolution Cytometry
J.C. Jones, A. Morales Kastresana, W.G. Telford, K.
McKinnon and J. Berzofsky. NCI, NIH, U.S.A.
B232 369
High-Resolution Noise Floor Analysis with LED
Pulser
J. Jones, A. Rosner, K. McKinnon, C. Ross, W.G.
Telford and J. Berzofsky. NCI, NIH and Beckman
Coulter Inc., Fort Collins, U.S.A.
B235 372
Generating Flow Profiles at Sea
C. Brownlee and G. van den Engh. Univ. of New
South Wales, Australia and Cytometry, Concrete, WA,
U.S.A.
Special
Lectures
OTHER TECHNOLOGY ADVANCES
B234 371
Ultrasensitive Detection of Serum Proteins Using Full
Assay Automation
L. Samsel, V. Dominical and J.P. McCoy. NHLBI,
NIH, U.S.A.
Congress
Overview
B224 361
Assessment of Measurement of Reticulated Platelets
in Normal Full-Term, Preterm Neonates and
Thrombocytopenic Neonates
K. Osada. Yuho Med. Fndn., Mitaka, Japan
B225 362
Culture of HSCs In Vitro According to GMPStandards
J. Mellet, C. Durandt and M. Pepper. Univ. of
Pretoria, South Africa
B226 363
Segmentation and Count of Corneal Cells Using
Median Filtering and Mathematical Morphology
K.K. Kuduvanagatta Venkatram and G. Srinivasa.
PESIT - BSC, Bangalore, India
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Index
Abstracts
REGENERATIVE MEDICINE
B243 380
Flow Cytometry as a Tool to Analyze the Impact of
Digestion Reagents on Primary Epidermal Cells
P. Langa, M. Pikula, V. Savkovic, A. Wardowska, J.
Zielinski, K. Kondej, A. Hedrych-Ozimina, A. Tarnok
and P. Trzonkowski. Med. Univ. of Gdansk, Poland,
Translational Ctr. for Regen. Med., Leipzig, and Univ.
of Leipzig, Germany
SIGNAL TRANSDUCTION
B244 381
Imaging Flow Cytometry-Based Assay for IRF-8
Expression
H. Minderman, K. O'Loughlin, O. Maguire, J.
Muhitch, T. Schwaab and S. Abrams. Roswell Park
Cancer Inst., Buffalo, U.S.A.
B245 382
Image Based Assessment of NFțB Nuclear
Translocation in Murine Cells with Imagestream
Cytometry
R. Kong, H. Pugsley, S. Friend and P. Morrissey.
Amnis part of MilliporeSigma, Seattle, U.S.A.
B246 383
An Experimental Pipeline Developed for the
Verification of Intracellular Phospho-Specific
Antibodies in Mass Cytometry
S-E. Gullaksen, S. Gavasso, A. Sulen, L. Bader and
B.T. Gjertsen. Univ. of Bergen, Haukeland Univ.
Hosp. and Helse Bergen, Norway
B247 384
Ovarian Carcinoma Ascites-Derived Extracellular
Vesicles Inhibit NFATsignaling in Antigen-Specific T
Cells
O. Maguire, G. Shenoy, R. Kelleher, J. Loyall, K.
Odunsi, P.K. Wallace, R. Bankert and H. Minderman.
Roswell Park Cancer Inst. and Univ. at Buffalo SUNY,
U.S.A.
SINGLE MOLECULE ANALYSIS AND TRACKING
B248 385
Counting and Sizing of Single DNA Fragments Using
High Sensitivity Flow Cytometry
M. Hussels, N. Bock, S. Dehnad, A. Devonshire, J.
Huggett, G. Jones, A. Gutteridge, G. Nixon and J.
Neukammer. Phys.-Tech. Bundesanstalt, Berlin and
LGC, Teddington, U.K.
SOLID TUMORS
B249 386
Proliferation and Polyploidization of Near-Triploid
Clones in Drug-Resistant Breast Carcinomas
B. Gerashchenko, J. Erenpreisa and K. Salmina.
Latvian Biomed. Res. and Study Ctr., Riga and R.E.
Kavetsky Inst. of Exptl. Pathol., Oncol. and and
Radiobiol., Kyiv, Ukraine
84
B250 387
Mass Cytometry Analysis of Pediatric Brain Tumors
M. Vaskova, D. Kuzilkova, F. Garcia N´Dua, D.
Thurner, T. Kalina and O. Hrusak. 2nd Fac. of Med.,
Charles Univ. in Prague and Univ. Hosp. Motol,
Czech Republic
STANDARDS AND CALIBRATION
B251 388
Optimization of 15-Color Panel Design Using the
FMO Controls
W. Liu, Y. Hao, X. Yang, Y. Zhou, X. Wu and L. Fang.
Becton Dickinson Med. Devices (Shanghai) Co Ltd.
and Beijing Ditan Hosp., China
B252 389
High Sensitivity Flow Cytometric Measurement of
Side-Scattered Light in the Submicron Size Range
W. Zhang, Y. Tian, C. Chen, L. Ma, S. Zhu and X.
Yan. Col. of Chem. and Chem. Engin., Xiamen Univ.,
China
B253 390
A Simple Method to Reliably Measure the Absolute
Number of Soluble Fluorochromes on a Flow
Cytometer
Y. Chen, G. Brittain and S. Gulnik. Beckman Coulter,
Miami, U.S.A.
B254 391
FlowQC: Pushing for a Global Solution to Monitor
Instrument Performance
C. Bispo, C. Andrade and R. Gardner. Inst.
Gulbenkian de Ciencia, Portugal
B255 392
Quantitative Comparison Study of Flow Cytometers
Using a Novel Ultra-Stable Calibration Light Source
K. von Volkmann, K. Feher, J. Kirsch, J. Popien and T.
Kaiser. APE Angewandte Physik & Elektronik GmbH
and DRFZ, Berlin, Germany
B256 393
Using a Pulsed Led to Characterize and Standardize a
Flow Cytometry Instrument
J. Wood and S.P. Perfetto. NIAID, NIH and Wake
Forest Sch. of Med., U.S.A.
B257 394
Flow Cytometry Study on the Relationship between
Cellular Uptake of Ag Nanoparticles and ROS
Production
J. Koh and T.H. Yoon. Hanyang Univ., South Korea
B258 395
Development of a Comprehensive Program for
Instrument Evaluation, Optimization, Comparison
and Monitoring in a Multi-Instrument Core Facility
C. Carswell Crumpton, D. Parks, M. Bigos and W.
Moore. Stanford Univ. Sch. of Med., U.S.A.
B272 409
Immunespace: Enabling Integrative Modeling of
Human Immunological Data
R. Sauteraud, L. Dashevskiy, G. Finak and R.
Gottardo. Fred Hutchinson Cancer Res. Ctr., U.S.A.
B276 413
CS&T and Fluorescence Control Beads for Flow
Cytometer Performance and Quantitation
M. Sharkey, E. Crowther and T. Chernenko. BD
Biosciences, U.S.A.
Index
85
Speaker/Author
B268 405
A Reliable Method for the Sorting and Identification
of LDHhhigh Cancer Stem Cells by Flow Cytometry
Z. Leng. North Sichuan Med. Col., China
Poster Session
Abstracts
B267 404
Cytometry - Metadata in XML and XHTML5
R. Leif and S. Leif. Newport Instruments, U.S.A.
Oral Session
Abstracts
B275 412
X-FISH: Analysis of Cellular RNA Expression Patterns
Using Flow Cytometry
A. Rieger, J. Havixbeck and D. Barreda. Univ. of
Alberta, Canada
Commercial
Tutorials &
Exhibits
TOOLS: CHEMICAL PROBES AND FLUORESCENT
PROTEINS
Scholars &
Emerging
Leaders
B274 411
Image Recognition Cell Dispensing System for Plating
of Single Cells and Monodisperse Spheroids
Y. Fujimura, T. Ino, T. Tanaka, J. Akagi, Y. Bansho, R.
Watanabe, M. Ishige and K. Takeda. On-chip
Biotechnologies Co. Ltd., Tokyo
Poster
Session
B273 410
Imaging Mass Cytometry — Quantitative
Multiparametric Imaging of Single Cells
O. Ornatsky, E. Swanson, Q. Chag, A. Bouzekri, T.
Closson, A. Loboda and V. Baranov. Fluidigm Canada
Inc., Markham, ON
Wednesday
15 June
TISSUE CYTOMETRY
B266 403
Achieving a Quantitative Optical Response in High
Resolution Surface Plasmon Resonance Imaging
M. Halter, A. Peterson, A.L. Plant and J. Elliott.
NIST,Gaithersburg, MD, U.S.A.
STEM CELLS
Tuesday
14 June
B263 400
Standardization Capabilities on the SA3800 Spectral
Analyzer
K. Futamura, K. Tahara, N. Nitta, T. Yamasaki, G.
Veltri, M. Dessing, M. Furuki and S. Catalano. Sony
Corp., Tokyo, Sony Biotechnol. Inc., San Jose, U.S.A.
and Sony Europe Ltd., Surrey, U.K.
B264 401
Characterizing Non-normal Distributions of
Transient Gene Expression in Mammalian Cells
P. McLean, C. Smolke and M. Salit. Natl. Inst. of Stds.
and Technol., Stanford and Stanford Univ., U.S.A.
B265 402
Benchmarking the Analytical Performance of a High
Content Imaging System
M. Halter, S. Titus and J. Elliott. NIST, Gaithersburg,
MD and NCATS, NIH, Rockville, U.S.A.
B271 408
Oscillations of Intracellular Calcium Concentration
and Neural Transcription Factor Expression Coding
Neural Differentiation Fate Determined by TimeLapse Imaging
H. Ulrich, R. Kageyama and T. Glaser. Univ. of Sao
Paulo, Brazil and Kyoto Univ., Japan
Monday
13 June
B262 399
Reagent-Driven Reconfiguration/Optimization of a
16-Parameter BD FACSARIA II SORP to Allow
Accurate Detection of Violet- and UV-Excited Sirigen
Dyes
A. Belkina and J. Snyder-Cappione. Boston Univ. Sch.
of Med., U.S.A.
Sunday
12 June
SYSTEMS BIOLOGY
Saturday
11 June
B261 398
Reference Cell for Enabling Standardization and
Quantification in Flow Cytometry
T. Chernenko, M. Sharkey and Y. Liu. BD
Biosciences, San Jose, U.S.A.
Special
Lectures
B269 406
The Use of Flow Cytometric Cell Sorting in the
Generation of Genomically Edited Human
Pluripotent Stem Cells
K. Haskell, K. Adusumilli, D.F. Gebhard, J. Stock and
S. Engle. Pfizer, Groton, CT and Vertex, San Diego,
U.S.A.
B270 407
Novel Flow Cytometric Approach for the Detection
of Adipocyte Sub-Populations during Adipogenesis
C. Durandt, F.A. van Vollenstee, C. Dessels, K.
Kallmeyer, D. de Villiers, C. Murdoch, M. Potgieter
and M.S. Pepper. Fac. of Hlth. Sci., Univ. of Pretoria,
South Africa
Congress
Overview
B259 396
Reference Measurement Procedures for
Concentration of Stained Cells
M. Hussels, M. Kammel, C. Divieto, J. Campbell, A.
Kummrow, A. Devonshire, J. Davies, T. Lekishvili, L.
Mortati, J. Huggett, M. Sassi, H. Parkes and J.
Neukammer. Phys.-Tech. Bundesanstalt (PTB Berlin),
INRiM., Torino and LGC, Teddington, U.K.
B260 397
Impact of UV Laser Power Output on Signal Intensity
and Quality of UV Dyes
M. Eich, A. Trumpp and S. Schmitt. Heidelberg Inst.
for Stem Cell Technol. and Exptl. Med. and DKFZ,
Heidelberg, Germany
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Index
Abstracts
TOXICOLOGY
B277 414
Effects of Iron Oxide Nanoparticles on Splenic
Lymphocytes of Male Wistar Rat
U. Singh Gaharwar and R. Paulrag. Sch. of Envrn.
Sci., Jawaharlal Nehru Univ., India
VACCINES
B278 415
Development and Statistical Modeling of an
Intracellular Cytokine Staining Baseline-Adjusted
Vaccine-Specific Response Criteria for Tuberculosis
G. Finak, K. Rutkowski, J. Fulp, D. Hokey, A.
Ginsberg, T. Evans and R. Gottardo. Fred Hutchinson
Cancer Res. Ctr., Seattle and Aeras, Rockville, MD,
U.S.A.
LATE-BREAKING ABSTRACTS
B279 416
Optimizing Proteomic Analysis of Small Numbers of
Sorted Cells
M. Cochran, W. Wojciechowski, K. Welle, S.
Ghaemmaghami and T. Bushnell. Univ of Rochester,
U.S.A.
B280 417
Search for the Optimal Dye for EV Staining
J. Lannigan, A. Görgens, A. Klibanov, C. Rudy, T. Lee,
B. Giebel and U. Erdbrügger. Univ. of Virginia, U.S.A.
and Univ. Hosp. Essen, Univ. of Duisburg-Essen,
Germany
B281 418
Characterization of Microparticles Generated after
Traumatic Brain Injury in Mice
Z. Zhao, Y. Tian, T. Hilton, X. Wu and J-F. Dong.
Bloodworks NW Res. Inst., U.S.A. and Tianjin Med.
Univ., China
B282 419
Enhanced Small Particle Detection on the BD
Facscelesta Flow Cytometry System
L. Yu, T. Van Den Broeck, P. Mognol and G. Reinin.
BD Biosciences, U.S.A. and BD Life Sciences,
Belgium
B283 420
The Measurement of Basophil Activation Using a
Flow Cytometry Kit for Application in Clinical Trials
S. Johnson, L. Brown and J. J. Stewart. Flow Contract
Site Lab. LLC, U.S.A.
B284 421
The Advantages of Fluorescent Barcoding of Human
Cells Using Flow Cytometry for High Throughput
Testing with an Application for Human Clinical Trials
R. Pal, L. Brown and J. J. Stewart. Flow Contract Site
Lab. LLC, U.S.A.
86
B285 422
The Activation Profiling of STAT in Human Whole
Blood Samples by Flow Cytometry with an
Application for Human Clinical Trials
S. Suhane, L. Brown and J. J. Stewart. Flow Contract
Site Lab. LLC, U.S.A.
B286 423
Withdrawn
B287 424
Noise Reduction for LSRII, Fortessa, and x20
Instruments
R. Lannigan, L. Nichols and E. Chase. Cytek
Biosciences Inc., U.S.A.
B288 425
Use of IR Laser to Excite and Avalanche Photodiode
to Detect IR and Near IR Dyes on the Cytoflex Flow
Cytometer
J. Tung, J. Xu, Y. Irwan, S. Harlacz, E. Anderson and
D. Fenoglio. Beckman Coulter Inc., U.S.A. and China
B289 426
WOLF Cell Sorter: A Microfluidic Fluorescence
Activated Cell Sorter
G. Narez, C. Ardila, Z. Mei, S. Cho, W. Alaynick and
J. M. Morachis. NanoCellect Biomed. Inc., U.S.A.
B290 427
Optical Phase Detection: A New Technology for
Nanoparticle Measurement
M. Yamamoto and J. P. Robinson. Miftek Corp. and
Purdue Univ., U.S.A.
B291 428
An Expanded Multiplexing Flow Cytometry Platform
G. Vacca. Kinetic River Corp., U.S.A.
B292 429
Using Violet Side Scatter to Detect and Resolve
Nanoparticles on the Cytoflex Flow Cytometer
G. Brittain, S. Gulnik and Y. Chen. Beckman Coulter
Inc., U.S.A.
B293 430
No-Lyse No-Wash New Strategy for CD34+ for
Absolute Cell Counting without Beads
L. Rico, J.A. Bradford, M. Ward and J. Petriz. Josep
Carreras Leukemia Res. Inst., Spain and Thermo Fisher
Scientific, U.S.A.
B294 431
Automated 3D Tissue Image Segmentation and
Quantification of Cellular Expression for Precision
Histo-cytometry
N. Samusik, Y. Goltsev and G. P. Nolan. Stanford
Univ., U.S.A.
B295 432
Cell-Mediated Cytotoxicity in “Untouched” Whole
Blood
L. Rico, M. D. García-Godoy, M. Ward and J. Petriz.
Josep Carreras Leukemia Res. Inst., Spain and Thermo
Fisher Scientific, U.S.A.
Monday
13 June
Tuesday
14 June
B308 445
Forster Resonance Energy Transfer Based Substrate
Peptide Inserted Bioprobes
K. Nichani, J. Li, M. Suzuki and J. Houston. New
Mexico State Univ., U.S.A. and Saitama Univ., Japan
Wednesday
15 June
B300 437
Allergic Response Predicted Using PBMC Subsets
with Mass Cytometry
W. Kennedy, J. P. Oliveria, M. Tran, M. Cook, A.
Johnson, M. Hughes, G. Gauvreau, K. McNagny and
F. Rossi. McMaster Univ. and Univ. of British
Columbia, Canada
Sunday
12 June
B299 436
Demonstration of Infrared Laser Applications Using
Beckman Coulter CytoFLEX S Flow Cytometer
Y. Irwan, E. Anderson, D. Fenoglio and J. Tung.
Beckman Coulter Inc., U.S.A.
Saturday
11 June
B298 435
The Immunophenotype Characteristics of 37 Cases of
CD45-Positive Mutiple Myeloma
B. Yue, S. Liu, Z. Li, F. Zhang and N. Wang. The First
Affiliated Hosp. of Zhengzhou Univ., China
B305 442
Engineered Mannose-Binding Lectin Variant (FcMBL)
Decreases Phagocytosis in THP-1-Derived
Macrophages
G. Cuneo, A. Chen, A. Waterhouse, M. Rodas, A.
Watters, M. Super and D. Ingber. Wyss Inst. for
Biologically Inspired Engin., Boston, U.S.A.
B306 443
Analysis the Immunophenotype of Acute Monocytic
Leukemia with HLA-DR Negative or Low Expression
B. Yue, Z. Li, F. Zhang, N. Wang and S. Liu. The First
Affil. Hosp. of Zhengzhou Univ., China
B307 444
A Method for In Situ Multiplexed Targeted Protein
Profiling of Circulating Tumor Cells
E. Gerdtsson, M. Pore, H. Sebastian, S. Wix, A.
Carew, S. Loboda, O. Ornatsky, J. Hicks and P. Kuhn.
Fluidigm Inc., Canada and USC Dornsife Col. of
Letters, Arts and Scis., U.S.A.
Special
Lectures
B297 434
Optimization of Imaging Flow Cytometry for In Vitro
Cytotoxicity Testing of Drugs against Plasmodium
falciparum
S. M. Geraldino and J. E. Lazaro. Univ. of the
Philippines Diliman
B304 441
Controlling Nanoparticle Cellular Uptake Kinetics by
Designing Their Shape Using DNA Origami
G. Cuneo, M. Bastings, N. Ponnuswamy and W. M.
Shih. Wyss Inst. for Biologically Inspired Engin.,
Boston and Dana Farber Cancer Inst., U.S.A.
Congress
Overview
B296 433
B-Cell Antigen Receptor Expression Governs
Signaling Potential and Protection from Apoptosis
K. Huse, C. Wogsland, H. Polikowsky, K. Diggins, J.
Wise, E. B. Smeland, J. Irish and J. Myklebust. Oslo
Univ. Hosp., Univ. of Oslo, Norway and Vanderbilt
Univ. Med. Ctr., U.S.A.
Oral Session
Abstracts
B311 447
Veri-Cells™ Leukocytes and PBMC (CD4 low),
Lyophilized Human Blood Cells Are Reliable Controls
for Flow Cytometric Assays
M. Rogers, M. Gordon, A. Divekar, X. Yang, D.
Williams, and C. Monell. BioLegend, U.S.A.
Commercial
Tutorials &
Exhibits
B303 440
Subcellular Localization by Conventional Flow
Cytometry
G. Brittain and S. Gulnik. Beckman Coulter Inc.,
U.S.A.
Scholars &
Emerging
Leaders
B309 22
Changes in Monocyte Intracellular mRNA and
Protein: Implications for the Development of
Atherosclerosis?
B. McFarlin, A. Henning and A. Venable. Univ. of
North Texas, U.S.A.
B310 446
High-Throughput Detection and Quantification of
Mitochondrial Fusion through Imaging Flow
Cytometry
A. Nascimento, J. Lannigan, and D. Kashatus. Univ. of
Virginia Sch. of Med., U.S.A.
Poster
Session
B301 438
Highly Multiplexed Imaging Cytometry by In Situ
Indexing
Y. Goltsev, N. Samusik, J. Kennedy-Darling, G.
Vasquez and G. Nolan. Stanford Univ., U.S.A.
B302 439
Correlating Internalization and Potency to
Accelerate Antibody Development and Discovery
J. Bradford, C. Langsdorf, B. Agnew, D. Beacham, J.
Berlier, K. Chambers, A. Dix, N. Dolman, K. Gee, M.
Janes, L. Smolenska and W. Zhou Thermo Fisher
Scientific, Eugene, OR and Pittsburgh, U.S.A.
Poster Session
Abstracts
Index
87
Speaker/Author
Congress
Overview
Sunday
12 June
Saturday
11 June
Special
Lectures
Sunday, June 12
Exploring Resolution Limits with
Cytek System Upgrades and QbSure
1245 – 1345 – Tahoma 5, TCC Level 3
Cytek Biosciences
46107 Landing Parkway
Fremont, CA 94538
Phone: 510-657-0102
Email: [email protected]
Web: www.cytekbio.com
Index
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Scholars &
Emerging
Leaders
Poster
Session
Wednesday
15 June
Tuesday
14 June
Monday
13 June
Presenter: Lisa Nichols, PhD
Flow cytometry users today benefit from being able to
choose from a variety of dyes that allow higher
parameter assays. Cytek Biosciences offers cytometer
upgrades that allow customers to leverage new
reagents for their experimental needs. In this tutorial,
we will present data from Cytek upgrades using both
the FACSCalibur (up to 14 colors) and Diva-based
instruments. Using our QbSure cytometer
performance validation software, we will discuss the
relationship between resolution limits and instrument
performance, with a focus on discrimination of dimly
fluorescent sample populations. Comparative analyses
will include sample data from UV laser upgrades, as
well as fluidic upgrades designed to increase system
stability and decrease noise. With a full suite of
options, Cytek upgrades can be a cost-effective way to
take advantage of the current reagent offerings, along
with improving cytometer capability and obtaining
high quality data.
Development of a PrimeFlow Assay
for Investigations into HIV Biology
major knowledge gaps remain. One such gap is the
complete characterisation of the specific CD4 T cell
subsets supporting HIV replication in vivo. To address
this question, we adapted the PrimeFlow assay for the
simultaneous detection of HIV mRNAs and proteins,
along with phenotypic surface and intracellular
markers expressed on CD4 T cells. The assay was
highly sensitive, allowing the detection of one HIVinfected cell per million CD4 T cells. This enabled the
characterisation of rare HIV-infected cells, isolated
directly ex vivo from HIV-infected patient samples.
HIV infection can be controlled by antiretroviral
therapy, however this does not represent a cure. The
virus persists in patients on therapy by “hiding” in a
silent form integrated into the patient’s DNA and can
rebound from this latent reservoir if therapy is
discontinued. We utilised the PrimeFlow assay to
quantify the size of the latent reservoir in the blood of
patients on treatment, as compared to other
established protocols. We will demonstrate the use of
this assay to monitor the efficacy, and understand the
mechanisms, of cure strategies.
From CAR-Ts to Checkpoint Inhibitors,
A Powerful NEW Platform for
Immuno-oncology…Introducing the
iQue Screener PLUS
1245 – 1345 – Skagit 4, TCC Lower Level
IntelliCyt
9620 San Mateo Boulevard, NE
Albuquerque, NM 87113
Phone: 669-300-6306
Web: www.intellicyt.com
Presenter: Joe Zock
eBioscience, an Affymetrix Business Unit
10255 Science Center Drive
San Diego, CA 92121
Phone: 858-642-2058
Fax: 858-642-2046
Web: www.eBioscience.com
Comparative Analysis of Cytometric
Technologies
Presenters: Amy E. Baxter and Daniel E. Kaufmann
Despite over 30 years of research, HIV/AIDS
represents a major global health problem, with over
40 million people infected worldwide. Technological
limitations have hampered research efforts – in part
because infected cells are very rare in vivo - and
88
Sony Biotechnology
1730 North 1st Street
San Jose, CA 95112
Phone: 408-352-4256
Web: www.sonybiotechnology.com
Presenter: Greg Veltri, PhD
Wednesday
15 June
Monday, June 13
Novel Laser Sources on Small Cytometer
Platforms
Presenter: Bob Balderas
Index
In this workshop, attendees will learn the cellular
BD Biosciences
2350 Qume Drive
San Jose, CA 95131
Phone: 408-421-9886
Web: www.bdbiosciences.com
Since the sequencing of the human genome,
biomedical research has made advancements in the
89
Speaker/Author
Presenter: William G. Telford, PhD
Poster Session
Abstracts
Presenter: Steve Offer
Optimizing Strategies for Reliable
Multicolor Flow Data: Moving to 50Color Flow Cytometry
Oral Session
Abstracts
Use of Real Time Cellular Analysis and
Flow Cytometry to Characterize Potential
Biomarkers of Aggressive Colorectal
Cancer
Commercial
Tutorials &
Exhibits
ACEA Biosciences Inc.
6779 Mesa Ridge Road, #100
San Diego, CA 92121
Phone: 858-724-0928
Web: www.aceabio.com
Scholars &
Emerging
Leaders
metastasis models developed to better understand the
molecular changes during colorectal cancer
metastasis; how the throughput and sensitivity of the
xCELLigence real-time cell analyzer and the
NovoCyte flow cytometer facilitated the testing of
specific parameters of candidate metastasis-associated
signatures. Also, we will describe the applications and
benefits of novel laser sources used in benchtop flow
cytometers.
Poster
Session
Novel Studies on Innovative
Technology Platforms
Tuesday
14 June
ChipCytometry is a high-content cytometry platform
combining the unsurpassed quantitative phenotyping
Monday
13 June
Presenter: Christian Hennig
Sunday
12 June
Zellkraftwerk GmbH
Bosestrasse 4
04109, Leipzig
Germany
Phone: 49 151 5238 5628
Web: www.zellkraftwerk.com
Saturday
11 June
Special
Lectures
ChipCytometry: A New 85-Plex
Immune Monitoring Panel, Exciting
Applications & An Update on the
Early Access Program
ability of flow cytometry with the unparalleled
information depth of microscopy. Besides some
features that are quite similar to conventional flow
cytometry, three technology features make
ChipCytometry an exciting technology for explorative
high-content analysis: 1. Long-term sample storage/
no sample consumption: ChipCytometry uses
microfluidic chips enabling biomarker-preserving
long-term storage of samples for a period of at least 24
months. Cell storage is particularly useful for precious
samples like patient samples, rare cells and
(multicenter) clinical trials because cells are not
destroyed during transport, storage and analysis and
can be stored for further tests exploring more and
more markers on these pre-analyzed samples. 2.
High-content cytometry: ChipCytometry enables
highly multiplexed biomarker assays. Zellkraftwerk
presents a 85-plex immune monitoring assay for
comprehensive human immune cell phenotyping, and
a 39-plex murine immune monitoring panel. 3. Tissue
Cytometry: ChipCytometry can work with as sorts of
cell specimens as well as with a broad range of tissue
types. The tutorial will give an overview on new
ChipCytometry applications and present case studies
from the Early Access Program. Innovators will be
invited to participate in the broadened Early Access
Program 2016 Join us for an introduction to the future
of “precious samples cytometry”. Lunch bags will be
served on a first come first serve basis.
Congress
Overview
Sony will present cases that describe how experiments
run on both Spectral Technology and conventional
flow. These case studies will clarify conditions when
data quality is advanced both in depth of information
as well as eliminating bias data when analyzing cell
populations and single cells. In addition, we will also
discuss how innovations in automation and software
improve visualization of single cell heterogeneity with
each multicolor analysis and sort while eliminating
manual steps – simplifying flow and making it more
approachable to a wider audience of researchers.
Poster
Session
Wednesday
15 June
Tuesday
14 June
Monday
13 June
Sunday
12 June
Saturday
11 June
Special
Lectures
Congress
Overview
measurement
of underlying molecular and
physiological mechanisms of complex biological
systems
and networks. Tools for molecular and cell
analysis have continued to evolve to
address new challenges and opportunities in many
different biological fields. Today, the BD
FACSymphony™ flow cytometer is capable of
analyzing up to 50 parameters, opening many
applications in genomics, proteomics, systems
immunology, and biology. The research opportunities
created will impact decisions in clinical diagnosis and
promote a deeper understanding of cancer biology,
vaccine development, and drug discovery.
As a result of the availability of new BD Horizon
Brilliant™ polymer fluorochromes, a study of receptor
density and expression, and the launch of the BD
FACSymphony high parameter cell analyzer, this year
we demonstrated practical 27-color flow cytometry.
In this presentation, we will discuss a systematic
strategy for successful panel design for high parameter
multicolor assays.
New Strategies to Monitor HIV
Infection and Therapy through a
Combined Cell Sorting and Droplet
Digital PCR Workflow
1245 – 1345 – Yakima 1, TCC Level 1
Bio-Rad Laboratories
2000 Alfred Nobel Drive
Hercules, CA 94547
Phone: 1-800-4BIORAD
Web: www.bio-rad.com
Index
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Scholars &
Emerging
Leaders
Presenter: Andrea Cossarizza, MD, PhD
Potent treatments for HIV infection can halt disease
progression by blocking production of the virus and
by inhibiting its integration into the DNA of host cells.
Response to therapy is typically monitored by
counting peripheral blood CD4+ T cells and by
measuring plasma viral load, which in most patients
becomes undetectable. However, several problems
remain, the first of which is how to monitor residual
viral activity and HIV reservoirs in the presence of
undetectable viremia. One approach is to measure
HIV DNA in peripheral blood mononuclear cells.
However, HIV infects to varying degrees different cell
populations such as naï ve and memory CD4+ cells or
monocytes. In addition, robust data are missing on the
relationship between the amount of virus present in
such cells and the actual progression of the infection,
including loss of the T cell pool. Thus, by linking flow
cytometry and cell sorting with a molecular biology
approach based on droplet digital PCR (ddPCR), we
can now combine information on the amount of virus
present in different cell populations (by quantifying
HIV DNA) and on the regenerative capability of the
90
immune system (by quantifying TREC), to provide
crucial guidance on optimal monitoring of HIV+
patients.
Single Cell Obsession: Workflow,
Discovery and Data Integration
FlowJo, LLC
385 Williamson Way
Ashland, OR 97520
Phone: 541-201-0022
Web: www.flowjo.com
Presenter: Mike Stadnisky, PhD
The single cell is the basic unit of disease and the
singular obsession we all share. But how do we start
using emerging technologies to gain deeper insight
into the 100 trillion cells of the human body, each
differentiated from a single cell, which in turn has
10,000 transcripts and 2-4 million proteins per cubic
micron?
In this tutorial, we will:
1) Share our collaborative work with multiple
research and bioinformatics groups which empower
anyone using FlowJo with the cool tools we have seen
at CYTO or read about in the literature.
2) Discuss a brand new workflow-driven data
management and integration ecosystem, which we
will then use to ...
3) Analyze a high-content flow cytometry data set
using spectral compensation, data reduction, and
ontology tools to name newly discovered populations.
4) Show how single cell phenotyping and gene
expression data can be integrated to map cellular
phenotypes with 40+ proteins and 10,000 transcripts.
From the Horses’ Mouth: Results and
Publications Using Amnis® Imaging
Flow Cytometry
MilliporeSigma
28820 Single Oak
Temecula, CA 92590
Phone: 951-514-4373
Web: www.emdmillipore.com
Presenters: Kathleen McGrath, PhD and Ziv Porat,
PhD
Dr. McGrath’s pioneering research focuses on how
the hematopoietic system is assembled, both during
embryonic development and when necessary for
repair in the adult. She describes overlapping waves
of stem cell emergence, each with different lineage
Special
Lectures
Miltenyi Biotec GmbH
Friedrich-Ebert-Strasse 68
NRW 51429
Germany
Phone: 49 2204 8306 3190
Web: www.miltenyibiotec.de
Saturday
11 June
Presenters: Stephen Elliman, PhD and Lisa O´Flynn,
PhD
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
A Comparative Study of the Novocyte
Flow Cytometer to Assess Biomarkers in
Rheumatoid Arthritis Research
Oral Session
Abstracts
Presenter: Andy Filby, PhD
Multicolor Analysis and Volumetric Cell
Counting of Clinical Samples for
Hematological and Immunological
Studies and Diagnosis on NovoCyte
System
Poster Session
Abstracts
Presenter: Wanmao Ni, MD
Index
91
Speaker/Author
ACEA Biosciences Inc.
6779 Mesa Ridge Road, #100
San Diego, CA 92121
Phone: 858-724-0928
Web: www.aceabio.com
Poster
Session
Wednesday
15 June
Practical Disease Studies Using High
Performance Benchtop Flow
Cytometer
Tuesday
14 June
Tuesday, June 14
Monday
13 June
We described a rare MSC type in human bone
marrow (CD271+CD362+ (ORBCEL-M™)), including
explorations of the therapeutic potential of these
MSCs in pre-clinical models of inflammatory disease.
The clonogenic potential (colony-forming unitfibroblasts, CFU-F) is commonly employed to assess
the sort performance and health of MSCs after
processing. Further, CFU-F is an important indication
of the potential therapeutic benefit and cost of
production of these cells for therapeutic applications
after expansion. Enriching stromal cells by plastic
adherence typically results in a CFU-F ratio of 1 MSC
in 30,000 cells, while four-color droplet-sorting of
CD271+CD362+ isolates stromal cells to a frequency
of 1 MSC in every 3-4 cells sorted. However, we
compared CFU-F frequencies of droplet-sorted cells
with the frequencies of cells purified on the
MACSQuant® TytoTM, using 2 colors for both systems,
and found substantially higher frequencies with the
MACSQuant Tyto. Aspects important for GMP
manufacturing were also substantially improved. We
now proceed with MACSQuant Tyto-processed cells
in early human clinical trials for studies of safety and
indications of efficacy in the repair of non-healing
ulceration secondary to chronic diabetes.
Sunday
12 June
Dr. Porat has been shepherding imaging flow
cytometry experiments at the Weizmann Institute for
the last five years, where ImageStream® data has
been generated for inclusion in more than 40
publications. Applications have included nuclear
localization and signaling kinetics, quantification of
mRNA granules in relation to life span in yeast,
measurement of clinically significant CD19-related
cell aggregation in blood cancers, identification and
characterization of rare hematopoietic stem cell
populations and actin rearrangements, bacterial
length measurement, ROS and lipid quantification in
ocean algae, and localization of antiviral peptides to
the fusion interphase, among others. In this workshop,
Dr. Porat will focus on two recent projects: one
studying viral factories during the infection cycle of a
nucleocytoplasmic large DNA virus (NCLDV) in
Acanthamoeba species, and a second elucidating the
mitotic reorganization and fragmentation of the Golgi
and the shuttling of ERK1c integrating Golgiregulating processes into a single coherent pathway.
Novel Aspects of Sorting in a Closed
System: The MACSQuant® Tyto™
Congress
Overview
and self-renewal potentials using imaging flow
cytometry. Using imaging flow cytometry, a unique
hematopoietic progenitor with potential for short term
production of erythroid and myeloid cells was
identified in embryonic stem cells and found to
function when transfused in adults. Dr. McGrath also
uses Amnis® applications to study the recovery of
hematopoietic stem cells and megakaryocytes
following radiation, addressing mitigation of short
term thrombocytopenia as well as long term stem cell
dysfunction.
Sunday
12 June
Saturday
11 June
Special
Lectures
Congress
Overview
In
this workshop attendees will learn about the critical
benefit of high performance multi-parameter, cell
counting
capabilities of a benchtop flow cytometer for
applications in disease studies such as rheumatoid
arthritis, and other hematological and immunological
diseases.
Novel Approaches to Simplifying
Sorting
BD Biosciences
2350 Qume Drive
San Jose, CA 95131
Phone: 408-421-9886
Index
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Scholars &
Emerging
Leaders
Poster
Session
Wednesday
15 June
Tuesday
14 June
Monday
13 June
Presenter: Joe Trotter
While flow cytometry core managers have certified an
impressively large number of researchers to operate
an analyzer independently, the number of people they
would trust to set up and run their cell sorter typically
lags far behind—often placing significant demand on
core lab staff. This tutorial will focus on new
hardware and software automation that simplifies and
streamlines the use of high- performance BD cell
sorter technology. By transferring the responsibility for
instrument setup and monitoring to the system, a user
with previous experience using a benchtop analyzer
can, with minimal training, become proficient at cell
sorting as well. In depth explanations of these behind
the scenes methods will be presented for flow
cytometry enthusiasts to understand and appreciate.
High Dimensional Cytometry in Small
Packages: Specialized Laser Sources in
Small-scale Cytometers & A Web-based
Software Concept to Support High
Content Cytometry Antibody Panel
Design
Beckman Coulter /LIH6FLHQFHV
/DNHYLHZ3DUNZD\6RXWKH'ULYH
,QGLDQDSROLV,1
3KRQH
:HEZZZEHFNPDQFRP
Presenters: William Telford, PhD and Michael
Kapinsky, PhD
footprint cytometers like the CytoFLEX. These
instruments can also be equipped with lasers beyond
the traditional blue/red/violet. In part one, Dr. Telford
will present data from CytoFLEX S cytometer
equipped with yellow 561 nm and near-UV 375 nm
laser sources, allowing analysis of red fluorescent
proteins, Hoechst side population, and the latest UVexcited phenotyping fluorochromes. More channels,
more lasers, and more fluorochromes mean more
possibilities, but also imposes challenges in multicolor
panel design. In part two, Dr. Kapinsky will discuss
an expert software system that enables lessexperienced cytometry users to design high content
panels based on embedded instrument configurations,
fluorochrome properties, and cell biology. Learn how
sensitivity in high content cytometry is related to
panel design, instrument characteristics, and antigen
co-expression patterns. See a way to translate the
seemingly complex and fuzzy problem of high
content panel design into a systematic, easy-to-use
software approach.
Mass Cytometry: Powering
Breakthroughs in Health and Disease
Fluidigm Corporation
7000 Shoreline Ct.
South San Francisco, CA 94080
Phone: 650-266-6033
Web: www.fluidigm.com
Defining the Phenotypic and functional
Immune Microenvironment in AML
Presenter: Evan Lind, PhD
Towards an Assay of Global Immune
Competence
Presenter: Holden Terry Maecker, PhD
Mass cytometry is a powerful research tool for
elucidating the mechanisms underlying human
disease and assessing treatment efficacy. Enabling
high-parameter studies with simple panel design,
mass cytometry has quickly become a mainstay at
multiple immune monitoring core facilities and
translational research laboratories around the world.
In this tutorial, two prominent researchers will share
how they successfully implemented mass cytometry
as a routine analysis tool in different settings: an
immune monitoring core facility involved in
multicenter studies and a research lab performing an
immune checkpoint inhibitor study with acute
myeloid leukemia (AML) patient samples.
Advanced optical, fluidic, and electronic technologies
have greatly reduced the size of flow cytometers with
almost no loss of functionality. Ten to fourteen color
high-dimensional analysis is now possible on small
92
Presenters: Brian McFarlin, PhD and Katherine Gillis
Introduction: In MS patients, no data exist on changes
in metabolic profiles of T cell subsets after activation
or differentiation, nor on their mitochondrial (mt)
functionality.
Scholars &
Emerging
Leaders
Methods: We studied 23 patients with different MS
forms: 2 inactive relapsing-remitting (RR), 15
secondary progressive (SP), 6 primary progressive
(PP), and 14 healthy controls (CTR). PBMC were
stained with a large panel of mAb; purified CD4 and
CD8 T cells were in vitro stimulated with antiCD3/CD28. Mt mass, mt O2- level, mt membrane
potential (MMP), GLUT-1 receptor and mTORdependent S6 ribosomal protein phosphorylation
were analyzed by an Attune NxT.
Commercial
Tutorials &
Exhibits
Index
Speaker/Author
93
Poster Session
Abstracts
Results: PP patients had high % of CD4+ TEMRA cells
and low % of CD8+ Tnaï ve cells vs. CTR. SP patients
displayed high % of CD8+ TCM cells. SP and PP
patients had more activated CD4+ and CD8+ T cells.
Patients with different forms of MS also showed
changes in S6 phosphorylation, GLUT-1 receptor
expression, mt mass, O2- production and MMP. Such
changes were more marked in patients with
progressive MS forms.
Oral Session
Abstracts
Presenters: Sara De Biasi, Andrea Cossarizza,
Marcello Pinti
Poster
Session
To test how strenuous exercise affects senescence
phenotypes in monocytes, blood samples were
Thermo Fisher Scientific
5791 Van Allen Way
Carlsbad, CA 92008
Phone: 800-955-6288
Web: www.thermofisher.com
Wednesday
15 June
Effect of Strenuous Aerobic Exercise on
Monocyte Telomere Length
1245 – 1345 – Yakima 1, TCC Level 1
Tuesday
14 June
PBMC isolated from blood samples from subjects who
have recently exercised were used as a source of
monocytes. Following treatment with staurosporine to
induce apoptosis, monocytes were phenotyped to
identify classic (CD14+/CD16(-)) and proinflammatory
(CD14+/CD16+) monocytes. For purposes of
comparing resistance to proapoptotic stimuli,
multichannel flow cytometry is simultaneously
employed to assess the degree of viability, early
phase, and midphase apoptosis using 7-AAD,
Annexin V, and a multicaspase reagent, respectively.
The findings of this work have implications for the
role of monocytes in muscle recovery, particularly
when exposed to damage-associated molecular
patterns (DAMP) that may induce apoptosis.
Monday
13 June
Effect of Muscle Damage/Oxidative Stress
on Monocyte Apoptosis
High Level of Activation and Altered
Mitochondrial Functionality in T Cells
from Patients with Progressive Forms
of Multiple Sclerosis
Sunday
12 June
Background
Within the last 15 years, flow cytometry technology
has advanced such that compact, high throughput,
multi-parameter flow cytometry is possible. Access to
such powerful instrumentation at the benchtop has
many unique benefits ranging from ease of
experimental design to contemporaneous data
analysis. The ability to quickly obtain biologically
relevant information and then adapt experiments ‘on
the fly’ is one of many advantages to cytometry
designed for the benchtop. The McFarlin laboratory is
a long-term user of MilliporeSigma’s Guava®
easyCyte platform, which uses microfluidics to
significantly reduce instrument footprint, buffer
consumption, and generation of biohazardous waste.
Over the past year their applied physiology lab has
worked with an easyCyte™ 12HT and has leveraged
its capabilities to complete novel research. In this
workshop, Dr. McFarlin will present work on
apoptosis and sensescence, two common measures of
cellular health.
Saturday
11 June
MilliporeSigma
28820 Single Oak
Temecula, CA 92590
Phone: 951-514-4373
Special
Lectures
collected from study subjects following 60 minutes of
cycling, and compared with pre-exercise samples.
Monocytes in PBMC isolated from blood samples
were again treated or not to induce apoptosis, and
CD14/CD16 staining used to resolve classic and
proinflammatory phenotypes. For these studies,
multiparameter flow cytometry was used to detect
signal from the senescence marker KLRG1 and from
fluorescent RNA probes for detection of telomere
length. Results of these investigations may be
significant to our understanding of how physical stress
influences monocyte functional capacity.
In summary, benchtop, multiparameter flow
cytometry has allowed the McFarlin laboratory to
explore unique, novel research questions that aim to
better elucidate the role of monocytes in systemic and
skeletal muscle health.
Congress
Overview
Exercising Benchtop Multiparameter
Flow Cytometry to Answer Unique
Cellular Research Questions
Conclusion:
Our data support the hypothesis that T
cells from MS patients with PP form tend to shift more
rapidly
and easily towards effector cells, and mediate
a rapid progression of the disease.
Monday
13 June
Sunday
12 June
Saturday
11 June
Special
Lectures
Congress
Overview
Wednesday, June 15
Exploiting the CytoFLEX Flow
Cytometer for Biological Applications
Beckman Coulter Life Sciences
5350 Lakeview Parkway Southe Drive
Indianapolis, IN 46268
Phone: 800-742-2345
Web: www.beckman.com
CytoFLEX has avalanche photodiodes (APD) in place
of photomultiplier (PMT) for the detection of photons.
Using biologically relevant samples (bacteria, cells
and protists) we investigated the impact of the APDs
and the relevance of the advantages gained. Using
calibration beads we compared PMT-type cytometers
to the CytoFLEX which clearly demonstrated that the
Index
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Scholars &
Emerging
Leaders
Poster
Session
Wednesday
15 June
Tuesday
14 June
Presenter: Karen Hogg, PhD
CytoFLEX was superior in its ability to resolve the 8- peak beads, especially in the red channels. Samples
containing cells which are small have peak and area
signals that correlate closely; for larger cells this
assumption is no longer valid. This was demonstrated
when investigating the host symbiont interaction
between Paramecium and Chlorella. Log peak data
provided qualitative data; however using CytoFLEX
log area we obtained quantitative data with enhanced
sensitivity and a 2-fold increase of staining index (Ex
640 nm Em 660 nm). CytoFLEX data from a diverse
range of biological applications including cell
proliferation, cell cycle and ploidy studies will be
shown. These will demonstrate a lower variance of
fluorescence and reduced fluorescence data spread.
And the positive impact of this with multicolour
panels with dim populations will be shown.
94
Congress
Overview
([KLELWRU6KRZFDVHV
Monday, June 13
Special
Lectures
Abcam
1850 – 1905, Exhibit Hall 4EF, WSCC
Level 4
De Novo Software
Take the Arghhh out of R with FCS
Express Version 6
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
FCS Express 6 takes the frustration out of high
dimensional flow analysis with many clustering
algorithms built right in, and new, direct integration
with R, for running any additional algorithms you like.
Integrated spreadsheets and regression analyses that
update in real time combine with our legendary ease
of use and high speed to take your flow analysis into
the next dimension(s). Take advantage of the latest
tools you have read about without having to be a PhD
in Bioinformatics! And first the first time ever, choose
your own operating system: FCS Express 6 runs
natively on the Mac as well as Windows, and both are
built with the same high standards, quality, and full
set of features.
Poster
Session
Wednesday
15 June
Assays implemented on Accellix platform
include: Sepsis diagnosis: CD64 Index, T cell
subsets including viability, Population
analysis of cells: T cells, B cells, NK cells &
monocytes and Sepsis-induced
immunosuppression: HLA-DR expression.
Tuesday
14 June
The Accellix multicolor (5 color) flow
cytometer enables complete automation in 3
steps: biochemical sample preparation in a
dedicated disposable cartridge, flow
cytometer reading & analytical data
processing utilizing a proprietary algorithm &
in-process software quality controls.
Interest in circulating miRNA biomarkers in plasma,
serum & exosomes continues to accelerate.
Conventional miRNA profiling assays cannot be used
with flow cytometers and require tedious RNA
purification using at least 100μl of precious clinical
sample per purification. They also cannot be used
with the many biobank samples that contain heparin.
Find out about our multiplex miRNA profiling assays
for flow cytometers which enable researchers to
profile miRNAs directly from just 20 ʅl of crude
biofluid. These assays use our Firefly® particle
technology which allows miRNA and cytokine
multiplexing on one technology platform.
Monday
13 June
LeukoDx plans to launch its first product for
the rapid diagnosis and monitoring of sepsis,
in the EU during 2016, utilizing its clinically
validated exclusive licensed CD64 Sepsis
Biomarker.
Use Crude Biofluids for miRNA and
Cytokine Multiplexing on your Flow
Cytometer with Firefly® Particles
Sunday
12 June
LeukoDx’s CE Marked Accellix™ platform is designed
to provide 24/7 flow cytometric capability at
moderate complexity level labs, the majority of
hospital labs, & ultimately at a CLIA waived level.
Accellix provides the answers physicians need with a
compact, tabletop device operated on location. It’s
simple and automated operation requires just a drop
of blood in a standardized, disposable cartridge to
return results in 20 minutes.
Saturday
11 June
LeukoDx/Accellix
Poster Session
Abstracts
Index
95
Speaker/Author
Congress
Overview
Tuesday, June 14
Special
Lectures
Melles Griot
1930 – 1945 , Exhibit Hall 4EF, WSCC Level 4
Intelligent Solutions for Life
IDEX Health & Science is the global authority in
fluidics and optics, bringing life to advanced
optofluidic technologies with our products, people
and engineering expertise. With deep applications
knowledge in cytometry applications, our portfolio is
unrivaled in breadth, quality, and performance. We
are respected worldwide for solving complex
problems and delivering complete path innovation for
analytical, diagnostic and biotechnology applications
for the life sciences market. As a genuine and
trustworthy partner, we solve our customers’ most
demanding challenges with the industry’s most
extensive portfolio of state-of-the-art components and
capabilities. Our vision of the complete path goes far
beyond just meeting our customers’ needs — it
anticipates them, with intelligent solutions for life.
Index
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Scholars &
Emerging
Leaders
Poster
Session
Wednesday
15 June
Tuesday
14 June
Monday
13 June
Sunday
12 June
Saturday
11 June
96
Congress
Overview
([KLELWRU/LVWLQJ
Company Name
Booth Number
Company Name
Booth Number
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
89 North ......................................................... 223
Abcam ............................................................ 725
ACEA Biosciences, Inc. ................................... 509
Affymetrix Inc. ................................................ 610
APE GmbH ..................................................... 824
Apogee Flow Systems ..................................... 721
Bangs Laboratories .......................................... 513
BD Biosciences ............................................... 318
Beckman Coulter Life Sciences ....................... 902
BioLegend....................................................... 401
Bio-Rad Laboratories ...................................... 802
Cedarlane ....................................................... 719
Cellix Limited ................................................. 723
Cobolt AB ....................................................... 812
Coherent, Inc. ................................................. 219
Cytek Biosciences Inc. .................................... 410
Cytognos S.L. .................................................. 921
Cytomark ........................................................ 826
Dako - Agilent Technologies .......................... 911
De Novo Software .......................................... 820
Enzo Life Sciences .......................................... 614
EXBIO Praha, a.s. ............................................ 211
Flow Contract Site Laboratory, LLC................. 925
FlowJo, LLC .................................................... 430
Fluidigm Corporation...................................... 602
FluoroFinder ................................................... 114
Hamamatsu Corporation ................................ 914
Hellma USA, Inc............................................. 413
iLab Solutions ................................................. 235
IntelliCyt Corporation ..................................... 134
Iridian Spectral Technologies .......................... 411
ISAC ............................................................... 439
Special
Lectures
Jackson ImmunoResearch Laboratories, Inc. ... 209
Kinetic River Corp. .......................................... 112
LASOS Lasertechnik GmbH ............................ 909
Leinco Technologies ....................................... 711
LeukoDx/Accellix ............................................ 907
Magsphere Inc................................................. 233
Melles Griot .................................................... 225
MilliporeSigma ................................................ 310
Miltenyi Biotec ................................................ 620
NanoCellect Biomedical, Inc. ......................... 231
NanoFCM Co. Ltd. .......................................... 919
Newport Corporation ...................................... 920
Nexcelom Bioscience...................................... 923
Omega Biosystems Incorporated ..................... 913
On-chip Biotechnologies Co., Ltd. .................. 110
Pavilion Integration Corporation ..................... 122
Photop Technologies Inc. ................................ 924
Propel Labs ..................................................... 808
RPMC Lasers, Inc. ........................................... 126
Sapphire North America .................................. 120
Seahorse Bioscience ........................................ 814
Shanghai Ruiyu Biotech Co. Ltd. ................... 1009
Sony Biotechnology Inc. ................................. 402
Spherotech, Inc. .............................................. 213
Stratedigm, Inc. ............................................... 302
Stratocore ........................................................ 709
Streck .............................................................. 713
Sysmex ............................................................ 102
Thermo Fisher Scientific .................................. 330
Union Biometrica, Inc. .................................... 409
Verity Software House ..................................... 414
Vortran Optical Subsystems .......................... 1012
ZELLKRAFTWERK ........................................... 910
Scholars &
Emerging
Leaders
Exhibit Hours
1800 - 2000
1030 - 1930
1030 - 2000
1030 - 1430
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Sunday, June 12
Monday, June 13
Tuesday, June 14
Wednesday, June 15
All exhibitors are welcome to attend the Exhibitor Town Hall Meeting on
Wednesday June 15, from 900 – 1000 in the showcase area of the Exhibit Hall.
Index
97
Speaker/Author
Poster Session
Abstracts
98
Congress
Overview
Exhibiting Companies
Special
Lectures
Disclaimer
Participation in the Exhibits Program does not constitute an endorsement by the International Society for
Advancement of Cytometry (ISAC) of the claims, products or services offered.
223
APE GmbH develops and manufactures measurement
devices and other accessories for ultrafast laser
systems. In 2015 APE expanded its product portfolio
with the quantiFlash®, an LED light source that can
be used for calibration purposes in flow cytometry.
Various versions of the quantiFlash® are available,
optimized for different applications and requirements.
APE is based in Berlin, Germany, and the
quantiFlash® is available for worldwide order.
Oral Session
Abstracts
Apogee Flow Systems
Commercial
Tutorials &
Exhibits
721
Unit 7 Grovelands Boundary Way
Hemel Hempstead HP2 7TE
United Kingdom
Phone: 44 208 123 6831
Web: www.ApogeeFlow.com
Poster Session
Abstracts
Index
Apogee Flow Systems Ltd is the market leader
in small particle flow cytometry (cell-derived
99
Speaker/Author
ACEA Biosciences provides high performance,
affordable flow cytometers, and real-time cell analysis
instrumentation for efficient monitoring of cell-based
assays including, cytotoxicity, migration/invasion,
apoptosis, and cardiotoxicity.
Plauener Strasse 163-165
Berlin 13053
Germany
Phone: 49 30 98601130
Fax: 49 30 986011333
Web: www.ape-berlin.de
Scholars &
Emerging
Leaders
6779 Mesa Ridge Road, Suite 100
San Diego, CA 92121
Phone: 858-724-0928
www.aceabio.com
824
Poster
Session
509
APE GmbH
Wednesday
15 June
Abcam plc is a leading provider of protein research
tools and services, with an unrivalled range of
products and expert technical support, enabling
scientists to analyse living cells at the molecular level
and improving the understanding of health and
disease. To find out more, come and speak to our
friendly booth staff.
eBioscience, an Affymetrix company, develops and
manufactures over 12,000 antibodies, proteins,
immunoassays and multiplex assays at ISO-certified
facilities worldwide. Focused on accelerating
immunologic discoveries with reagents to measure
gene and protein expression in single cells, we
provide innovative solutions to researchers and
clinicians looking to answer questions driving today's
life science communities. Partner with the industry
leader of translational science.
Tuesday
14 June
330 Cambridge Science Park
Cambridge CB4 OFL
United Kingdom
Phone: 44 1223 696000
Web: www.abcam.com
Monday
13 June
725
Sunday
12 June
Optical components provide precise color separation
and signal purity for applications such as fluorescence
microscopy, flow cytometry, confocal or multiphoton
microscopy. BP/LP/SP * Multiband * Notch * Dichroic
Mirrors * Polychroic Mirrors * UV/VIS/NIR * AR
Coatings * Hot/Cold Mirrors * ND/AG/AL Mirrors *
Laser Grade and more, engineered and manufactured
by a team of employee-owners committed to bringing
you the finest optical filters, filter sets and optics
solutions.
ACEA Biosciences
610
3420 Central Expressway
Santa Clara, CA 95051
Phone: 408-731-5000
Fax: 408-731-5380
Web: www.affymetrix.com
1 Mill Street, Unit 285
Burlington, VT 05401
Phone: 802-881-0302
Web: www.89north.com
Abcam
Affymetrix Inc
Saturday
11 June
89 North
Special
Lectures
Congress
Overview
microvesicles,
bacteria, large virus) for which it offers
its Micro model with up to 3 lasers, 12 optical
detectors
and an autosampler. For
conventional cell applications (mammalian cells,
yeast, plant cells) Apogee offers its Universal model
which is a compact, aggressively priced flow
cytometer again with up to 3 spatially separate lasers
and 11 optical detectors.
Saturday
11 June
Bangs Laboratories
9025 Technology Drive
Fishers, IN 46038
Phone: 317-570-7020
Fax: 317-570-7034
Web: www.bangslabs.com
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Index
Abstracts
513
Bangs Laboratories, Inc. produces fluorescence
microsphere standards to help the research and
clinical community conduct studies and evaluate data
in an easier, more precise manner. Our products set
the standard for quality control and quantitation in the
fields of flow cytometry, fluorescence microscopy,
image analysis, and other fluorescence analytical
applications. Superior Customer and Technical
Service complement Bangs’ extensive product
offerings.
BD Biosciences
318
2350 Qume Drive
San Jose, CA 95131
Phone: 877-232-8995
BD Biosciences, a segment of Becton, Dickinson and
Company, is one of the world’s leading businesses
focused on bringing innovative tools to life science
researchers and clinicians. Its product lines include:
flow cytometers, cell imaging systems, monoclonal
antibodies, research reagents, diagnostic assays, and
tools to help grow tissue and cells.
Beckman Coulter Life Sciences
902
5350 Lakeview Parkway South Drive
Indianapolis, IN 46268
Phone: 800-742-2345
Web: www.beckman.com
Flow Cytometry – Research / Life Science Offering the
broadest range of cellular analysis systems in the
world, Beckman Coulter provides a variety of
reagents, instruments and software to meet the diverse
needs of today’s research laboratories. Our extensive
line of reagents include human and non-human
antigens, signal transduction and immunotoxicology.
100
BioLegend
401
9727 Pacific Heighs Boulevard
San Diego, CA 92121
Phone: 858-455-9588
Web: www.biolegend.com
BioLegend develops and manufactures world-class,
cutting-edge antibodies and reagents at an
outstanding value. The broad product portfolio
includes flow cytometry, cell biology, and functional
reagents for research in immunology, neuroscience,
cancer, and stem cells. Product development is
accomplished through technology licensing,
collaborations, and internal R&D. Custom services
include assay development, sample testing, and
conjugation. BioLegend is certified for ISO 9001:2008
and ISO 13485:2003.
Bio-Rad Laboratories
802
255 Linus Pauling Drive
Hercules, CA 94547
Phone: 510-741-4486
Web: www.bio-rad.com
Depend on Bio-Rad for tools, technologies and
expertise to enable genomic and proteomic analysis.
Bio-Rad provides instrumentation and reagents for
droplet digital PCR, conventional and real-time PCR,
amplification reagents and primers, flow cytometry,
xMAP technology, cancer biomarkers,
electrophoresis, blotting-systems, chromatography,
imaging, cell counting, cell imaging and antibodies.
Cedarlane
719
1210 Turrentine Street
Burlington, NC 27215
Phone: 336-513-5135
Fax: 336-513-5138
Web: www.cedarlanelabs.com
CEDARLANE® (ISO 9001, ISO 13485 registered)
specializes in manufacturing and supplying high
quality reagents and temperature sensitive shipping
solutions. We offer one of the most extensive product
listings in the world, both domestically and
internationally. Delivering Today’s Innovations for
the Science of Tomorrow™
Cellix Limited
723
Unit 1, Longmile Business Park Longmile Road
Dublin 12
Ireland
Phone: 353-1-4500-155
Fax: 353-1-4500-158
Web: www.cellixltd.com
De Novo Software
820
Index
101
Speaker/Author
De Novo Software specializes in producing high
quality cytometry data analysis software. Our flagship
product, FCS Express 5, is a full feature solution for
Flow Cytometry data. FCS Express 5 Image Cytometry
introduces the same support and flexibility for
Poster Session
Abstracts
400 North Brand Boulevard, Suite 850
Glendale, CA 91203
Phone: 213-814-1240
Fax: 213-814-1240
Web: www.denovosoftware.com
Oral Session
Abstracts
Produktionsvej 42
Glostrup 2600
Denmark
Phone: 0045 51676125
Web: www.dako.com
Commercial
Tutorials &
Exhibits
46017 Landing Parkway
Fremont, CA 94538
Phone: 510-657-0102
Fax: 510-657-0151
Web: www.cytekdev.com
410
911
Scholars &
Emerging
Leaders
Dako - Agilent Technologies
Coherent Inc. is one of the world's leading suppliers
of laser-based solutions offering reliability, cost, and
performance advantages for the widest range of
commercial and scientific research applications.
Founded in 1966, Coherent designs, manufactures
and markets laser systems and components, laser
beam measurement and control equipment as well as
leading-edge beam forming and beam guidance
systems for manufacturers and scientific researchers
across the globe.
Cytek Biosciences Inc.
Whiteleaf Business Centre 11, Little Balmer
Buckingham Buckinghamshire MK18 1TF
United Kingdom
Phone: 44 01280827460
Web: www.cytomark.com
Poster
Session
5100 Patrick Henry Drive
Santa Clara, CA 95054
Phone: 408-764-4000
Web: www.coherent.com
826
Wednesday
15 June
219
Cytomark
Tuesday
14 June
Coherent, Inc.
Cytognos S.L. is a biotechnology company based in
Salamanca (Spain) dedicated to the design and
development of new reagents, software and
techniques that provide innovative solutions in the
flow cytometry field. Our products are developed in
close cooperation with international universities,
research centers and innovative companies in order to
rapidly obtain the best solutions for our customers.
Our team is devoted to satisfy our customer needs and
therefore we offer highly qualified technical support.
Monday
13 June
Cobolt AB (Stockholm, Sweden) has, since year 2000,
been committed to development and supply of
innovative laser products that meet or exceed the
market’s expectations concerning performance,
quality and robustness. Through continuous
technology development, customer orientation and an
ISO-certified quality management system, Cobolt has
become a preferred supplier of lasers to major
manufacturers of analytical instrumentation
equipment and leading research labs.
Polí gono La Serna, Nave 9
Santa Marta de Tormes Salamanca 37900
Spain
Phone: 34923125067
Web: www.cytognos.com/
Sunday
12 June
Vretenvagen 13
Solna
Sweden
Phone: 46 8 54591230
Fax: 46 8 54591231
Web: www.colbot.se
921
Saturday
11 June
812
Cytognos S.L.
Special
Lectures
Cobolt AB
Cytek provides the flow cytometry tools scientists
need at prices they can afford accompanied by cost
effective service programs. Customize a high-powered
cytometer from a menu of industry standard platforms
at a fraction of the cost of a new system. Or, select an
upgrade package to expand the capabilities and
extend the useful life of your existing cytometer.
Finally, our comprehensive service plan options allow
you to select coverage that aligns with your needs and
resources.
Congress
Overview
Cellix develops microfluidic technologies for
diagnostics; integrating and miniaturizing sample
detection and preparation techniques on-chip. At
Cyto, Cellix will exhibit the new Inish Analyzer for the
first time. It combines microfluidics and advanced
electronics with impedance detection on-chip = labelfree flow cytometry; no fluorescent dyes/stains
required. Inish is a fast and reliable cell and particle
analyzer. Applications include personalized
healthcare, Food & Beverage analysis and the Agribiotech sector.
imaging
data files. FCS Express combines a user
friendly modern interface with powerful analysis
tools,
visualization capabilities and sophisticated
presentation features, making it the tool of choice for
thousands of researchers needing quick results from
their data
Enzo Life Sciences
Sunday
12 June
Monday
13 June
Tuesday
14 June
Enzo Life Sciences, Inc. is organized to lead in the
development, production, marketing, and sales of
innovative life science research reagents worldwide.
With over 30 years’ experience, we are a proven
leader in labeling and detection technologies across
research and diagnostic markets. Our Cellestial®
assays and probes have been optimized for the most
demanding imaging applications including
fluorescence microscopy, flow cytometry, and high
content screening where consistency and
reproducibility are essential.
EXBIO Praha, a.s.
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
211
Nad Safinou II 341
Vestec 25242
Czech Republic
Phone: 420 261 090 595
Fax: 420 261 090 660
Web: www.exbio.cz
EXBIO Antibodies is a dynamic biotechnology
company focused on design, development and
manufacture of highest quality flow cytometry
products (antibodies and kits) for IVD and RUO
application, as well as on custom services at
affordable prices. Besides world-wide distribution of
its products under EXBIO label, the company sells
many antibody reagents also on OEM basis.
Flow Contract Site Laboratory
925
18311 Bothell Everett Highway, Suite 180
Bothell, WA 98034
Phone: 425-821-3900
Fax: 425-821-3925
Web: www.fcslaboratory.com
Oral Session
Abstracts
Index
Abstracts
614
10 Executive Boulevard
Farmingdale, NY 11735
Phone: 631-694-7070
Fax: 800-942-0430
Web: www.enzolifesciences.com
Saturday
11 June
Special
Lectures
Congress
Overview
Flow Contract Site Laboratory (FSC Lab) is a
specialized Contract Research Organization (CRO)
which offers expert services focused on GLP and nonGLP flow cytometry assays for research and
development, nonclinical and clinical drug
development. The management of our organization
has resolved, as part of our diversification objective,
102
to seek out partners who are interested in utilizing our
capabilities to reduce existing work load while
increasing flexibility and expanding their capabilities.
FlowJo, LLC
430
385 Williamson Way
Ashland, OR 97520
Phone: 541-201-0022
Fax: 541-482-3153
Web: www.flowjo.com
FlowJo is the next generation of flow cytometry
analysis software. It handles your most ambitious
projects with a high-level drag-and-drop user
interface. Based on a patented experiment-based
analysis paradigm, FlowJo intelligently handles
protocols containing multiple tubes (any FCS files)
with different samples stained with different reagent
sets.
Fluidigm Corporation
602
7000 Shoreline Court, Suite 100
South San Francisco, CA 94080
Phone: 650-266-6033
Web: www.dvssciences.com
We strive to partner with customers to pursue truth in
the complex biological world. Our core microfluidic
technology provides rapid, simplified and elegant
workflows for high-throughput qPCR or nextgeneration sequencing library preparation. Whatever
the application, we never compromise; rather, we
deliver cost-effective, easy-to-deploy, easy-to-scale
and easy-to-use workflows that produce the highquality data your lab demands. Let’s partner. Engage
with us at fluidigm.com.
FluoroFinder
114
10835 Dover Street, Suite 300
Westminster, CO 80021
Phone: 720-583-0974
Web: www.fluorofinder.com
Simplifying the complexity of building and iterating a
flow cytometry panel, the free FluoroFinder tool and
first class service offers unparalleled support and
resources to member core facilities. Members receive
additional benefits and support to drive efficiencies
and resource savings. Saving thousands of research
days, and millions of dollars, for a research institution,
every year, FluoroFinder is the fastest way to build a
flow cytometry panel. Become a member facility of
the free FluoroFinder Service today.
914
413
872 West Baltimore Pike
West Grove, PA 19390
Phone: 610-869-4024
Fax: 610-869-0171
Web: www.jacksonimmuno.com
103
Index
Jackson ImmunoResearch provides highly adsorbed
secondary antibodies for flow cytometry and other
applications. Our comprehensive range of conjugates
includes biotin and fluorophores, such as FITC, R-PE,
Speaker/Author
209
Poster Session
Abstracts
9620 San Mateo Boulevard NE
Albuquerque, NM 87113
Phone: 408-963-6113
Web: www.intellicyt.com
134
Jackson ImmunoResearch
Laboratories, Inc.
Oral Session
Abstracts
IntelliCyt Corporation
The International Society for Advancement of
Cytometry (ISAC) serves a multidisciplinary
community by leading technological innovation,
scholarship, and the exchange of knowledge in the
quantitative cell sciences to advance the impact of
cytometry in meeting current and emerging
challenges in the live, biomedical, and physical
sciences. Please visit the ISAC booth to learn more
about the society and CYTO U, meet society
representatives such as the executive director,
education manager or the editor-in-chief of Cytometry
Part A, and discover how ISAC can help you advance
your career.
Commercial
Tutorials &
Exhibits
iLab Solutions provides core facility management
software to streamline requests, track usage, and
automate billing. The web-based solution supports
the entire shared service work flow, from scheduling
and request approvals through communication, billing
and reporting. Integration available with financial
system such as SAP, PeopleSoft, Banner, and Lawson.
www.ilabsolutions.com
9650 Rockville Pike
Bethesda, MD 20814
Phone: 301-634-7454
Web: www.isac-net.org
Scholars &
Emerging
Leaders
Ten Post Office Square Floor 8
Boston, MA 02109
Phone: 617-297-2805
Web: www.ilabsolutions.com
439
Poster
Session
235
ISAC
Wednesday
15 June
iLab Solutions
Founded in 1998, Iridian Spectral Technologies Ltd.
specializes in the design and manufacture of optical
interference filters for various purposes within the
photonics industry. Iridian offers its customers a wide
range of standard and custom filters as well as coating
services. Iridian is a global supplier with headquarters
in Canada.
Tuesday
14 June
Hellma Analytics is the world’s leading manufacturer
of custom flow channels for Cytometry and a reliable
partner both for scientists and for instrument
manufacturers. Sophisticated equipment and trained
staff guarantees quality flow channels from
prototyping to large scale production. Flow channel
dimensions start as low as 50ʅm. State of the art
features like cones to optimize hydrodynamic
focusing, thin windows to fit large NA objectives,
application of focusing lenses etc can be integrated at
the highest precision. Hydrodynamic modeling is also
available to assist in design creation.
2700 Swansea Crescent
Ottawa, ON K1G 6R8
Canada
Phone: 613-741-4513
Fax: 613-741-9986
Web: www.iridian.ca
Monday
13 June
80 Skyline Drive
Plainview, NY 11803
Phone: 516-939-0888
Web: www.hellmausa.com
411
Sunday
12 June
Hellma USA, Inc.
Iridian Spectral Technologies
Saturday
11 June
Hamamatsu Corporation is the North American
subsidiary of Hamamatsu Photonics K.K. (Japan), a
leading manufacturer of devices for the generation
and measurement of infrared, visible, UV light and Xrays. These devices include photomultiplier tubes,
photodiodes, image sensors, cameras, and light
sources. Hamamatsu Photonics also offers specialized
systems for select applications.
Special
Lectures
360 Foothill Road
Bridgewater, NJ 08807
Phone: 908-231-0960
Web: www.hamamatsu.com
IntelliCyt enables rapid and cost effective screening
and profiling of physiologically-relevant model
systems using cell and/or bead-based assays. Our
easy-to-use hardware, software, and reagent systems
are optimized to work together to conserve precious
samples, use less reagent, and to minimize time-toanswer to enhance productivity and preserve the
biology of interest.
Congress
Overview
Hamamatsu Corporation
APC,
PerCP and Alexa Fluors®. Anti-Human, Mouse,
Rabbit and Rat antibodies are available among others,
with
specificities including whole Ig, F(ab’)2 fragment,
Fcɶ fragment, μ chain and Mouse subclasses. We also
offer conjugated Streptavidin and Purified IgG
controls.
Kinetic River Corp.
Sunday
12 June
Monday
13 June
233
1993 Loctus Street
Pasadena, CA 91107
Phone: 626-584-9446
Fax: 626-795-0797
Web: www.magsphere.com
LASOS Lasertechnik GmbH
Melles Griot
Wednesday
15 June
Tuesday
14 June
Magsphere Inc.
We provide and support high quality monosized
microspheres. Specifically designed for cytometry
application includes NIST traceable particle size
standards, count standards, flow cytometry QC
alignment standards, linearity beads and bright
fluorescent nanobeads for tracking application.
Particle based immunoassays such as lateral flow
rapid tests, turbidity, slide agglutination etc. can count
on Magsphere Inc, ISO9001, for supplying quality,
consistent and cost effective beads. OEM welcome.
909
Franz-Loewen-Strasse 2
Jena Thuringia 07745
Germany
Phone: 49 3641 2944 0
Fax: 49 3641 2944 300
Web: www.lasos.com
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
LeukoDx dramatically increases access to complex
cell-based diagnostics testing. Accellix, our tabletop
diagnostic device with single-use assay cartridges,
dramatically expands access to flow cytometry
capabilities by replacing previously complex testing
with a user-friendly automated instrument that can be
used by any medical practitioner, nursing staff, or
junior lab tech on a 24/7 basis.
Kinetic River is a biophotonics design and product
development company focused on flow cytometry
instrumentation. NEW LIVE DEMO: the BeamGenie, a
compact device that stabilizes a laser beam and CVs,
automatically compensating for thermal
misalignment. The Potomac is a completely
customizable analyzer (1 to 7 lasers, 4 to 20
detectors), built for flexibility and modularity. The
Danube is the only fluorescence lifetime flow
cytometer on the market able to resolve
multiexponential decays – with sub-nanosecond
resolution.
Leinco Technologies
711
410 Axminister Drive
Fenton, MO 63026
Phone: 636-230-9477
Web: www.leinco.com
LeukoDx/Accellix
3 Hamarpe Street
Jerusalem
Israel
Phone: 972-2-674-4422
Web: www.accellix.com
104
225
2051 Palomar Airport Road, 200
Carlsbad, CA 92011
Phone: 760-438-2131
Fax: 760-438-5208
Web: www.cvimellesgriot.com
LASOS develops and manufactures solid state and gas
lasers in the UV-visible to NIR/IR spectrum with
special focus on OEM applications in Biophotonics,
Cytometry and Holography. Within the superb
geographic footing in Jena, LASOS is well placed to
channel the latest in scientific and technological
process into cutting edge products and services.
Oral Session
Abstracts
Index
Abstracts
112
897 Independence Avenue, Suite 4A
Mountain View, CA 94043-2357
Phone: 650-269-0726
Web: www.KineticRiver.com
Saturday
11 June
Special
Lectures
Congress
Overview
Melles Griot, a part of IDEX Health & Science, designs
and manufactures Optical Systems, Optomechanical
Shutters, Lasers, and Laser-based light engines. By
working seamlessly with the customer from start to
finish, we deliver high-value engineered solutions for
Analytical Instrumentation, Bio-Instrumentation,
Defense and Semiconductor Equipment applications.
Melles Griot provides the perfect balance of
performance, reliability, and manufacturability for
OEM applications worldwide.
MilliporeSigma
907
310
290 Concord Road
Billerica, MA 01821
Phone: 800-225-3384
MilliporeSigma, the U.S. life science business of
Merck KGaA, Darmstadt, Germany, is comprised of
the legacy EMD Millipore organization and SigmaAldrich Corporation; supporting research,
development and production of biotech and
pharmaceutical drug therapies. With a portfolio of
620
919
Omega Biosystems is proud to introduce its newest
product, the Vulcan imaging flow cytometer, at
CYTO2016. By combining flow cytometry with
world-record speed fluorescence imaging, this
revolutionary instrument features an unmatched
combination of cellular throughput and spatial
resolution, which provides an unprecedented level of
flexibility and precision in cellular analysis. Come
visit us at booth 913 to learn more and witness the
future of flow cytometry.
On-chip Biotechnologies Co., Ltd. 110
Poster Session
Abstracts
204 Venture Port, 2-24-16 Naka-cho
Koganei-city Tokyo 184-0012
Japan
Phone: 81-42-358-0461
Web: www.on-chip.co.jp
Index
105
Speaker/Author
On-chip Biotechnologies Co., Ltd is a Japanese Bio
venture and has developed a compact cell sorter, Onchip Sort, which adopted a disposable microfluidic
Oral Session
Abstracts
NanoFCM provides a powerful platform for the
multiparameter characterization of NPs (7-1000 nm)
and viruses at the single-particle level. Light scattering
is used for the measurement of nanoparticle size and
size distributions. Fluorescence detection is used to
analyze the chemical properties of nanoparticles.eb:
www.nanofcm.com
10316 Bannockburn Drive
Los Angeles, CA 90064
Phone: 401-439-0159
Web: www.omegabiosystems.com
Commercial
Tutorials &
Exhibits
Zhaori Building 215 Longhushan Road Siming
District
Xiamen Fujian 361005
China
Phone: 86 13459026961
Omega Biosystems Incorporated 913
Scholars &
Emerging
Leaders
NanoFCM Co. Ltd.
Nexcelom’s Cellometer and Celigo image cytometry
products are designed for cell analysis in life science
and biomedical research. The Cellometer instruments
cover basic cell counting, viability and cell-based
assays with 20uL of sample. The Celigo is a high
throughput micro-well image cytometer. Products are
placed in thousands of laboratories globally.
Poster
Session
NanoCellect's WOLF Cell Sorter uses a closed fluidpath microfluidic cartridge that enables easy-to-use
cell sorting and avoids biohazard and shear stress
issues associated with traditional FACS. A major
advantage is the elimination of sample contamination
between runs and increased cell viability after low
pressure sorting. Plus, the WOLF Cell Sorter is
aerosol-free—avoiding containment systems.
360 Merrimack St. Bldg. 9
Lawrence, MA 01843
Phone: 978-327-5340
Email: info@nexcelom
Web: www.nexcelom.com
Wednesday
15 June
6404 Nancy Ridge Drive
San Diego, CA 92121
Phone: 951-966-4963
Web: www.nanocellect.com
923
Tuesday
14 June
231
Nexcelom Bioscience
Monday
13 June
NanoCellect Biomedical, Inc.
Newport’s Corion Optical Filters and Opticon
Replicated Optics are key enablers in a wide variety
of biomedical instruments including RT-PCR Systems,
Cytometers, DNA Analyzers, In Vivo Imagers,
Microarray/Microplate Readers, Confocal and Epifluorescence microscopes, FTIR spectrometers, and
many more. For more information, please visit our
website at www.newport.com/corion-opticon
Sunday
12 June
Miltenyi Biotec provides products that advance
biomedical research and cellular therapy. Our
innovative tools support research from basic research
to translational research to clinical application. Our
more than 25 years of expertise includes immunology,
stem cell biology, neuroscience, and cancer. Miltenyi
Biotec has more than 1,500 employees in 25
countries.
1791 Deere Avenue
Irvine, CA 92606
Phone: 949-863-3144
Web: www.newport.com
Saturday
11 June
Friedrich-Ebert-Strasse 68
Bergisch Gladbach 51429
Germany
Phone: 49 2204 83063190
Web: www.miltenyibiotec.de
920
Special
Lectures
Miltenyi Biotec
Newport Corporation
Congress
Overview
more than 300,000 products, we provide the research
tools, instrumentation and reagents, facilitating
complete workflow solutions for cellular and
analytical analysis, critical for the success of our
customers.
chip
to separate cells. This novel technology enables
damage-free sorting of cells by gentle liquid pulse
flow.
Furthermore, On-chip Sort permits the use of
any liquid as sheath, for instance, culture media and
sea water for optimal cell condition. Oil can also be
used for collection of water-in-oil emulsion droplets.
Pavilion Integration Corporation 122
2380-F Qume Drive
San Jose, CA 95131
Phone: 408-453-8801
Fax: 408-453-8802
Web: www.pavilionintegration.com
PIC’s WhisperIT diode-based lasers cover the UV,
Visible, and IR wavelength range and utilize a
patented technology that creates a beam with
excellent mode quality, low noise under all
conditions, and low speckle, but at a substantially
reduced cost, footprint, and power consumption
compared to ion or DPSS lasers. PIC customizes
its laser products to be quickly and seamlessly
integrated into biotechnology instrumentation to
ensure optimum performance.
Photop Technologies Inc.
Poster
Session
Scholars &
Emerging
Leaders
Photop Technologies, subsidiary of II-VI Corp.
(NASDAQ:IIVI), is a leading photonics designer and
integrated manufacturing company on Fiber Optics,
Precision Optics, Projection and Display Optics,
DPSS Laser, Crystal Materials, and other Photonics
Products.
Propel Labs
808
345 E. Mountain Avenue
Fort Collins, CO 80524
Phone: 970-295-4570
Fax: 970-372-5664
Web: www.propel-labs.com
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Index
Abstracts
924
253 Fuxin East Road
Fuzhou, Fujian 350014
China
Phone: 8659188052884
Web: www.photoptech.com
Wednesday
15 June
Tuesday
14 June
Monday
13 June
Sunday
12 June
Saturday
11 June
Special
Lectures
Congress
Overview
Propel Labs is a team of flow cytometry experts who
specialize in cutting edge technology. Following
development of the Avalon Cell Sorter, Propel
partnered with Bio-Rad Laboratories who now offers
the system as the S3e High Performance Bench-Top
Cell Sorter. Propel Labs also offers MoFlo and CyAn
upgrades including Co-Lase Towers and NanoView
for small particle detection.
106
RPMC Lasers, Inc.
126
203 Joseph Street
O’Fallon, MO 63366
Phone: 636-272-7227
Web: www.rpmclasers.com
Innovative solid-state lasers and systems for Life
Science and Measurement applications. - LaserBoxx
monolithic DPSS, available at 532, 553 and 561nm
from 25 up to 500mW in very compact, industry
standard package (100x40x32mm). - LaserBoxx Laser
Diode modules at 375nm, 405nm, 445nm, 473nm,
488nm , 515nm , 594nm, 638nm, 660nm, 705nm,
730nm, 785nm. - LBX-4C Cost effective and modular
up to 4 wavelengths combiner for laboratory use.
Sapphire North America
120
795 Highland Drive
Ann Arbor, MI 48108
Phone: 855 256-9433
Fax: 734 468-0906
Web: www.sapphire-usa.com
Sapphire North America distributes innovative Life
Science research products from specialty
manufacturers from around the globe. Sourcing the
planet for scientists, Sapphire North America provides
laboratories and purchasing groups a convenient onestop procurement resource. Sapphire North America
offers antibodies, biochemicals, assay kits, probes,
proteins, and other products for research in cancer,
cell biology, immunology, inflammation, and
neurochemistry.
Seahorse Bioscience
814
16 Esquire Road
North Billerica, MA 01862
Phone: 978-671-1600
Web: www.seahorsebio.com
Seahorse Bioscience is a part of Agilent Technologies.
Seahorse XF metabolic analyzers and stress test kits
are the industry standard for measuring cell
metabolism. XF Extracellular Flux Analyzers enable
the simultaneous measurement of the two major
energy pathways of the cell – mitochondrial
respiration and glycolysis – in live cells, in real time.
Shanghai Ruiyu Biotech Co. Ltd 1009
Bldg. C3-301, 6000 Shenzhuan Road
Songjiang District
Shanghai
PR China
Phone: +86 21 62299622
Web: www.countstar.cn
402
302
330
46360 Fremont Boulevard
Fremont, CA 94538
Phone: 510-979-5000
Web: www.thermofisher.com
Thermo Fisher Scientific is the world leader in serving
science. Our mission is to enable our customers to
make the world healthier, cleaner and safer. Through
our Invitrogen, Applied Biosystems, and Gibco,
brands, we help customers accelerate innovation and
enhance productivity. Visit Booth #330 for details.
Index
107
Speaker/Author
21 rue Loigny la Bataille
Chartres 28000
France
Poster Session
Abstracts
709
Thermo Fisher Scientific
Oral Session
Abstracts
Stratocore
Sysmex, a global leader in hematology, offers a broad
range of flow cytometry solutions for research and
biotech. Debut at Cyto: a comprehensive line of
Sysmex fluorescent monoclonal antibodies targeting
cancer research. The CyFlow® Cube cytometer is
enhanced with new software and offers up to 8
optical parameters/ 3 lasers plus UV LED. The flexible
CyFlow Space cytometer offers up to 5 light sources.
For research use only.
Commercial
Tutorials &
Exhibits
Stratedigm manufactures configurable, scalable, and
upgradeable flow cytometry solutions that fit every lab
and every budget. Users can configure their S1000EXi
cytometer up to 4 lasers and 24 parameters.
Complimenting any S1000EXi or SE520EXi cytometer,
the Stratedigm automation suite provides automated
reagent mixing, digital temperature control,
sterilization, and 100% automated runs of up to 320
plates. Stratedigm – truly, cytometry without
compromise.
577 Aptakisic Road
Lincolnshire, IL 60069
Phone: 224-543-9331
Web: www.sysmex.com
Scholars &
Emerging
Leaders
6541 Via Del Oro Suite A
San Jose, CA 95119
Phone: 408-884-4029
Fax: 408-351-7700
Web: www.stratedigm.com
102
Poster
Session
Stratedigm, Inc.
Sysmex
Wednesday
15 June
Spherotech manufactures a variety of microparticles
for flow cytometers. These particles are used for
calibration, alignment, multiplexing, compensation,
absolute counting and drop delay determination.
Specifically, the calibration, alignment, and drop
delay particles are used extensively for QC and long
term performance tracking. In addition, Spherotech
has particles for confocal fluorescence microscopy.
Streck develops and manufactures hematology,
immunology and molecular biology products for
clinical and research laboratories. Recognized as a
worldwide leader in cell stabilization, Streck focuses
on the development of quality control and diagnostic
products. New developments include flow cytometry,
body fluids and urinalysis product lines.
Tuesday
14 June
27845 Irma Lee Circle, Unit 101
Lake Forest, IL 60045
Phone: 847-680-8922
Fax: 847-680-8927
Web: www.spherotech.com
7002 South 109th Street
Omaha, NE 68128
Phone: 800-843-0912
Web: www.streck.com
Monday
13 June
213
713
Sunday
12 June
Spherotech, Inc.
Streck
Saturday
11 June
Sony Biotechnology analyzers, sorters and imagers
incorporate advanced technologies and intuitive
functionality to bring support for scientific discovery
to a new level. See how spectral technology in our
analyzers delivers high sensitivity, as it simplifies
application design and workflow. See imaging
differently with our 2015 SLAS award winning Cell
Motion System. Its real time, cell observations and
quantification is built on high speed video
microscopy and motion vector analysis.
Stratocore is a leader in Resource Management. Our
software PPMS, in development since 2003, is used
by thousands of clients in over 60 research institutions
worldwide. PPMS is scalable and affordable;
providing flexible tools that adapt to the changing
needs of any facility. It optimizes productivity,
increases revenue and provides Resource Managers
real time control.
Special
Lectures
1730 North First Street
San Jose, CA 95112
Phone: 800-275-5963
Fax: 408-352-4130
Web: www.i-cyt.com
Phone: 1-678-827-2673/+33 184 16 10 92
Web: www.stratocore.com
Congress
Overview
Sony Biotechnology Inc.
Sunday
12 June
Saturday
11 June
Special
Lectures
Congress
Overview
U
nion Biometrica, Inc.
84 October Hill Road
Holliston, MA 01746
Phone: 508-893-3115
Web: www.unionbio.com
Union Biometrica Large Particle Flow Cytometers
automate the analysis and sorting of objects too
big/fragile for traditional cytometers, e.g., large
cells/clusters, cells in/on beads and small model
organisms (10-1500 micron diameter). The new LP
Sampler module can aspirate these samples from
multiwell plates and deliver them intact to the
BioSorter. The new VAST BioImager system automates
cellular-level imaging of 2-7 dpf zebrafish larvae.
Verity Software House
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
910
Bosestrasse 4
Leipzig 04109
Germany
Phone: 4915152385628
Fax: 49 341 94089071
Web: www.zellkraftwerk.com
ChipCytometry: 83-plex Cytometry Assay &
Instrument Chipcytometry is a new cytometry
platform enabling the multiplex analysis of more than
80 markers from a single cell. Chipcytometry makes it
possible to store cells for at least 24 months while
maintaining full biomarker integrity. Cells can be
biobanked, shipped, analyzed and re-analyzed for an
unlimited number of times enabling true collaborative
cytometry projects of research groups working on
different continents. www.zellkraftwerk.com
Verity Software House, an industry leader in flow
cytometry software development, offers a unique
combination of innovative software for flow cytometry
and unparalleled technical and customer support.
ModFit LT, WinList, and GemStone form an
unbeatable combination. We have recently released
V-Comp, a fully automated compensation system.
Please stop by and see us in booth 414, and learn
more about data modeling and automating your
analysis process. Without Verity, it’s just software.
1012
21 Goldenland Court #200
Sacramento, CA 95834
Phone: 916-283-8208
Web: www.vortranlaser.com
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
414
ZELLKRAFTWERK
45A Augusta Road P.O. Box 247
Topsham, ME 04086
Phone: 207-729-6767
Fax: 207-729-5443
Web: www.vsh.com
Vortran Optical Subsystems
Index
Abstracts
`4
409
Vortran Laser Technology, Inc. is one of the fastest
growing providers of the highest quality laser
solutions. Our products range from OEM laser
modules up to fully integrated photonic sub-systems
in wavelengths from 375nm to 830nm and powers
from 1mW to 400mW which maximize performance
and profitability for our customers. The experience
and capabilities of our engineering will expand your
product performance while reducing costs allowing
your focus to be on the biology and overall system
performance.
108
Oral Abstracts
1
Considerations for Integrating Flow Cytometry with
Single Cell Transcriptomics
a. Local and global intensity thresholding
b. Thresholding based on object size
3. Separation of clustered objects
b. Shape-based object separation
ImmunoTechnology Section, Vaccine Research Center,
NIAID, NIH, Bethesda, MD, United States
c. Additional a priori knowledge to improve object separation
Carolina Wahlby , Lee Kamentsky , Anne E. Carpenter
1
Department of Information Technology, Uppsala
University, Uppsala, Sweden, 2Imaging Platform, Broad
Institute of Harvard and MIT, Cambridge, MA, United States
1. A concise overview or summary of the tutorial, what will be
covered/discussed
Patricia Simms1, Peter Lopez2, Michele Black3
1
FACS Core Facility, Office of Research Services, Loyola
University Chicago, Maywood, IL, United States,
2
Department of Pathology, Office of Science & Research,
New York University, School of Medicine, New York, NY,
United States, 3Cell Analysis Facility, Department of
Immunology, University of Washington, Seattle, WA,
United States
Course Details or Outline:
1. Overview of changes that require SRL management paradigm
shift
Index
b. Image enhancement
109
Speaker/Author
2. Regulation of instrument usage and maintenance, and assuring
quality data
Poster Session
Abstracts
Course Objectives: The paradigm of Shared Resource Laboratory
(SRL) management has changed over the last few years and
potentially more so in flow cytometry than in other types of SRLs.
Previously, day to day management of an SRL primarily consisted of
instructing users, overseeing quality control, use and monitoring the
quality of data produced. Now, instruments with varied levels of
capabilities, unregulated access to those instruments, generation of
large complex data sets and a new generation of incoming scientists
have changed the landscape of SRL management. These instrument
improvements, as well as upgrades in reagents and development of
novel flow cytometry assays also necessitate additional educational
programs, for users and investigators. This tutorial will address
managing a facility with these new issues in mind. In the first
section, implementation and enforcement of regulations for
instrument maintenance of user-run instruments and satellite sites
will be presented. Also covered will be the identification and
remedial actions for difficult users and assuring generation of
quality data. In the second section, data analysis packages that are
available will be assessed. Generation of big data requires not only
commercially available software but also more complex data
analysis programs. This section will offer insight as to how to
choose the best software program for the scientific question at hand
and the necessary facility personnel training needed to use R-based
software. Finally, the new generation of investigators requires
different methods of interaction than previous generations.
Millennials arrive with their own set of strengths and weaknesses.
Suggestions to take advantage of their computer and social media
skills while providing mentorship not only in the science, but also
in institutional compliance and independence will be discussed in
the third section.
Oral Session
Abstracts
a. Illumination correction
3
Shared Resource Management – A Changing
Paradigm for the 21st Century
Commercial
Tutorials &
Exhibits
1. Basics/Overview of image pre-processing
c. Exporting measurements
Scholars &
Emerging
Leaders
b. Measuring intensity features
Poster
Session
2. What will the student leave this session with (after participating
in this tutorial, the student should have…Object detection is the
first and often most crucial step in any image based analysis
approach when measurements are to be extracted from objects
such as individual cells in an image. Robustness and accuracy of
object detection depends on many factors, such as noise, variability
in staining efficiency, clustering of objects, and background
illumination variations. Automated image analysis approaches for
object detection can be optimized in many different ways, and the
number of different parameters that can be tweaked is often
overwhelming. During this tutorial we will focus on algorithms and
parameter settings for object detection in the free and open source
CellProfiler software. We will go through thresholding and
watershed segmentation, including different options for preprocessing, image enhancement and background illumination
correction. We will also look at the details of separating adjacent
cells based on shape and intensity. We will explain how the
algorithms and settings are presented in CellProfiler, and discuss
strategies for parameter optimization using a number of real
examples. We will also discuss pixel classification as a preprocessing step for segmentation, and show how measuring objects
is a simple task once objects are accurately detected.After
participating in this tutorial, the students should be able to
approach their own image analysis challenges using CellProfiler,
and have a good grasp on how to optimize settings for accurate
object detection. They will also know how to extract and export
measurements such as size, shape, position and counts of objects to
quantitatively answer questions related to variations in cell
morphology as observed by microscopy or image flow cytometry.
a. Measuring size and shape features
Wednesday
15 June
Course Objectives:
5. Feature measurements and data export
Tuesday
14 June
2
b. Identifying sub-regions and associating related objects
Monday
13 June
2
Configuring Accurate Cell Detection in Images Using
CellProfiler
a. Identifying cytoplasms using nuclei as input
Sunday
12 June
Course Details/Outline: Overview of why transcriptomics might be
valuable in cytometry. Methods for stimulation of cells,
purification/sorting of cells, QC of assay, and performing pre- and
on-chip amplifications. Data Analysis. Summary/Conclusions
4. Identification of regions of interest associated with other objects
Saturday
11 June
Course Objectives: In recent years, technical advances have made
it possible to obtain transcriptomic data (i.e., mRNA expression)
from single cells. This presentation will be focused on using this
technology for studies of T-cells, and will discuss: a) stimulation of
cells, b) purification and sorting of single cells, c) considerations for
selecting mRNA targets, and qualifying assay primers/probes, d)
procedures and workflow for performing the assay, and e) data
analysis considerations. We will also discuss the power and
limitations of transcription-based approaches to cytometry.
2
Special
Lectures
a. Intensity based object separation
Pratip K. Chattopadhyay
1
Congress
Overview
2. Methods of intensity thresholding
Congress
Overview
3. Selecting appropriate data analysis program and finding facility
training
4.
Successful professional relationships with next generation
scientists
Special
Lectures
5. Summary/Conclusions
4
Validation – the Key to Translatable Flow Cytometry
in the 21st Century
Index
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Scholars &
Emerging
Leaders
Poster
Session
Wednesday
15 June
Tuesday
14 June
Monday
13 June
Sunday
12 June
Saturday
11 June
Virginia Litwin1, Teri Oldaker2, Cherie Green3
1
Hematology/Flow Cytometry, Covance Central Laboratory
Services, Indianapolis, IN, United States, 2Flow Cytometry,
Genoptix Medical Laboratory, a Novartis Company,
Carlsbad, CA, United States, 3Flow Cytometry Biomarker
Development Sciences, Genentech, Inc., a Member of the
Roche Group, San Francisco, CA, United States
Course Objectives: This Tutorial will cover all aspects of analytical
method validation for flow cytometry (methods and instruments).
The target audience for this course is everyone who is using flow
cytometry and wants to generate high quality data (i.e. basic
researchers, academia, pharma, biotech, clinical laboratories,
environmental labs on boats, you name it). Okay, you get the idea,
everybody!!!!The course will begin with an introduction to the
various regulated environments and then an overview of instrument
qualification and analytical method validation. We will define each
of the instrument qualification and method validation parameters,
and how each are addressed in flow cytometry. We will also
discuss how to interpret the validation data once it is generated and
how the validation data influences the final use of the assay.After
participating in this tutorial, students will have an understanding of:
What instrument qualification and analytical validation are and
why they are important;
The difference between assay development, optimization and
validation;
5
Writing, Publishing and Reviewing: Advice, Tips and
News from Cytometry A
Attila Tarnok1, Vera Donnenberg2, Anar Murphy3
1
University Leipzig, 2University of Pittsburgh, Pittsburgh, PA,
United States, 3Wiley-Blackwell, Hoboken, NJ, United
States
Course Objectives: Scientific journals require certain quality
standards from manuscripts to be acceptable for further reviewing
and publication. There are some very common reasons why a
paper gets reviewed and accepted or rejected. This tutorial aims to
highlight all major aspects of manuscript writing, submission and
communication with the reviewers, points out what can (and very
often does) go wrong and how to do it right in order to improve
your chances to get your paper published. Special emphasis will be
taken to focus on the needs for publishing cytometry data in
biomedical and technical oriented journals such as Cytometry Part
A. The process will be shown from the Eitors, publishers and
reviewers point-of-view.
Course Details: How to write a good manuscript: Here the most
important aspects of writing a good manuscript and the most
common mistakes made in writing a manuscript will be explained
and discussed. Manuscript processing and reviewing process: The
processing of manuscript within the journal will be presented and
discussed from the editors, reviewers and publishers perspective.
Course Outline:
How to write a good manuscript
General aspects for good manuscript writing (Attila Tarnok)
Basics of manuscript processing
Selection of the right Journal
Cytometry Part A overview
The performance specifications of assay validation;
Author guidelines from EiC`s perspective (specific aspects of
cytometry data presentation; MIFlowCyt, OMIP, Repositories)
The statistical analysis and interpretation of validation data.
Writing Review articles and Communications to the Editor
Outline: Regulatory Environments and Myth Busters; Nonregulated; CLIA/CAP; GLP;GMP; ISO Commonalities in Regulatory
Environments
Editing Special Issues and Sections and and Commentaries
Method Validation Introduction:
Good manuscript writing: the Publisher’s perspective (Anar
Murphy)
Scope: We will discuss why we should validate a method and the
differences between assay characterization, qualification and
validation.
Manuscript writing tips
Terminology: We will discuss how the precise definition of basic
validation terms differs slightly in the various regulatory
environments and more so when applied to flow cytometry.
Instrument Qualification:Importance of characterization of
performance;Installation and performance qualification;Crossinstrument, cross-site standardization;Maintaining longitudinal
stability
Applying the Iterative Approach:Questions and Considerations:
sample type; result to be reported (% positive, phenotype, abs
count, intensity); intended use;Using the Iterative Approach:
development -->optimization--> validation-->implementation-->
repeat (?)
Just Do It: A Deep Dive in to Validation; Bioanalytical Data
Categories;What impacts each validation parameter (accuracy,
precision, sensitivity, specificity, stability, reference intervals);
When is each validation parameter needed?
Tying It All Together: Data interpretation; Documentation; Next
steps/assay utility
110
Scientific authorship, ethical aspects, conflict of interest.
How to make your article popular: Search Engine Optimization tips
Reviewing and manuscript processing
Best praxis for manuscript reviewing: The Associate Editor and
Reviewer perspective (Vera Donnenberg)
Evaluating overall scientific quality, accuracy and relevance of the
submission (cover letter, abstract and manuscript)
Detailed technical evaluation of the manuscript (abstract,
introduction, materials and methods, results, discussion and
conclusion)
Making reviewer/AE recommendation and EiC decision
Open access and other policies (Anar Murphy):
Open Access
Copyright and Licenses
Software policy
Conclusions, Perspectives and Specific Programs. (Attila Tarnok)
Joe Trotter1, Geoffrey Osborne2, Charles Pletcher3
Course Objectives: Provide the attendee with helpful theoretical
and practical understanding applicable to various cell separation
scenarios common to most current laboratory
needs/workflows/protocols.
Course Outline:
A basic introduction to IC:
How do we define IC as a cytometric discipline?
What are the underlying principles of IC?
Key descriptive terminology/language in IC
When and how should IC be deployed to ask a defined cytometric
question?
What are the major stages in an IC experimental pipeline?
IC system/platform selection
Tuesday
14 June
Image acquisition: Key considerations
How to obtain the best overall results for purity and yield goals
given the nature of the former and the reality of the latter
7
Image Cytometry for the Flow Cytometrist: When
Image Really is Everything
Data presentation
Application Examples:
Static IC-based system
Flow-based IC system
Summary/Conclusions and further sources of information.
Poster
Session
Attendees will walk away with a comprehensive view of current
electrostatic cell separation technology as well as an understanding
of how they might go about applying it in the best way for their
applications.
Image analysis and feature extraction (manual and automated)
Wednesday
15 June
Technical aspects with some do’s and don’ts will be covered, as
well as selected examples of some novel applications that have
been enabled by the technology and provide a good template for
success.
8
Laboratory Infrastructure: Moving and Designing a
Flow Cytometry Laboratory
Kevin L. Holmes1, Nancy C. Fisher2, Stephen P. Perfetto3
2
1
Flow Cytometry Core Facility, Faculty of Medical Sciences,
Newcastle University, Newcastle upon Tyne, United
Kingdom, 2De Novo Software, Glendale, CA, United States
Index
111
Speaker/Author
Biosafety considerations – Kevin Holmes will discuss the basis and
practice of BSL-2 lab design including critical considerations for
BSL-3 (reference BSL-3 chapter). Aerosols, containment thereof and
the latest recommendations for monitoring containment will also be
discussed.
Poster Session
Abstracts
Course Objectives: A concise overview or summary of the tutorial,
what will be covered/discussed, The panel will discuss biosafety,
instrumentation and infrastructure considerations for the
development of a new flow cytometry core laboratory. What will
the student leave this session with (after participating in this tutorial,
the student should have…The workshop will provide lab directors
and managers a set of relevant considerations for interacting with
institutional facilities personnel and offer some solutions about
planning and moving a core facility.
Oral Session
Abstracts
Flow Cytometry Section, Research Technologies Branch,
NIAID, National Institutes of Health, Bethesda, MD, United
States, 2Flow Cytometry Core Facility, Department of
Microbiology & Immunology, University of North Carolina
at Chapel Hill, Chapel Hill, NC, United States, 3Core Flow
Cytometry Facility, Vaccine Research Center, National
Institutes of Health, Bethesda, MD, United States
Commercial
Tutorials &
Exhibits
Course Objectives: The word “Cytometry” is a fusion of two Greek
words, “kytos” meaning “basket/cell” and “metria” meaning to
“measure”. Cytometry itself can be conducted in many ways but
whatever the chosen approach it almost always involves measuring
the properties of single cells/particles from within large populations
with the sole aim of decoding the heterogeneity inherent to all
biological systems. While Non-Image-based Flow Cytometry (NIFC)
is a powerful, multi-parameter, high-throughput cytometric
technology with widespread applications it cannot provide the
spatial/morphometric information often essential to addressing
certain key biological questions. As such a number of image-based
cytometric approaches exist that can be grouped into a relatively
broad field known as “Imaging Cytometry” (IC). This tutorial is
primarily aimed at cytometrists who have a background in NIFC
and want to understand the basic principles of IC and how it can be
pivotal in decoding biological heterogeneity at the cellular level.
The course will provide a generic definition and the underlying
principles of IC as well as outlining the key stages in a typical IC
experimental pipeline. Application examples will be included
where appropriate in order to provide context to the participants. A
recurring theme throughout the tutorial will be to compare and
contrast IC with NIFC in order to provide a foundation for
1
Scholars &
Emerging
Leaders
Andrew Filby , David Novo
Consider attending future IC-based tutorials at Cyto or other
conferences that cover more advanced elements of IC to build on
the foundations provided by this tutorial.
QC and biological controls
How samples tend to behave
1
Be able to appreciate the major steps in an IC experimental pipeline
and what the main challenges to success could be.
Monday
13 June
How cell sorters work
Decide if, when and how IC should be deployed within their
individual domains in order to address key biological questions.
Sunday
12 June
Course Details or Outline: The cell sorter and its ability to rapidly
separate viable cell fractions into pure populations is very often a
critical stage within both research and clinical application
workflows designed to separate a wide variety of very specific cell
phenotypes for various purposes; to obtain rare transfected cells for
culture, correlate cell phenotype and gene expression, create
product for human regulatory T cell therapy, and a host of other
applications. Several of the more important aspects of the
technologies involved, how they may be used and leveraged will
be presented after an initial discussion of cell sorting fundamentals.
Understand the basics principles/language of IC.
Saturday
11 June
Advanced Research Group, R&D, BD Biosciences, San
Jose, CA, United States, 2The Queensland Brain Institute /
The Australian Institute for Bioengineering and
Nanotechnology, The University of Queensland, Brisbane,
Australia, 3University of Pennsylvania School of Medicine,
Philadelphia, PA, United States
After attending this tutorial, it is envisaged that participants will be
able to:
Special
Lectures
1
introducing IC as a potentially new concept to the attendees by
building on their existing knowledge/expertise.
Congress
Overview
6
Cell Sorting: Fundamentals and Selected Novel
Applications
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Designing and Moving a Flow Cytometry Shared Resource – Nancy
Fisher will share her experience the layers of design considerations
and the importance of cross-functional communication during a
laboratory
move. The presentation will expand on BSL-2 lab design
from Kevin with air flow design, APM vs Ball-in-the-wall, options
for house air pressure for sorters (wet cart as back up and reduces
heat generated from machine, compressors), and lab furniture
design (size, weight bearing capacity).
Jennifer Wilshire1, Tomas Baumgartner2
This talk wll focus on practical considerations and steps for starting
a cytometry-related device, software, or service company. Gary
Durack will share some of his experiences as a cytometry
technology entrepreneur, angel investor, and founder of multiple
companies. Aspects of the lean startup model (made popular by
Steven Blank and Eric Reiss) that apply to the cytometry technology
entrepreneur will be shared, as well as, experiences from the NSF
and NIH I-Corp programs. The tutorial will cover key steps to
forming and funding a cytometry technology startup, and will
include advice on how to rapidly build your startup company’s
value. Topics will include intellectual property management,
strategic partnerships, SBIR/STTR funding, and product realization.
This talk will serve as an introduction to those interested in
entrepreneurship or simply working at a startup company.
Hopefully this tutorial will also provide encouragement to our new
generation of cytometry entrepreneurs.
1
Other nagging details that creep in will be discussed, such as move
quotes, Insurance quotes (replacement value), moving lab logistics:
communication, timing, biosafety and workflowBuilding and
Expanding Flow Cytometry Instrumentation – Steve Perfetto will
provide insight for purchasing new cytometers/expanding cores –
new instrument integration, transitioning. He will discuss measuring
instrument panel compatibility, using the LED-pulser. He will also
discuss efforts in bead standardization through a new NIST
program.
9
Forensic Flow Cytometry - Multiparameter
Research and Development, STEMCELL Technologies,
Vancouver, BC, Canada, 2Flow Cytometry Core Facility,
Memorial Sloan Kettering Cancer Center, New York, NY,
United States
Course Objectives: This tutorial will take an active learning
approach to multicolor flow cytometry. Come ready to participate!
Starting with the fundamentals (adding more colors never made bad
flow data better) we will use a “forensic” approach to illustrate
potential underlying problems. Test your knowledge – see if you
can identify the flow crime in each case study.In the second part of
the tutorial, a step-by-step process for multicolor panel design will
be outlined for three categories of experiments (<6 colors, 6-10
colors, 11+ colors). The complexity increases with more colors but
we will lay out the steps for building a panel of any size. During the
panel design workshop, the audience will pair off and tackle
designing an 11 multicolor panel together in order to practice
applying these steps. After participating in this tutorial, attendees
will have a step-by-step guide to multicolor panel design and
hands-on experience with applying those steps in order to yield
good flow data.
Scholars &
Emerging
Leaders
Take care of fundamentals – forensic case studies
TitrationFcR block
Dead cells / Viability StainingCompensation controls
Panel design approach
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Step-by-step <6 colors
Index
Abstracts
analytics and IoT. Many of these products will find their way to
market through efforts of a new generation of cytometry technology
entrepreneurs. In this rapidly changing landscape, an
entrepreneurial career path can be attractive and rewarding for
professionals with the ideas, skills, education, and experience to
found a new company.
Step-by-step 6-10 colors
Step-by-step 11+ colors
Build your own multicolor panel
Audience pairs will tackle designing an 11 color panel
10
Founding and Launching a Cytometry Startup
Company
Gary Durack
The TEKMILL Inc., Champaign, IL, United States
Course Objectives: Forty years after its birth, cytometry is entering
another period of rapid technology commercialization. The
development of new sensors and microfluidic devices, plus the
realization of innovative Point-of-Care diagnostic systems is
creating a significant, world-wide demand for new cytometry
devices, research assays, and cytometry-related, clinical
diagnostics. These innovations also drive opportunities for Big Data
112
History of Cytometry Entrepreneurship
Lean Startup Ideas
Purpose of startup
Value propositions
Customer segments
Business model canvas
Cytometry-specific Opportunitites
Practical Steps
Organization
Funding
Execution
Visual and Leadership
Expectations and Risk
Challenge
11
Imaging Bioinformatics for High Content Screening
of 3D Cell Culture Models
Bahram Parvin1,2
1
Department of Biomedical Engineering, University of
Nevada, Reno, Reno, NV, United States, 2Biological System
Engineering, Lawrence Berkeley National Laboratory,
Berkeley, CA, United States
Course Objectives: The current trend in high content screening
(HCS) has been the utilization of more complex model systems that
mimic both structural and functional properties of cells in vivo.
Complex model systems have been the main motivation behind the
development of imaging bioinformatics systems for phenotypic
screening, with the main drivers for the utility of these biological
systems being high fecundity and a short life span. Similarly, 3D
cell culture models have emerged as effective systems to study
tumor initiation, biological processes, and reversion properties with
respect to therapeutic targets. In contrast to 2D cell culture models,
3D cell culture models (i) have different patterns of development
and can be functional, (ii) respond differently to therapeutic targets,
and (iii) have different patterns of gene expression. Hence, the 3D
cell culture model is a potential model system to bridge between
the 2D model systems and the animal studies. In some systems, 3D
colonies are imaged using elegant specialized microscopy and
profiled using 2D image analysis and global intensity
Kevin Holmes1, Hank Pletcher2, Simon Monard3,
Stephen P. Perfetto4
Microfluidic cell sorting mechanisms
Conclusion and future directions
Have you ever been asked to talk about cytometry to students or
adults with little science background? Have you ever declined this
request because you were not sure how to present the concepts in a
way that non-scientists would understand? This tutorial will provide
examples of make-it-yourself/hands-on activities for introducing
cytometry to audiences with varying scientific backgrounds.
448
Introducing Cytometry through Hands-On Activities
Juliane P. Hill1, Alexis Conway2, Dan Callen3, Reitha
Weeks4, Tami Caraballo5, Jan Chalupny4, Sherree Friend6
1
Immuno Technology Section, Vaccine Research Center,
NIAID, NIH, Bethesda, MD, United States, 2Roswell Park
Cancer Institute, Buffalo, NY, United States, 3Coherent Inc.,
Wilsonville, OR, United States, 4Shoreline Community
College, Seattle, WA, United States, 5Glacier Peak High
School, Shoreline, WA, United States, 6Amnis/EMD
Millipore, Seattle, WA, United States
Have you ever been asked to talk about cytometry to students or
adults with little science background? Have you ever declined this
request because you were not sure how to present the concepts in a
way that non-scientists would understand? This tutorial will provide
examples of make-it-yourself/hands-on activities for introducing
cytometry to audiences with varying scientific backgrounds.
Course Outline:
This tutorial will also provide a forum in which to discuss with
experts in the field, specific scenarios that operators or core facility
managers encounter.
Rotate through four activity stations:
After participation in this tutorial the attendee should have a clearer
understanding of the principles and practices of biosafety as it
pertains to flow cytometry, in particular cell sorting. Additionally,
the attendee will have a list of resources to aid in risk assessment
and the development of Standard Operating procedures in their
own lab.
Antibodies
Science through theater: watch a short, humorous play depicting
key features of flow cytometry
Presenting cytometry concepts
Discussion and application of activities in a high school setting
Discussion of play: cytometry concepts and value of drama in
science education
Index
Wrap-up and resource review
Summary/Conclusions
113
Speaker/Author
Open forum to discuss specific issues
Image cytometry
Poster Session
Abstracts
Discussion of scenarios that might be encountered
Lasers and fluorescence
Oral Session
Abstracts
Basics/Overview Biosafety Principles and Practices in Flow
Cytometry for sorters and analyzers
Introduction/overview: teaching science and cytometry to students
and adults
Commercial
Tutorials &
Exhibits
This tutorial will provide a summary of biosafety principles as they
apply to flow cytometry and cell sorting, with emphasis on the
2014 ISAC Cell Sorter Biosafety Standards.
Scholars &
Emerging
Leaders
Course Objectives:
Cell detection units
Poster
Session
1
Flow Cytometry Section, Research Technologies Branch,
NIAID, National Institutes of Health, Bethesda, MD, United
States, 2University of Pennsylvania, Philadelphia, PA,
United States, 3Walter & Eliza Hall Institute of Medical
Research, Victoria, Australia, 4Core Flow Cytometry Facility,
Vaccine Research Center, National Institutes of Health,
Bethesda, MD, United States
Microfluidic cell focusing strategies
Wednesday
15 June
12
Flow Cytometry Biosafety
Overview of microfluidic flow cytometry and cell sorter
Tuesday
14 June
Case studies
Monday
13 June
Complexities and solutions associated with quantitative analysis of
3D cell culture modelsDemonstration of BioSig3D features
Course Objectives: The tutorial session will cover the unique
features, technical details, and applications of micro-flow
cytometers and cell sorters. Technical aspects including device
fabrication, cell focusing methods, detection strategies, and sorting
mechanisms will be discussed in detail. After participating in this
tutorial, the attendees are expected to have a thorough
understanding of the field.
Sunday
12 June
Imaging bioinformatics for an HCS design for 3D cell culture
models
Department of Engineering Science and Mechanics, Penn
State University, University Park, PA, United States
Saturday
11 June
Basics/Overview of complexities associated with an HCS
experiment
Tony Huang
Special
Lectures
13
Microfluidic Flow Cytometry and Cell Sorting
Congress
Overview
measurements. In this tutorial, we will show how the 3D cell
culture systems can be screened when they are imaged in 3D
volume using confocal microscopy. Attendees will be introduced to
BioSig3D, which is an open source imaging bioinformatics system
for analyzing 3D data. BioSig3D provides an end-to-end solution
for designing high content screening assays, based on colony
organization that is derived from segmentation of nuclei in each
colony. The system provides visualization of raw and processed 3D
volumetric data for quality control, and integrates advanced
bioinformatics analysis, and consists of multiple computational and
annotation modules that are coupled together with a strong use of
controlled vocabularies to reduce ambiguities between different
users. It is a web-based system that allows users to design an
experiment by defining experimental variables, upload a large set of
volumetric images into the system, analyze and visualize the
dataset, and either display computed indices as a heatmap, or
phenotypic subtypes for heterogeneity analysis, or download
computed indices for statistical analysis or integrative biology.
BioSig3D will be used to profile baseline colony formations with
several experiments: (i) morphogenesis of a panel of human
mammary epithelial cell lines (HMEC), (ii) heterogeneity in colony
formation using an immortalized non-transformed cell line, and (iii)
morphogenesis of colony formation in response to the
environmental perturbation (e.g., stiffness, radiation). These
experiments reveal intrinsic growth properties of well-characterized
cell lines that are routinely used to study biological processes.
Congress
Overview
14
The Journey from Concept to Commercialization: A
Daunting
Task with High Rewards for Entrepreneurs
Index
Abstracts
Oral Session
Abstracts
Commercial
ial
Tutorials &
Exhibits
ts
Scholars &
Emerging
Leaders
Poster
Session
Wednesday
15 June
Tuesday
14 June
Monday
13 June
Sunday
12 June
Saturday
11 June
Special
Lectures
Janette Phi
Chief Business Officer, IntelliCyt, Albuquerque, NM, United
States
Strategic and tactical decisions must be made at multiple points on
the journey from the birth of an idea to commercialization. The
selection of the type of investors from the different categories of
investors, can provide insight into decision-making, including not
only the usual stage distinction but also the investors' experience
and knowledge affecting investor opinions on the company and the
management team. The expectations of investors in entrepreneurial
life sciences companies, from initial business plan formulation and
presentation to post-investment management and board issues, can
be daunting. The strategy of learning from customers over the
lifetime of the company, is critical from first product launch to
market prominence. Creating a global commercialization strategy
that enables a company to “look” big while working as a lean
startup. Continuing innovation around the core technology,
maintaining close relationships with customers, and thinking and
acting globally can lead a company from startup to market
prominence. Insights and challenges will be shared from
experiences on various companies such as AmCell, Quantum Dot,
Guava, ForteBio and IntelliCyt.
15
Advancing the Art and Science of Flow Cytometry
through Advanced Materials
Brent Gaylord
Biosciences, Research Reagents, Becton Dickinson, San
Diego, CA, United States
Awarded the 2000 Nobel Prize in Chemistry, the discovery and
development of conductive polymer materials was recognized as a
seminal innovation, rooted in the ability to blend the properties of
conductive materials with those of plastic or polymeric materials,
classically characterized as insulators. Insightful and pioneering
work in physics and synthetic chemistry gave rise to semiconducting materials which were readily tunable and solution
processable, sparking a number of commercial enterprises looking
to generate flexible displays, better TVs, printable circuits and low
cost photovoltaics. Over time, key scientists realized the unique
optical properties of these materials could alternatively be exploited
in novel and highly sensitive chemical and biological detection
formats. Sirigen and its Brilliant dye technology evolved from this
branch of research tracing its roots back to the late 1990’s at UC
Santa Barbara. This presentation will trace the journey of taking an
early stage technology from initial development in an academic
environment, through commercialization as a small entrepreneurial
start-up and eventual integration within a large commercial
organization, providing a good case study for some of the key
technical (solubility, conjugation) and business (licensing, funding,
strategic direction) challenges encountered along the way. As with
any venture, academic or commercial, building the right team,
focusing on the right problems, finding the right partners and
maintaining a strong conviction proved critical to the eventual
success of this journey.
In the case of Sirigen, a particularly difficult yet defining decision,
after having spent several years pursuing other options, was
ultimately to redirect and focus efforts on the field of flow
cytometry. Guiding us along this new path were two clear value
propositions critical to this field: greater brightness and more
colors. It was clear that the extended pi-conjugation inherent to
these materials could be used to create fluorescent reporters with
exceptional light harvesting abilities, a key factor in delivering high
brightness. As chemists we also recognized we could adapt or tune
their molecular structures to create a wide range of colors to enable
greater multiplexing (deeper phenotyping, etc.). A major challenge,
however, was figuring out how to solubilize these hydrophobic
macromolecules and reproducibly attach them to biological
molecules such as monoclonal antibodies. The output of this work
114
has already started to transform the field of flow cytometry by
successfully revitalizing the violet laser, repurposing the ultra-violet
laser and potentially redefining the landscape of the blue and
yellow-green lasers. Excitingly the journey has not ended. As we
will demonstrate this ever-expanding pallet of bright colors is not
only driving higher parameter analysis with greater resolution of
biological populations but also enabling systems with less
compensation and greater ease of use
16
Quantitative Physiology of Living Single-Cells: Subtle
Pleasures and Future Promise
Roger Brent
Basic Science-Brent Lab, Fred Hutchinson Cancer Research
Center, Seattle, WA, United States
In eukaryotes, numerous cellular processes (including cell
signaling) depend on cause-and-effect chains of molecular events
involving proteins. Key classes of molecular events include
changes in protein association (oligomerization state), changes in
posttranslational modification, changes in conformation, changes in
subcellular localization, and changes in amount of expressed
reporter gene product. Understanding the quantitative function
(quantitative physiology) of such processes is benefiting from the
ability to use signals from fluorescent protein derivatives to quantify
specific molecular events in such chains in single living cells.
Starting about a decade ago, development of low-measurementerror (high precision) single-cell quantification has now enabled
characterization of the dose-response relationship at many levels of
a yeast signaling pathway, providing data that disproves particular
models of how dose response is regulated (1, 2, 3, and Andrews et
al., submitted). Development of single cell quantification has also
allowed identification of previously unknown cell physiological
states (3), defined (for example) by cell-to-cell differences in the
ability to send signal through different pathways and by differences
in general cellular ability to express genes into proteins. In the
multicellular animal Caenorhabditis elegans, some of these celllevel states affect consequential phenotypes displayed by the whole
organism (5).
Here, I'll review the above developments. Depending on progress
of work, I will also describe in more detail:
a) Improvements in applied mathematical tools to quantify new
physiological state variables, including measurements of absolute
differences in gene expression power, from single cell
measurements.
b) Improved means to decrease measurement error for single- cell
quantification in live C. elegans.
c) Use of single-cell measurements to generate hypotheses about
order of events and causation for whole-organism phenotypes, and
to generate new physiological state biomarkers to stratify
organismic populations for later outcomes such as resistance to
oxidative damage.
References:
1. Yu, R., Gordon, A., Colman-Lerner, A., Benjamin, K.R., Pincus,
D., Serra, E., Holl, M., Brent, R. (2008) Nature, 456, 755-761
2. Bush, A., and, Colman-Lerner, A. (2013). Biophys J. 104(3):727736
3. Mendenhall, A. R., Tedesco, P. M., Johnson, T. E., and Brent, R
(2015). PLoS ONE 10(5): e0124289
4. Colman-Lerner, A., Gordon, A., Serra, E., Chin, T., Resnekov, O.,
Endy, D., Pesce, G. and Brent, R. (2005) Nature. 437, 699-706
5. Mendenhall, A., Driscoll, M., and Brent, R. (2016) Aging Cell 15,
4-13
19
Highly Multiplexed Simultaneous Detection of
Proteins and RNAs in Single Cells
David Hedley
Pier Federico Gherardini1, Andreas P Frei1, Felice Alessio
Bava1, Eli Zunder2, Elena W Y Hsieh3, Shih-Yu Chen1,
Garry P Nolan1
PLAYR was validated in multiple biological systems and showed a
range of detection from highly abundant down to transcripts that
are expressed at ~10 copies per cell. Moreover we compared our
technology with RT-qPCR, thus confirming that PLAYR can be used
to reliably quantify relative changes in transcript abundance across
different biological conditions.
Index
Macrophages are crucial regulators of the immune system that
interact with tumor cells in the cancer setting to promote disease
115
Speaker/Author
Insitute of Molecular Life Sciences, University of Zurich,
Zurich, Switzerland, 2University Hospital Zurich, Zurich,
Switzerland
Poster Session
Abstracts
1
Oral Session
Abstracts
Daniel Schulz1, Stephane Chevrier1, Hartland Jackson1,
Zsuzsanna Varga2, Bernd Bodenmiller1
Commercial
Tutorials &
Exhibits
20
Simultaneous Imaging of Proteins and Transcripts in
Cells by Highly Multiplexed Imaging Mass Cytometry
Scholars &
Emerging
Leaders
In conclusion PLAYR expands high-throughput deep phenotyping
of cells beyond protein epitopes to include RNA expression. We
believe that such an approach will help to define new cell
populations that share patterns of temporally or spatially regulated
gene expression. The elevated sample throughout makes PLAYR
particularly useful in clinical settings and in cohort studies.
Moreover protein co-detection enables the study of posttranscriptional regulation and the relationship between signaling
and gene expression with single cell resolution.
Poster
Session
We demonstrated the potential of PLAYR by measuring the
expression of cytokine genes in human peripheral blood
mononuclear cells after stimulation with lipopolysaccharide.
Current methods for measuring cytokine production in single cells
by flow cytometry require the use of protein secretion inhibitors, so
that cytokine proteins can accumulate in the cells for detection with
specific antibodies. This setup precludes the study of cellular
communication based on paracrine signaling, and may introduce
artifacts. Conversely, PLAYR measures the abundance of cytokine
transcripts directly, while simultaneous staining with antibodies
against surface markers is used to delineate different cell
populations.
Wednesday
15 June
Extracellular vesicles (EVs) have emerged as a novel biomarker for
diagnosis and prognosis of human diseases, including cancer,
infection, and metabolic disorders. Analyzing EVs in routine
clinical settings, however, still remains a difficult task, due to the
lack of adequately sensitive and fast assay platforms. Our laboratory
has been developing new assay systems optimized for on-site EV
analysis. This presentation will discuss two representative systems:
i) microfluidic filter that enrich EVs in-flow, and ii) a nanoplasmonic sensor for EV protein detection. The filter system is a
microfluidic cartridge capable of fast and on-chip vesicle isolation;
it uses size-based sonophoresis to selectively enrich EVs. The
plasmonic chip, termed nano-plasmonic exosome (nPLEX), is
developed for high-throughput EV protein profiling. The detection is
based on extraordinary optical transmission through periodic
nanoholes. The strategy provides an ideal sensing scheme for EVs,
as the sensor probing depth (< 200 nm) could be matched to EV
size. The developed systems have been applied to isolate and
profile cancer-derived EVs in patient samples. Our results suggest
the potential of using EV analysis to monitor tumor progression and
treatment responses. Further clinical investigations are now
underway to rigorously evaluate the EV’s clinical utility.
To enable detection of expression signatures specific to individual
cells we developed PLAYR (Proximity Ligation Assay for RNA), a
technology for highly multiplexed quantification of transcripts in
single cells by flow- and mass-cytometry. PLAYR is compatible
with standard antibody staining of proteins, thus allowing
simultaneous quantification of up to 40 different mRNAs and
proteins. Given the multiplexing capabilities of the CyTOF mass
cytometer, we consider this technology to be complementary to
single-cell RNAseq in providing information on a selected panel of
genes, but with a throughput of hundreds or thousands of cells per
second and a very low cost per sample. Moreover, cells are fixed at
the beginning of the protocol and no reverse transcription is
required, thus minimizing experimental variability due to sample
processing or PCR amplification bias.
Tuesday
14 June
Center for Systems Biology, Massachusetts General
Hospital, Boston, MA, United States
Measurements of gene expression are a fundamental tool to
understand how genetic networks coordinately function in normal
cells and tissues and how they malfunction in disease. The most
commonly used methods (e.g. qPCR, microarrays or RNA-seq) are
bulk assays that only measure the average expression in a sample.
As such they cannot detect expression differences between different
cellular populations in a complex sample.
Monday
13 June
Hakho Lee
Stanford University, Stanford, CA, United States,
University of Virginia, Charlottesville, VA, United States,
3
University of Colorado Denver, Aurora, CO, United States
2
Sunday
12 June
18
Nanotechnology Platforms for Extracellular Vesicle
Analysis
1
Saturday
11 June
Imaging mass cytometry (IMC) was recently introduced by
Bodenmiller and co-workers, and is now being developed as a
commercial instrument by Fluidigm Canada. Unlike the original
CyTOFTM, IMC uses a pulsed UV laser to ablate tissue sections
labelled with mass-tagged antibodies, and a stream of inert gas to
transfer material to the mass cytometer. Computer-generated
images provide anatomical detail comparable to 20x light
microscopy. Because IMC can be applied to routine paraffin
sections, it lends itself to the study of solid tumours, as well as a
wide range of complex processes occurring in normal and diseased
tissues. Pancreatic cancers have a complex microenvironment,
characterized by an intense stromal reaction and by a pronounced
tendency towards the development of hypoxia. It is thought that
tumour-stroma interactions and reprogramming within the hypoxic
environment play a major role in their aggressive growth and
metastasis. These anatomical features, which are underappreciated by whole genome sequencing or fluidics-based
cytometry, can be dramatically revealed using IMC’s unique ability
to co-register 40+ antibodies plus a range of heavy elementcontaining histochemical probes, and extracted using advanced
computational techniques for segmentation and image analysis. To
explore this potential in a clinical context, we are applying IMC to
a large cohort of surgical samples from patients given the hypoxia
tracer pimonidazole prior to pancreatectomy, and to their
corresponding primary xenografts. Of particular interest is the
identification of alterations in metabolism and growth regulation in
relation to the hypoxic microenvironment, and their relationships to
whole genome sequencing data from this patient cohort and patient
outcome. Our goals are: 1) identify challenges and solutions
applying IMC to clinical samples; 2) build standardized IMC
protocols, with appropriate computational and bioinformatics tools,
that can provide a rapid turnaround assessment of key regulatory
pathways in a clinical setting; and 3) integrate this with standard
clinical and genomics-based features in order to inform treatment
decisions for individual patients.
Special
Lectures
Princess Margaret Hospital, Toronto, ON, Canada
Congress
Overview
17
High Content Analysis of Pancreatic Cancers Using
Imaging Mass Cytometry
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
al
Tutorials &
Exhibits
Oral Session
Abstracts
Index
Abstracts
progression. Imaging mass cytometry (IMC) is a novel technology
that currently allows for simultaneous visualization of 50 spatially
resolved markers with subcellular resolution. In tissues, IMC allows
the
detection of macrophages of diverse phenotypes in the context
of health and disease, but cannot measure cytokines and
chemokines that communicate signals between cells. To date,
(multiplexed) imaging approaches mainly rely on antibodies,
however, the addition of RNA detection allows for functional
characterization of gene regulatory networks with simultaneous
detection of antibody readouts such as signaling pathway activity,
of proteins of interest that are lowly expressed, quickly turnover, or
for which no antibodies are available.
Here, we established novel protocols that enable us to
simultaneously measure RNA, proteins, and protein modifications
in a multiplexed manner by IMC. Therefore, we adapted available
protocols for RNA detection and used metal labeled
oligonucleotides to enable mass cytometric detection. For
validation of the approaches, macrophage phenotypes and RNA
expression profiles were first determined from primary cultures and
macrophages isolated from tumors using suspension mass
cytometry and then, when grown on glass slides after staining and
imaging using IMC. Using both protein and RNA markers, the
phenotypic and functional characterization of macrophages in vitro
and tumor associated macrophages ex vivo revealed novel
macrophage subpopulations that are enriched for tumor promoting
properties. Finally, IMC of breast cancer tissue samples quantified
tumor cell subtypes and their interactions with macrophages
expressing distinct cytokines and chemokines in the tumor
microenvironment.
In summary, we show that combined detection of proteins and
RNA fully harnesses the power of IMC and allows for spatially
resolved, multiparametric studies of the tumor microenvironment.
CD45RA- CD197+) after exposure to different experimental
conditions. The data showed that T-bet and Eomes mRNA were
found to be expressed in all cellular subsets. While Eomes protein
was constitutively expressed at a low level in all CD8+ T cell
subsets, T-bet protein was found to be expressed mainly in the Teff
and Tem cells. The number of CD8+ T cells that were positive for Tbet protein was 8.5% ± 0.9% when unstimulated, increased by
approximately 5 fold when exposed to IL-2, nearly 8 fold when
exposed to anti-CD3 and anti-CD28, and about 10 fold when
exposed to anti-CD3, anti-CD28, and IL-2. For Eomes protein
expression, the number of positive cells in CD8+ T cell subsets was
35.8 ± 4.8% when unstimulated, and increased by approximately 2
fold when exposed to IL-2. When stimulated with anti-CD3 and
anti-CD28, or in combination with IL-2, the number of CD8+ cells
positive for Eomes protein increased by 3 fold.
Conclusions: Unlike typical experiments in which the expression
levels of mRNA and protein are evaluated by separate methods, our
data demonstrate in a single assay that while all CD8+ T cell
subsets may produce T-bet and Eomes mRNA, not all of the
transcripts were translated into durable protein expression; and that
the protein expression of T-bet is found mainly in the Teff and Tem
cells. The expression of T-bet and Eomes proteins can also be
induced by using anti-CD3 and anti-CD28, IL-2, or a combination
of both stimuli. This study presents the possibility of simultaneously
elucidating the kinetic expression profiles of mRNA and protein for
T-bet and Eomes during T cell polarization when exposed to
different stimuli.
23
Label-Free Separation of Neuroblastoma Tumor Cells
from Mononuclear Cells Using an AcoustophoresisBased Microfluidic Chip
21
Simultaneous Determination of mRNA and Protein
Expression Profiles of T-bet and Eomes in Stimulated
T Lymphocytes by Flow Cytometry
Franziska Olm1, Anke Urbansky2, Thomas Laurell2,
Stefan Scheding1
Kah Teong Soh, Joseph D. Tario, Jr., Paul K. Wallace
Background: High-dose chemotherapy and transplantation of
autologous peripheral blood progenitor cells (PBPC) are an
established treatment for children with high-risk neuroblastoma.
However, survival rates are poor which may partly be due to graftcontaminating tumor cells that can contribute to relapse. Current
methods to remove tumor cells from the graft (“purging”) are time
and labor consuming, expensive or insufficient. Herein, a novel
acoustophoretic microfluidic chip-based technology, using
ultrasound for gentle and efficient cell separation, was established
aiming to develop a proof-of-principle for label-free purging of
neuroblastoma-contaminated PBPC products. The acoustic standing
waves generated in the microfluidic channel act differently on cells
depending on their acoustophysical properties, such as size, density
and compressibility, thus enabling the separation of different cell
types.
Department of Flow and Image Cytometry, Roswell Park
Cancer Institute, Buffalo, NY, United States
Introduction: T-box-expressed-in-T-cells (T-bet) and Eomesodermin
(Eomes) are transcription factors which govern T cells’
differentiation and maturation during immune responses. Upon
activation via the T cell receptor or through cytokine stimulation,
the concerted modulation of T-bet and Eomes in T cells can
promote the generation of a broad repertoire of T cell subsets. Each
of the cellular subset has distinct functions, such as the production
of cytolytic mediators or cytokines to elicit immunostimulatory or
immunomodulatory effects. The objective of this study is to
simultaneously measure and correlate the mRNA and protein
expression levels of T-bet and Eomes in CD8+ T cells in response to
different stimulation conditions using a single assay. In this regard,
the Branched DNA technique enables the elucidation of mRNA and
protein expression profiles on a per cell basis, as opposed to a bulk
analysis.
Methods: Human peripheral blood mononuclear cells (hPMBCs)
were isolated from TRIMA leukoreduction filters using a density
gradient centrifugation technique. T cells from prepared hPBMCs
were activated for a total of 4 days using anti-CD3 or anti-CD28
mAbs, with the addition of IL-2 on the third day. Thereafter, cells
were harvested and subjected to cell surface labeling using mAbs,
were fixed and permeabilized with proprietary buffers, were
labeled for intracellular transcription factors, and then exposed to a
Branched DNA procedure to label the T-bet and Eomes mRNA.
RPL13A ribosomal RNA was selected as the positive control to
identify cells that had successfully incorporated mRNA target
probes during the hybridization process.
Results: The mRNA and protein expression levels of T-bet and
Eomes were studied in CD8+ naï ve T cells (Tn; CD45RA+
CD197+), effector T cells (Teff; CD45RA+ CD197-), effector memory
T cells (Tem; CD45RA- CD197-), and central memory T cells (Tcm;
116
1
2
Laboratory Medicine, Lund University, Lund, Sweden,
Biomedical Engineering, Lund University, Lund, Sweden
Study Design and Methods: For the acoustophoretic separation,
neuroblastoma tumor cells (cell line) were spiked into mononuclear
cells (MNCs) isolated from blood or MNC isolated from PBPC
samples, under defined conditions. Following separation, fractions
were analyzed for recovery, purity and viability of MNCs and tumor
cells, respectively. The influence of different voltages applied on
the main separation transducer on the separation of the different
cell types, as well as different cell concentrations and tumor
cell/MNC ratios on separation efficiency were investigated. T-cell
proliferation assays were performed to investigate a possible impact
of the acoustic separation on T-cell function.
Results: Using a concentration of 1 × 106 cells/ml and a tumor
cell/MNC ratio of 1:1, 92 ± 3.5 % of MNCs were recovered, while
tumor cells were depleted by nearly 2 log (97.4 ± 3.8 %). At a
tumor cell/MNC ratio of 1:1000, 87.9 % of the MNCs could be
recovered, while tumor cells were depleted to undetectable levels.
Cell type distribution analysis after separation showed that
especially lymphocytes were recovered in the target side fraction,
while granulocytes and monocytes tended to be collected in the
central waste fraction outlet. Also, stem and progenitor cells were
recovered in the target fraction, while tumor cells were removed
Joseph D'Silva1, Robert Austin2, James Sturm1
Electrical Engineering, Princeton University, Princeton, NJ,
United States, 2Physics, Princeton University, Princeton, NJ,
United States
Poster Session
Abstracts
Index
117
Speaker/Author
imec, Leuven, Belgium, 2Physics Department, Katholieke
Universiteit Leuven, Leuven, Belgium
Oral Session
Abstracts
1
[4] Z. Liu et al., Biomicrofluidics, 2013, 7, 011801.
Commercial
Tutorials &
Exhibits
[3] X. Ding et al., Proc. Natl. Acad. Sci. U. S. A., 2014, 111,
12992–12997.
Chengxun Liu1, Dries Vercruysse1, Koen de Wijs1,2,
Alexandra Dusa1, Shengping Mao1, Kamil Blaszkiewicz1,
Yuqian Li1, Bivragh Majeed1, Xavier Rottenberg1, Deniz
Sabuncuoglu Tezcan1, Paru Deshpande1, Liesbet Lagae1,2
Scholars &
Emerging
Leaders
[2] DW Inglis et al., Journal of Micromech. Microeng., 2011, 21,
054024.
26
A High Throughput Bubble Jet Microfluidic FACS
Poster
Session
[1] J. D’Silva et al., Lab Chip, 2015, 15, 2240–2247.
Conclusions: We have developed a platform to add to the arsenal
of biosensor development techniques that have largely relied on
relatively low-throughput rational design for their optimization. The
ability to introduce large-scale mutations and quickly identify
improved variants will improve our understanding of which
mutations lend increased resolution and tunable kinetics of the
individual constructs leading toward improved rational engineering
of genetically encoded biosensors. This method can be more
broadly applied to the development of other molecular sensors with
fluorescent readout on similar timescales.
Wednesday
15 June
References:
Results: Present work has utilized Zn2+ FRET sensors consisting of a
FRET pair, ECFP and mVenus, linked by a mutated Zap1 zinc
binding domain (ZBD). Two such cytosolic sensors, ZapCV2 and
ZapCV5, differ by only 2 mutations of the Zn2+ coordinating ligands
in the ZBD. They have dynamic ranges of 2.1 and 1.6, respectively,
and are readily resolved and separable with > 90% accuracy.
Additionally, sensors tagged to mitochondria or the endoplasmic
reticulum show response in flow, opening up the possibility of
library screening in tagged organelles. Generally, sensors showing
at least a 10% FRET ratio change are resolvable on our instrument.
Time delays from 100 msec-10 sec are attainable through
modification of the microfluidic geometry and input pressures.
Sorting rates of 5-20 s-1 are achievable, allowing for mutant libraries
of 105 clones to be sorted for response to Zn2+.
Tuesday
14 June
The DLD chip consists of an array of micro-posts, which interact
with the fluid and cells in a deterministic (not random) fashion, to
harvest cells above a certain size. At high flow rates, cells can be
compressed against the micro-posts in the DLD array, causing these
cell to not be harvested. The effect is especially severe for leukocyte
harvesting because leukocytes are more easily compressed [3]. By
reducing the shear strain on cells caused by the fluid flow,
asymmetric triangular posts have been shown to prevent the
compression of cancer cells compared to the conventional circular
micro-posts [4]. We find these posts allow efficient leukocyte
harvesting at high flow rates as well, up to ~100 ȝL/min. However,
the asymmetric posts also result in significantly more (>10-100X)
displacement of erythrocytes into the product (contaminating the
leukocytes) than symmetric posts at a flow velocity corresponding
to Re of 15, leading to a tradeoff of high leukocyte yield vs. low
erythrocyte contamination. A new post design is presented which
reduces the hydrodynamic lift effects causing the erythrocyte
contamination but still allows high leukocyte harvesting yield. With
this new diamond post design, we then demonstrate capture of
83% of leukocytes from ~4 mL of blood with only 0.02% of
erythrocytes displaced into the product in less than 45 minutes
using a single DLD array. Integration of multiple (10) DLD arrays on
a single chip will enable throughput of ~50 mL/hr.
Methods: Microfluidic devices are manufactured using standard
soft-lithography techniques. HeLaS3 cells expressing a Zn2+
biosensor flow into a Y-junction for rapid mixing with Zn2+
followed by introduction to a water-in-fluorinated-oil droplet
generator. Epifluorescence is collected on a home-built microscope
from at least two fluorescence interrogation points spatially and
temporally separated by a controllable delay. Sorting of droplets is
actuated by dielectrophoresis triggered by a sorting algorithm on a
microcontroller on the basis of signal intensity and the magnitude
of the time-resolved FRET ratio change.
Monday
13 June
Deterministic lateral displacement (DLD) arrays are microfluidic
devices that offer continuous-flow separation of particles suspended
in a fluid based on size and have been applied to separation of
large cells, such as cancer cells or leukocytes, from blood without
the background of erythrocytes, platelets, and plasma. Recently, a
method to inhibit clot formation in DLD arrays has allowed for
separation of 86% of PC3 cancer cells from 14 mL of blood in less
than one hour using a single DLD array [1]. DLD arrays have also
been applied to harvesting of leukocytes from small (~50 ȝL)
volumes of blood with a capture efficiency >90%, using typical
microfluidic flow rates (<10 ʅL/min per DLD array) [2]. Highthroughput separation of leukocytes from large volumes of blood is
more challenging than separation of cancer cells because
leukocytes are both smaller and more compressible. In this talk, by
novel post engineering, we demonstrate for the first time DLD
harvesting of leukocytes at high volumes and flow rates (~4 mL of
blood at ~100 ȝL/min in a single DLD array), high leukocyte yield
(~85%), and low erythrocyte contamination (< 0.02%).
Background: Förster resonance energy transfer (FRET)-based
genetically encoded fluorescent biosensors are immensely useful
biochemical tools for in vivo study, but often underperform with
respect to the resolution and kinetics necessary to accurately report
on the concentrations and dynamics of their analyte. In particular,
fluorescent protein FRET sensors for the detection of Zn2+ have a
less than 2-fold dynamic range in most cellular compartments, as
well as lacking the kinetics required to report on all biologically
relevant timescales (i.e. msec synaptic release and uptake of Zn2+ in
zincergic neurons). The optimization of these sensors is often timeintensive: hindered by the overwhelming combination of possible
amino acid substitutions that could lead toward improved range
and timing of a response. Conventional flow cytometry is not
designed for msec to sec time-resolved kinetic measurements. Here,
we present an instrument to screen and sort FRET-based sensors for
dynamic range and time-resolved response.
Sunday
12 June
1
1
Chemistry and Biochemistry, University of Colorado,
Boulder, CO, United States, 2Chemical Engineering,
University of Colorado, Boulder, CO, United States,
3
JILA/NIST, Boulder, CO, United States
Saturday
11 June
24
Microfluidic Post-geometry Design for HighThroughput (100 ȝL/min) Harvesting of Leukocytes
from Blood Using Deterministic Lateral
Displacement Arrays
Brett Fiedler1, Steven Van Buskirk2, Amy Palmer1, Ralph
Jimenez1,3
Special
Lectures
Conclusion: These results provide proof-of-principle evidence that
neuroblastoma tumor cells can be sorted from MNC preparations
with high recovery and purity. Thus, acoustophoresis is a promising
technology for PBPC processing, which provides the basis to
develop novel, label-free and improved tumor cells purging
procedures for future clinical use.
25
A Time-Resolved Microfluidic Flow Cytometer and
Sorter for FRET-Based Biosensors
Congress
Overview
with the waste. T-cell proliferation assay showed that the
proliferation capacity of sorted lymphocytes was not affected by the
acoustic sorting.
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Index
Abstracts
Despite its success in fundamental research, fluorescence activated
cell sorting (FACS) is facing some challenges in clinical applications
such as cell therapy or rare cell sorting, due to high cost, demand of
operator
proficiency and safety concerns with aerosol-induced
sample contamination. The unmet needs from clinical applications
motivated efforts to develop microfluidic sorting technologies.
Previously, we demonstrated the bubble jet flow sorting principle.
Here we will share our efforts on the high throughput microfluidic
fluorescence activated cell sorter (μFACS) with integrated optical
waveguides and bubble jet sorters in a multi-channel chip.
Technology: Within a few cm2 chip footprint, our μFACS device
consists of 5 parallel sorting channels. Three key components are
integrated into every channel: (a) a SiN optical waveguide network
through which light is coupled into the chip, subsequently
distributed to every channel and finally projected onto flowing cells
to generate fluorescence excitation, (b) a thin film metal heater
which fires a rapid liquid jet flow for cell sorting by nucleating
hundreds of micro vapor bubbles, and (c) a precise microfluidic
channel network that supports simultaneous cell flow focusing for
all 5 channels. The integration of the three key components enables
every single channel to operate as an independent high speed
automated FACS.
Results: The on-chip light coupling, distribution and focusing was
realized with a microfabricated SiN waveguide. With a high
precision coupling structure and waveguide, the light in-coupling
efficiency reached > 30% and transmission loss was < 1dB/cm. At
the measurement spot a dotted grating coupled out the light from
the waveguide in a broad, even beam that illuminated passing cells.
A jet flow cell sorting cycle took up to 100 μs, corresponding to
max. sorting rate of 10,000 cells/sec. Experimentally, the chip was
used to sort fluorescent beads at a speed of 4,000 cells/sec/channel
(or 20,000 cells/sec per chip). The performance of our μFACS
device was also validated on biological samples. In one test, CD14fluorescently labeled monocytes were sorted on chip with 90%
yield and 99% purity. Cell viability was confirmed by multi-marker
staining. In another test, rare MCF7 tumor cells were spiked into
peripheral blood mononuclear cells and sorted in the μFACS chip
with 91.6% yield and 58.6% purity.
Taken together, our results demonstrate the amenability of our
μFACS device to diverse applications, including those requiring
high throughput, yield and purity as well as rare cell sorting
applications. Additionally, our device has a number of advantages
over conventional FACS, such as no potentially hazardous aerosolformation, disposability and low cost, while offering comparable
sorting rates to conventional instruments.
The authors thank ERC (SCALPEL Consolidator Grant 617312) and
KU Leuven IOF (IOFHB/09/037) for financial support.
27
Generation of Stem Cell-Like Memory T Cells from
Naive Precursors for Adoptive Cell Transfer
Immunotherapy
Alessandra Roberto1, Veronica Zanon1, Karolina
Pilipow1, Enrico Lugli1,2
1
Laboratory of Translational Immunology, Humanitas
Clinical and Research Center, Rozzano (MI), Italy,
2
Humanitas Flow Cytometry Core, Humanitas Clinical and
Research Center, Rozzano (MI), Italy
Human CD8+ T memory stem cells (TSCM) are the least
differentiated memory T cell population in humans. These cells
have demonstrated superior reconstitution capacity and anti-tumor
immune responses compared to other naï ve and memory subsets
both in humanized mice and following adoptive transfer in
humans. However, their paucity in the peripheral blood limits
clinical applications. We improved recent method to generate large
numbers of these cells, which are suitable for the study of memory
T cell differentiation in humans as well as for adoptive T cell
transfer. We used polychromatic fluorescence-activated cell sorting
(FACS) to obtain highly-purified CD8+ naï ve T cell precursors
(CD45RA+CD45RO–CCR7+CD27+CD95–) which were subsequently
118
stimulated with anti-CD3/CD2/CD28 antibody-coated beads at
different bead:cell ratios, in the presence of different cytokine
cocktails. High-content phenotypic, functional and proliferative
analysis at 7 to 10 days following stimulation determined the
acquisition of the TSCM identity. We determined the following: i) IL7 and IL-15 in the culture are necessary to arrest differentiation and
induce the TSCM cell phenotype; ii) anti-CD2 must be removed from
stimulation, otherwise differentiating naï ve T cells will
preferentially acquire a central memory T cell phenotype in the
presence of the same cytokines; iii) a 0.5 bead:cell ratio is
necessary to induce the TSCM cell phenotype (i.e.,
CD45RA+CD45RO–), while increased stimulation (1:1) induce the
expression of CD45RO; iv) the final progeny of TSCM cells expanded
up to 100-fold the original number, depending on the donor; v) the
obtained TSCM cells were able to self-renew following IL-15
homeostatic stimulation and vi) preferentially engrafted
immunodeficient NSG mice following adoptive transfer when
compared to naturally-occurring CD45RO+ memory T cells.
Parallel experiments revealed that the same naï ve T cells
stimulated in the presence of anti-CD3/CD2/CD28 antibody-coated
beads plus IL-2 and IL-12 differentiate into CD45RA–
CD45RO+CCR7–CD27+/–CD95+ effector T cells. The obtained cell
populations can be further modified with lentiviral vectors
encoding shRNAs to identify molecular mechanisms of memory T
cell differentiation or adoptively-transferred in vivo to be tested in
preclinical models of immunodeficiency and anti-tumor immune
responses.
28
The IL-21 Signaling Pathway Is Enhanced in RA B
Cells and Has the Potential to Alter Development
and Cytokine Production in RA B Cells
Elizabeth Dam, Jane Buckner
Translational Research, Benaroya Research Institute, Seattle,
WA, United States
Background: B cells have been implicated in the development of
rheumatoid arthritis (RA) primarily through their autoantibody
production but also by their production of pro-inflammatory
cytokines and their actions as antigen presenting cells. IL-21 plays a
central role in B cell maturation and can induce antibody and
cytokine production by B cells. In this study, we examined the
response of B cells to IL-21 from RA subjects to determine if
alterations in this response contribute to perturbations in B cell
development and function in RA.
Methods: RA subjects were age, race and gender matched to
healthy controls. In addition, RA subjects were excluded if they
were on a biologic at the time of draw. Initial flow cytometry was
conducted utilizing surface staining for detection of the IL-21
receptor (IL-21R). Further studies utilized methanol based phosphoprotein staining to detect changes in pSTAT3 levels via flow
cytometry following IL-21 stimulations at various concentrations
and time points. To understand the molecular mechanisms involved
in the altered signaling in RA B cells, we examined mRNA
expression in B cell subsets with an RNA flow cytometry assay.
However, during our troubleshooting we observed that certain
conjugates bound the probes for detection of mRNA resulting in a
false positive detection of the probe. Once we excluded these
antibodies from our detection, we were able to observe mRNA
levels of IL-21R, SOCS1 and SOCS3.
Results: Flow cytometric studies of PBMC from a cohort of RA and
healthy subjects demonstrated an increase in the expression of the
IL-21 receptor (IL-21R) on RA B cells as compare to those of healthy
controls. Additionally, we observed a positive correlation between
the IL-21R surface expression and the phosphorylation of STAT3 in
response to IL-21. Utilizing RNA flow cytometry, we determined
that IL-21R mRNA levels were enhanced in RA B cells and
correlated with IL-21R protein expression. We were able to make
these observations comparing mRNA and protein levels in the same
cells allowing for a unique perspective on expression of the IL-21R.
When we measured SOCS1 and SOCS3 mRNA levels, which are
both negative regulators of the IL-21 signaling pathway, we
observed no changes in their expression. Analysis of the Akt
pathway demonstrated enhanced total FOXO1 protein expression
1
Background: Stroke-induced immunosuppression (SIIS) leads to
severe complications in stroke patients, however, functional
alterations of T lymphocytes are poorly described following acute
ischemic stroke (AIS).
1
School of Medicine, Cardiff University, Cardiff, United
Kingdom, 2Biostatus Ltd, Shepshed, United Kingdom,
3
Institute of Cancer Therapeutics, University of Bradford,
Bradford, United Kingdom, 4BioStatus Ltd, Shepshed,
United Kingdom
Index
119
Speaker/Author
Background: Tumour microenvironment hypoxia facilitates disease
progression and therapeutic resistance. Hypoxia sensors can be
used to explore critical changes in cell behavior in the hypoxic
tumour environment, such as checkpoint/proliferation modulation
and inhibition of cell death pathways. However, the hypoxic
experience of cells may be complex, resulting in heterogeneous cell
populations undergoing varying degrees of population expansion,
Poster Session
Abstracts
Oral Session
Abstracts
Conclusions: Kv1.3 channel inhibition might have beneficial
therapeutic consequences, since within hours after the insult, it
might prevent excessive tissue injury through the inhibition of CD8
cells, while at one week after the insult, it may improve the
Paul Smith1,2, Marie Wiltshire1, Klaus Pors3, Laurence
Patterson3,4, Rachel Errington1,4
Commercial
Tutorials &
Exhibits
Results: Our results indicate that the Ca2+ influx kinetics is altered
in the Th2 and CD8 subsets in AIS which may play a role in the
development of SIIS. Specific inhibition of Kv1.3 channels
decreased the calcium influx of cytotoxic CD8 cells in the 6 hour
group and that of immunosuppressive Th2 cells in the 1 week
group. The expression of Kv1.3 channels is also altered after AIS.
31
Profiling Cellular Experience of Hypoxic
Environments
Scholars &
Emerging
Leaders
Leaders
For evaluation a specific software developed at our laboratory,
FacsKin was used. Analysis was based on the calculation of a
double-logistic function. Parameter values were also calculated,
such as the AUC, which corresponds to the sum of [Ca 2+]cyt
increase, Maximum which reflects the highest amount of [Ca2+]cyt
and Time to reach maximum, representing how soon the peak is
reached.
Altered lymphocyte potassium channel inhibitory patterns,
regulators of calcium influx kinetics, might contribute to the
development of age-related changes of T cell function. The lower
level of activation of Th1 cells in the elderly upon the same stimuli
may play an important role in the increased susceptibility to
infections in this age group.
Poster
Session
Methods: We took peripheral blood samples from 12 AIS patients
within 6 hours and one week after the insult and 12 age-matched
healthy individuals. Peripheral blood mononuclear cells (PBMCs)
were isolated, divided to 3+1 vials and were incubated with the
following conjugated antibodies: anti-CD4 PE-Cy7, anti-CD8 APCCy7, anti-CXCR3 APC and anti-CCR4 PE. The extra vial was also
incubated with anti-Kv1.3 channel FITC. PBMCs were loaded with
Ca2+ sensitive Fluo-3 and Fura Red dyes and two vials were treated
with selective blockers of Kv1.3 and IKCa channels, while the third
was used as control. Measurements were conducted on a BD
FACSAria flow cytometer. At the beginning a 2 min baseline of the
cytoplasmic Ca2+ level was recorded, then PBMCs were activated
by the addition of phytohemagglutinin and recording of cell
fluorescence data was continued for 15 min in a kinetic manner.
AUC of calcium influx was higher in Th1 cells of adults, however,
its extent decreased again with aging. Importantly, these changes
were not detected in Th2 cells, where the pattern of calcium influx
kinetics is similar throughout all investigated age groups. AUC and
Max values were also higher in CD8 cells of adults compared to
children. MGTX had a more pronounced inhibitory effect on
calcium influx in Th2 cells, while in Th1 cells the same was true for
TRAM in the 25-40 ys and 45-55 ys groups. Calcium influx of CD8
cells were inhibited to a similar extent by both applied inhibitors in
these groups, and had no effect in the elderly.
Wednesday
15 June
We aimed to characterize Ca2+ influx kinetics in major lymphocyte
subsets (CD4, Th1, Th2, CD8) in AIS patients without infection 6
hours and one week after the CNS insult. We also assessed the
sensitivity of the above subsets to specific inhibition of the Kv1.3
and IKCa1 lymphocyte K+ channels.
We isolated peripheral lymphocytes from blood samples taken from
healthy individuals of the above groups (n = 9, 9, 10, 10, 12,
respectively). We evaluated calcium influx kinetics following
activation with phytohemagglutinin in Th1 (CD4+ CXCR3+), Th2
(CD4+ CCR4+) and CD8 cells using Fluo-3 and Fura Red calcium
sensitive dyes. We also assessed the sensitivity of the above subsets
to specific inhibition of the Kv1.3 and IKCa1 potassium channels by
margatoxin (MGTX) and triarylmethane (TRAM), respectively. Flow
cytometry analysis was performed in a kinetic manner (BD
FACSAria). Data acquired from the measurements were evaluated
using an algorithm based on the calculation of a double-logistic
function for each recording (the FacsKin method). Specific
parameter values describing each function, such as Area under the
curve (AUC), Maximum value (Max) and Time to reach maximum
(tmax) were also calculated and used to compare individual
measurements in an objective manner.
Tuesday
14 June
The transient increase of the cytoplasmic free calcium level [Ca2+]cyt
is a key signal transduction mechanism in the process of
lymphocyte activation. Kv1.3 and IKCa1 lymphocyte potassium
channels are important regulators of the maintenance of calcium
influx. Thus the relationship between the influx of calcium and the
efflux of potassium makes the activation and cytokine production of
T lymphocytes sensitive to pharmacological inhibition of Kv1.3 and
IKCa1 channels.
Adaptive immunity and T cell function are affected by aging.
Calcium influx patterns, regulated by Kv1.3 and IKCa1 potassium
channels, influence T cell activation. We aimed to compare
calcium influx kinetics in CD8, Th1 and Th2 cells in human
peripheral blood samples obtained from five different age groups
(cord blood, 10-15 ys, 25-40 ys, 45-55 ys, 60-75 ys).
Monday
13 June
1st Department of Obstetrics and Gynecology,
Semmelweis University, Budapest, Hungary, 2Department of
Neurology, Szent János Hospital, Budapest, Hungary,
3
Semmelweis University, Budapest, Hungary
1
First Dept of Obstetrics, Semmelweis University, Budapest,
Hungary, 2Birmingham Women's Hospital, Birmingham,
United Kingdom, 3Second Dept of Obstetrics, Semmelweis
University, Budapest, Hungary
Sunday
12 June
Anna Bajnok1, András Folyovich2, Csaba Orban3,
Gergely Toldi1
Gergely Toldi1,2, Szonja Kollár3, László Berta1, János Rigó
Jr1
Saturday
11 June
29
Kv1.3 Lymphocyte Potassium Channel Inhibition as a
Potential Novel Therapeutic Target in Acute
Ischemic Stroke
30
Impact of Aging on Calcium Influx and Potassium
Channel Characteristics of T Lymphocytes
Special
Lectures
Conclusions: Together these findings suggest a mechanism by
which the IL-21/IL-21R pathway may be contributing to alterations
in B cell development and function in RA and point to a role for
therapeutics targeting this pathway in these individuals.
inflammatory response through the inhibition of Th2 cells, thus
reducing the clinical consequences of SIIS.
Congress
Overview
in RA B cells with high IL-21R expression. This suggests that both
STAT3 and Akt signaling pathways are enhanced in RA B cells that
express elevated IL-21R levels. We found that in vitro differentiation
of naï ve B cells to plasma cells was enhanced in RA B cells and
that these plasma cells are IgM+ and produce increased quantities
of IL-6.
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Methods: The in vitro effects of hypoxia (pO2 10-30 mm Hg; 1-3%
oxygen) on metabolism were studied for 4-5 day exposure periods.
A549, U-2 OS and a panel of cell lines were analyzed by confocal
imaging and flow cytometry for cell cycle redistribution, arrest and
viability using DRAQ5™, CyTRAK Orange™ and DRAQ7™
profiling respectively. HypoxiTRAK™ activation (BioStatus Ltd, UK)
to its intracellular far-red fluorescent product (Ex488nm or
Ex633nm/Em>695nm) was assessed by flow cytometry and HPLC
analysis.
Results: Irreversible conversion of HypoxiTrak™ under hypoxia to a
persistent intracellular metabolite was confirmed. Normoxic A549
cells showed minimal toxicity towards HypoxiTrak™ while under
hypoxia there was a dose- and pO2 level-dependent cell cycle
arrest. Long-term arrest correlated with the accrual and persistence
of the far-red fluorescent metabolite in A549 cells under hypoxia.
3D hypoxic lung cell line cultures revealed locations of hypoxic
cellular experiences. Reduced activation in U-2 OS cells correlated
with a reduced arrest. Results from a cell line panel suggest that
HypoxiTrak™ can reveal differences in the modulation of cell death
pathways through its unique dual action.
32
Detection of HIV RNA Expression in Peripheral
Blood from HIV-Infected Study Participants by
RNAFlow Cytometry
Christopher Baker, Alice Tan, Joseph (Mike) McCune,
Elizabeth Sinclair, Jeffrey Milush
Index
Abstracts
Oral Session
Abstracts
Scholars &
Emerging
Leaders
Conclusion: HypoxiTrak™ can report the both the degree of
hypoxic experience, hypoxic fraction and the proliferative
contribution of hypoxic cells within a heterogeneous cell
population. This sensor provides a new tool for the quantification of
hypoxic fractions within in vitro cellular systems qualified for CYP
expression. The information gathered informs models for
molecular-targeted therapies that have to account for dynamic
changes in hypoxic fractions and their contributions to tumour
progression. (PS, LP & RE are directors of BioStatus Ltd).
Commercial
Tutorials &
Exhibits
Poster
Session
confounding conventional biomarker analysis. We propose an
approach to the analysis of the dynamics of such cell populations
involving the use a hypoxia sensor that, upon activation in hypoxic
environments,
can yield a persistent fluorescent reporter product,
quantifying the recruitment of a hypoxic fraction over extended
periods, while the product simultaneously halts cell proliferation in
activating cells. The new probe HypoxiTRAK™ is non-toxic but can
be activated by the cytochrome P450s monooxygenase family of
proteins (CYPs) under hypoxia. Importantly, the degree of specific
CYP expression can vary in different cell line models and tumour
tissues. Given that the target CYPs for HypoxiTRAK™ include those
expressed in extra-hepatic tissues such as the lung, we have used
the human A549 (p53 wt) lung cancer to profile the dynamics of
HypoxiTRAK™ activation and the impact of activation on the cell
cycle. The concept was further tested in human U-2 OS
osteosarcoma (p53 wt) cells, which show reduced CYP expression
compared to A459, to explore the linkage between hypoxic
experience and proliferation.
Medicine- Division of Experimental Medicine, UCSF, San
Francisco, CA, United States
Despite the success of antiretroviral therapy (ART) in reducing
replicating Human Immunodeficiency Virus (HIV) to below
detectable levels, a population of cells that contain integrated HIV
DNA has proven to be highly resistant to decay over time and
elimination by aggressive ART. This very small reservoir of cells is
capable of rapidly producing a rebound in viremia if therapy is
interrupted. Measurement of the HIV reservoirs that persist during
ART is one of the most significant challenges facing the current HIV
cure efforts. Understanding how many HIV-infected cells exist in
the body and how many of these cells contain infectious virus is
key to designing strategies to decrease the size of the infectious
virus reservoir, to monitoring the effectiveness of these strategies,
and to make progress towards a cure. Current approaches designed
to measure the latent reservoir either do not provide single cell
resolution or are variable, laborious, and low throughput. To
address these limitations, we have developed a flow cytometry-
120
based assay that detects HIV RNA (referred to as RNAFlow) at the
single cell level in primary blood specimens. RNAFlow detection of
HIV RNA demonstrated a high concordance in an in vitro infection
system using a red fluorescent protein reporter virus in primary
CD4+ T cells. In order to detect the rare HIV RNA+ cells in primary
peripheral blood mononuclear cells (PBMC) from HIV-infected
study participants, we undertook numerous optimization steps to
improve the sensitivity and specificity of this technology. Currently,
the assay provides for a maximum 60,000-fold amplification of the
HIV RNA signal using RNA probes that span the entire 10,000 base
pair HIV genome. This results in 2.5-3 logs of fluorescence between
HIV RNA negative and HIV RNA+ cells. We can detect HIV RNA+
cells in stimulated PBMC from HIV-infected study participants with
low-level viremia, with little or no background found in HIV
negative controls. We determined the HIV RNA+ cells are CD4+ T
cells and that HIV RNA expression inversely correlates with CD4
expression on the cell surface. Current HIV cure strategies aim to
eradicate the latent viral reservoir using agents to shock the latent
virus into expression such that the immune system and ARTs can
kill the HIV producing cell. We have screened seven compounds
under different concentrations and combinations and observed
direct T cell receptor stimulation provides the optimal conditions to
induce HIV expression from latently infected cells. Importantly, we
can also measure the amount of infectious virus present in PBMC
before stimulation by inducing HIV expression in the presence and
absence of ARTs. In summary, we have developed a sensitive, highthroughput flow cytometry-based assay capable of detecting HIV
RNA in primary cells from infected study participants. This assay
will provide a robust platform on which to screen potential latency
reversing compounds to purge the HIV reservoir in infected
persons.
33
Increasing the Sensitivity of Functional Flow Assays
by Magnetic Pre-enrichment of Antigen-Specific
Lymphocytes
Elke Bergmann-Leitner1, Kevin Beck2, Fouzia Farooq1
1
Malaria Vaccine Branch, WRAIR, Silver Spring, MD,
United States, 2Miltenyi Inc, Silver Spring, MD, United
States
The lack of known immune correlates of protection has been a
major hurdle for the development of effective vaccines against
many diseases. Improved readout methods and advanced
technologies for the characterization of rare antigen-specific
responses induced by protective vaccines are urgently needed to be
able to identify correlates of protection. However, the analysis of
rare antigen-specific cells by flow cytometry is problematic and
typically requires large sample sizes in order to exceed the
detection threshold and achieve statistical significance of the
measured response. Previously, we and others demonstrated that
pre-enrichment of antigen-specific cells based on activation
markers such as CD154 can dramatically increase the signal to
noise ratio and serve as the basis of flow cytometric panels with a
high sensitivity. Originally, the pre-enrichment based on magnetic
bead separation could only be applied to samples which were
stained for cell surface markers. In an effort, to broadly extend this
technology to functional assays such as the detection of cytokines
by intracellular staining of cultured cells - an analysis commonly
employed for the characterization of vaccine-induced T cell
responses - we determined the feasibility of enriching fixed and
permeabilized samples. This experimental series included
polyclonally- as well as antigen-specifically stimulated CD4+and
CD8+ T cells. The data demonstrate that magnetic bead enrichment
(either manually or automated) can be used to significantly increase
the sensitivity of intracellular staining assays. Despite the fixation
and permeabilization no artefacts were induced and the proportion
of responder cells within the lymphocyte population was not
skewed. This new application is a highly useful tool for the in-depth
characterization of pathogen-specific cells and holds the promise to
assist in the identification of immune mechanisms mediating
protection against infectious diseases.
extract salient morphological features, and classify cell types in an
objective and precisemanner.
German Rheumatism Research Center (DRFZ) Berlin, Berlin,
Germany
Hartland Jackson1, Denis Schapiro1, Zsuzsanna Varga2,
Bernd Bodenmiller1
Commercial
Tutorials &
Exhibits
Conclusions: Applied to a clinical setting, highly multiplexed and
quantitative IMC improves patient classification and as such
promises that IMC could support pathologists in the future.
37
Identification and Classification of the Malaria
Parasite, Plasmodium falciparum Using Imaging Flow
Cytometry
Ziv Porat1, Elya Dekel2, Anna Rivkin2, Neta Regev2
Index
121
Speaker/Author
Malaria is the most devastating parasitic disease caused by the
unicellular protozoa of the Plasmodium genus, and is responsible
up to a million deaths each year. Plasmodium falciparum (Pf) and
P. vivax are responsible for most clinical cases of malaria in
humans. Pf life cycle is complex, and the transmission of the
parasite between humans via mosquitos involves a remarkable
Poster Session
Abstracts
1
Biological Services, Flow Cytometry Unit, Weizmann
Institute of Science, Rehovot, Israel, 2Biological Chemistry,
Weizmann Institute of Science, Rehovot, Israel
Oral Session
Abstracts
Scholars &
Emerging
Leaders
With Complex Morphology by Imaging Flow Cytometry Assessment
of morphology is critical for identification of different cell types. It
also plays a vital role in evaluating the health of the cell. However,
accurately classifying complex morphologies such as sickle cells,
diatoms, and spermatozoa can be a challenge due to the
heterogeneous shapes within a cell. To overcome this challenge,
we used multispectral imaging flow cytometer, ImageStream, to
acquire brightfield, side scatter, and fluorescent images of various
cells. We developed computational framework for automatically
analyzing thousands of cellular images to accurately identify
different cellular and subcellular components. Our novel
framework performs multispectral segmentation by iteratively
optimizing model fitting of different cellular parts. Using these
accurate segmentation masks for cells and subcellular parts,
advanced shape features such as contour curvature and bending
score can be extracted to detect complex morphologies such as
fragmented or detached cells, stretched or pointed cell boundary
etc. Using the segmentation algorithms, we created automated
classifiers to rank and select most discriminative shape based
features for classifying the cells into subpopulations. We
demonstrated the efficacy of our proposed framework on boar
semen samples by accurately classifying various sperm morphology
defects. Taking advantage of the ability of imaging flow cytometers
to acquire images at high speed and resolution and using the
proposed analysis framework with specific statistical modeling, we
observed above 90 percent sensitivity and specificity in detecting
abnormal morphology, thus demonstrating our ability to accurately
Poster
Session
MilliporeSigma, Seattle, WA, United States
Wednesday
15 June
Alan Li, Shobana Vaidyanathan
Results: IMC precisely reproduced and quantified current clinical
markers and in combination with an additional set of nearly 40
antibodies IMC enhanced the identification of unique cell
phenotypes, cell morphologies, cellular microenvironments thereby
elucidating inter and intra-tumour heterogeneity in great detail.
Correlating these cell types and tissue features with clinical data
identified cell populations that correspond to specific clinical
outcomes and increased patient risk. In direct comparison with
current clinical measurements, IMC improved patient stratification
and risk assessment. In summary, we highlight that IMC reproduces
current pathology scoring and ultimately improves patient
classification by providing quantitative information of dozens of
biomarkers.
Tuesday
14 June
35
Multispectral Shape Characterization and
Classification of Cells
Methods: Here, we used IMC combined with a novel
computational pipeline called miCAT to measure the single cell
features and common cell interactions of hundreds of breast cancer
patient samples. Breast cancer tissues representing the full range of
grade, subtype and metastatic progression with multiple cores from
each patient were organized on tissue microarrays. Tissue
microarrays were stained for current clinical markers; common
oncoproteins; epithelial, stromal and immune cell types; and
phospho-signaling events in one panel consisting of 44 antibodies
and measured by IMC. Multidimensional images were segmented
to produce spatially resolved single cell data. These features and
those of cellular microenvironments were correlated with clinical
parameters using miCAT.
Monday
13 June
Collectively, usage of silver NPs extends the analytical capacity of
mass cytometry staining panels by one, prospectively two,
additional parameters, well suitable for cellular targets of low
abundance.
Background: Current breast cancer classification lags far behind our
understanding of the molecular pathology of the disease. Pathology
scoring of breast cancer and subsequent patient risk assessment
depend mainly on the visual assessment of a few tissues stains such
as Hormone Receptors or HER2. Markers are measured
individually, not in relation to each other, and not in a quantitative
manner and cannot discern the molecular changes or cell types that
result in diverse tumor heterogeneity. Imaging Mass Cytometry
(IMC) addresses these problems by combining classic
immunohistochemistry techniques with tissue laser ablation and
mass cytometry to simultaneously quantify up to 50 markers with
sub-cellular resolution.
Sunday
12 June
For this reason, we explored the application of elemental silver
nanoparticles (NPs) for reporting specific cellular targets on human
lymphocytes in a mass cytometry assay. We demonstrate the
successful use of streptavidin-coated silver NPs of different sizes for
the specific detection of human cell surface antigens such as CD4,
CD8, CD45RO, CD16 and CD244 via biotinylated primary
antibodies. Signal intensities (SI) elicited by cell-bound silver NPs
were sufficient to enable detection of the low abundant antigen
CD25 at an SI of ~200 on T cells in comparison to an SI of ~20 by
lanthanide-based detection. Further, silver NPs were compatible
with standard mass cytometry staining protocols for PBMC and
WBC and showed low background signals in control assays. The
detection of silver NPs, containing both 107Ag and 109Ag at natural
abundance, does not overlap or interfere with the detection of any
other metal isotopes and further development may lead to the
application of isotopically purified silver NPs.
1
Institute of Molecular Life Sciences, University of Zurich,
Zurich, Switzerland, 2Institut für Klinische Pathologie,
University Hospital Zurich, Zurich, Switzerland
Saturday
11 June
The development of mass cytometry (CyTOF® technology) has
pioneered a new era of multiparametric single-cell analysis. Cells
and cellular networks can now be studied at an unprecedented
depth and complexity. Current instrumentation allows for the
theoretical detection of up to 135 parameters, but the availability of
metal tags suitable for labeling specific probes limits analyses to
~40 parameters, curtailing exploitation of the full analytical
capacity of mass cytometers.
Special
Lectures
Axel Ronald Schulz, Silke Stanislawiak, Sabine
Baumgart, Andreas Radbruch, Andreas Grützkau, Henrik
E. Mei
36
Multi-parametric, Single Cell Measurements by
Imaging Mass Cytometry Improves Classification of
Breast Cancer Patients
Congress
Overview
34
Application of Silver Nanoparticles for the Specific
Detection of Cellular Antigens in Mass Cytometry
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Index
Abstracts
series of morphological transformations. The human blood stage is
responsible for all disease pathology; the parasites undergo asexual
multiplication inside the red blood cell (RBC) where the parasites
mature
through the ring, trophozoite and schizont stages. During
the asexual cycle a small portion of the parasite population develop
into sexual forms (gametocytogenesis), thus ensuring parasite
transfer to the mosquito host.
Flow cytometry (FC) methods were previously shown to detect PfiRBCs, in live or fixed cells, using DNA stain Hoechst (HO) and
RNA stain, Thiazole Orange (TO). Here, by using Imaging Flow
Cyotmetry (IFC) we demonstrate a clear detection of each of the PfiRBC life stages. This technique allows multi-channel, high
resolution imaging of individual parasites, as well as detailed
morphological quantification of Pf-iRBCs cultures, and be
combined with additional stainings. We developed an improved
classification probing of the blood stages, both asexual and sexual.
This method provides an accurate quantification and robust
measurement of parasitemia profile and can serve as a valuable tool
in malaria research and drugs screen assays.
38
Multiplex Intravital Detection of Germinal Center in
Murine Lymph Node with Wave-Mixing Two-Photon
Microscopy
Asylkhan Rakhymzhan1, Ruth Leben1, Andreas Acs2,
Thomas Winkler2, Robert Guenther1, Peggy Mex1, Laura
Oehme3, Zoltan Cseresnyes1, Anja Hauser3,4, Raluca
Niesner1
1
Biophysical Analytics, Deutsches Rheuma
Forschungszentrum, Berlin, Germany, 2Division of
Genetics, Nikolaus-Fiebiger-Zentrum fuer Molekulare
Medizin, Erlangen, Germany, 3Immunodynamics,
Deutsches Rheuma Forschungszentrum, Berlin, Germany,
4
Charité – University Hospital, Berlin, Germany
Background: Immunity is a highly concordant system where many
different cellular and molecular players are involved. Modern
imaging techniques apply multiplex detection to visualize over 30
colors of cellular labels per ex vivo experiment. During in vivo
experiments the time constraints are very strict, thus making it very
challenging to detect more than three spectrally distinct
chromophores [1]. As a bright illustration, germinal center (GC)
immune reaction is a complex process that requires more than four
colors to be detected simultaneously. The GC immune reaction
represents all aspects of an adaptive immune response, including
highly specialized communication between proliferating antigenspecific B cells, T follicular helper cells and the resident follicular
dendritic cells. In order to study the mechanisms of such complex
processes in vivo, it is necessary to simultaneously observe all these
cells that have been labeled by various fluorophores.
Methods: We carry out experiments using two-photon laser
scanning microscopy combined with NIR and IR femtosecond
lasers. Using wave-mixing of the laser pulses, achieved by
spatiotemporal synchronization of the component laser beams, we
produce three separate light sources that can excite multiple sets of
fluorophores with improved efficiency. Consequently, the wavemixing excitation technique allows us to extend the number of
simultaneously observable fluorophores to six. Multicolor
fluorescence detection is often hindered by spectral bleed-through.
In order to reduce this effect, here we propose a spectral unmixing
method based on the analysis of information recorded by all
fluorescence channels, thus defining so-called “fingerprints”. Our
method differs from conventional unmixing approaches, where
only the reference channel signal is used for such correction.
Results and Conclusions: Here we demonstrate the simultaneous
intravital detection of six chromophores (Hoechst, eGFP, Kusabira
Orange, CMTPX Red, QDots 655, and Atto680) in murine lymph
nodes. Each chromophore labels a distinct cell type, which have
specialized functions during the GC immune reaction. In
preparation of the multiplex intravital experiments, we have
performed preliminary six color measurements using an in vitro
122
system of isolated murine splenocytes, as well as an ex vivo
experiment with explanted murine lymph nodes. We also propose a
testing system of five fluorescent proteins (FP), CFP, eGFP,
mOrange2, mKate2, and NIR, expressed by HEK cells. This testing
system of multiple sets of FPs can serve as a preliminary step for
transgenic mouse imaging, such as the Brainbow 3.0 mouse. Our
approach of multiplex detection will be able to shed light on many
important aspects of immune processes, which currently cannot be
addressed using traditional confocal and flow cytometry
techniques.
[1] P. Mahou; M. Zimmerley; K. Loulier, et. al., "Multicolor twophoton tissue imaging by wavelength mixing", Nat. Methods, vol.9,
No.8, 815-818 (2012)
39
Characterization and Standardization of Cytometers
for EV Analysis
Joanne Lannigan1, John P. Nolan2
1
2
Scintillon Institute, San Diego, CA, United States
Commercial flow cytometers designed to measure lymphocytes are
challenged to measure Extracellular Vesicles (EVs), which are 100
times smaller and 10,000 to 1,000,000 times dimmer than cells. In
addition, with a wide variety of instrument designs, assay
approaches, and measurement sensitivity, it can be difficult to
compare results between instruments, assays, and labs.
Recently three societies; International Society of Extracellular
Vesicles (ISEV), ISAC, and the International Society of Hemostasis
and Thrombosis (ISTH), joined forces to begin to address some of
major issues surrounding measuring EVs. This workshop is intended
to address the issues of instrument characterization and
standardization as part of ISAC’s contribution to the tri-society
initiative. Key questions to be addressed are: What methods are
available for characterizing instrument and assay performance?
What are the benefits/advantages of different approaches for
determining instrument and assay sensitivity? How can we begin to
report results in a way that is meaningful and standardized across
multiple labs? What tools/standards do we need in order to move
forward in making these efforts economical and easier to do?
Discussions will include what is currently available, what is being
done by those currently measuring EVs, and what do we need to do
this better.
40
Therapeutic Cell Sorting
Grace Chojnowski1, Christopher Choi2, Christopher
Wiwi3
1
Flow Cytometry and Imaging Core Facility, QIMR
Berghofer Medical Research Institute, Brisbane, Australia,
2
Rosewll Park Cancer Institute, Buffalo, NY, United States,
3
Celgene Cellular Therapeutics, Warren, NJ, United States
Intended Audience: Clinicians, core managers and clinical
researchers who are involved in development of cellular therapy
and immunotherapy to help in the treatment of diseases such as
leukaemias, graft versus host disease and diabetes.
Problem Focus and Key Questions: Fluorescent Activated Cell
Sorting has been a technology that has advanced medical research
over the last 6 decades. The increasingly complex phenotypic
description of cell populations has made high-speed droplet sorting
the cell selection method of choice in many research settings. There
is significant promise to utilizing specific cell populations for
therapeutic use but high-speed droplet sorting brings a series of
challenges when used in the clinic. The difficulties in clinical cell
sorting typically concern (1) sample throughput, i.e. processing
time, (2) sterility and safety, (3) ease-of-use (4) obtaining feasible
cell numbers of viable cells and (5) cost. A variety of technologies is
available to address the limitations in cell sorting but no single
technology addresses all. Progress towards clinical cell sorting
41
SRL Workshop: Communicating Up and Down
1
2
Michele Black , Rachael Walker
2
University of Washington, Seattle, WA, United States,
Babraham Institute, Cambridge, United Kingdom
1
Specifically, the workshop will focus on a few key areas: tools for
scientific data sharing and dissemination and how they are being
used in the context of automated data processing, traditional and
new data representation models in flow and image cytometry, as
well as statistical measures of quality and measures of effect size
used in design and optimization of singe-cell phenotypic assays and
screens.
Over the last decade the field of single-cell analysis has become
progressively more dependent on access to computing facilities,
data sharing infrastructure, use of databases, and implementation of
systems for data munging/wrangling, curation, annotation, quality
control and mining. Recent discussions regarding reproducibility of
biological research have brought to the forefront the issues of
quality control, statistical analysis, and scientific robustness.
Therefore, the topics of this workshop are aligned with the
requirements to implement scientific data management, improve
reporting of scientific findings, and provide public access to
research results, as set by federal funding agencies (AHRQ, NIH,
FDA, NOAA, USDA and others).
123
Index
Desired Outcomes: The presenters will demonstrate and discuss the
key elements the single-cell data analysis ecosystem (experimental
design, data management, communication of analysis), best current
Speaker/Author
In this workshop, we aim to demystify the concept of single-cell
data analysis pipeline, discuss what informatics tools exist and how
they can be employed in day-to-day practice, and advise how to
concisely report reproducibility/quality of implemented assays. The
workshop will be formatted with four brief presentations that are
composed of 1) introduction 2) demonstration of an informatics tool
or a statistical method, 3) discussion with the participants, and 4)
conclusion. Presentations will be brief, limited to approximately 1015 minutes. Five minutes before the workshop ends, the organizers
will restate the covered issues, recap the conclusions and outline
future action items. These will be summarized and posted on-line.
Poster Session
Abstracts
Agenda: Dr. Vera Donnenberg will give a brief overview of the
problem: Identification of cellular components within
heterogeneous human tumors. Dr. Albert Donnenberg will briefly
present an example of high dimensional flow cytometry on human
Format/Agenda: The reliance on large scale experiments has
greatly increased the role of data science (a.k.a. big data, data
mining, machine learning, pattern recognition, etc.) in biological
and clinical research. State-of-art single cell analysis, whether
performed by flow or image cytometry (high-content screening)
employs a plethora of informatics and statistics tools ranging from
various visualization methodologies, through feature selection and
discovery, linear and nonlinear dimensionality reduction, to
unsupervised and supervised learning.
Oral Session
Abstracts
Overview: Human tumors are complex and heterogeneous:
epithelial, mesenchymal, stromal and perivascular-endothelial and
immune cells can be observed in both metastatic lesions and
primary tumors. This workshop will consist of a survey of current
research applications with emphasis on flow-based proteomic
marker analysis, cell subset quantification, immuno-fluorescent
imaging, tissue culture and in vivo xenograft generation and cell
sorting for molecular characterization. The discussion will be
accompanied by practical suggestions for disaggregation of tissues
into single cell suspensions, the technical pitfalls encountered when
performing multi-dimensional flow cytometry, the use of supervised
classification to extract markers from cytometric data and analytical
strategies for multivariate data where number of samples is smaller
than the number of measured parameters. The workshop will also
address data display and how to clearly and parsimoniously convey
information generated from cytometric analysis.
Highlight challenges and solutions for quality assurance and
scientific reproducibility in cytometry (flow and imaging) assay
development
Commercial
Tutorials &
Exhibits
Cardiothoracic Surgery, Univ of Pittsburgh, SOM,
Pittsburgh, PA, United States, 2Medicine, Univ of Pittsburgh,
SOM, Pittsburgh, PA, United States, 3Institute of Molecular
Life Sciences, University of Zurich, Zurich, Switzerland
Educate the community on access to free and paid tools for data
visualization, analysis, and rapid scientific communication
Scholars &
Emerging
Leaders
Vera Donnenberg1, Albert D. Donnenberg2, Bernd
Bodenmiller3
Give an overview of the infrastructure and current ecosystem for
sharing single-cell data analysis and algorithms (e.g. scientific
workflow as well as public and private repositories)
Poster
Session
42
Tumor Heterogeneity
Problem Focus and Key Questions:
Wednesday
15 June
By the end of the workshop attendees will have ideas on which
communication methods that are most effective depending on the
personnel involved and nature of the issue to be resolved.
Intended Audience: anyone working with tons of flow data,
including flow and image cytometry practitioners, clinical flow
cytometrists, high-content screening system operators, as well as
existing and potential computational collaborators of this target
audience
Tuesday
14 June
The scenarios will include dealing with communication via live
dialogue, email or written communications and situational
examples that are common in laboratory environments where
communication styles can impact the outcome in a positive or
negative way.
Purdue University, West Lafayette, IN, United States,
Cytobank, Inc., Mountain View, CA, United States,
3
Stanford University, Stanford, CA, United States
2
Monday
13 June
This workshop is aimed at all Shared Resource Laboratory (SRL)
staff and will focus on common issues that require effective
communication to resolve. How communication is approached
can be dependent on the hierarchy of the parties involved. The
workshop will make use of the collective experience of the
audience and will involve group work. Scenarios will be discussed
within groups on how to best deal with situations including:
Communicating with users, conversations with your core staff to
ensure they’re providing consistent service, how to handling
complaints and how to deal with staff issues. The workshop will
also discuss methods for effectively discussing conflict with other
members of the SRL staff, when and how to get support of your
supervisor.
1
Sunday
12 June
1
Bartek Rajwa1, Tiffany J. Chen2,3
Saturday
11 June
Desired Outcomes: The workshop is intended to find concrete
answers to the four key questions specified above.
43
Mining and Modeling of Biomedical Data: Issues,
Approaches and Building Consensus
Special
Lectures
Format/Agenda: The workshop will be formatted with a few brief
presentations that are composed of 1) the need/utilities for
therapeutic cell sorting2) technology presently available and what
is still needed, 3) accreditation and regulations internationally, 4)
discussion and conclusion.
tumor tissue. Dr. Bodenmiller will present examples of single cell
and tissue mass cytometry. These short presentations will be
followed by a plenary discussion and Questions and Answers from
the audience.
Congress
Overview
requires a reassessment of traditional approaches to integrate
technologies into a device that suits the workflow of a clinical
environment. There are also different national and international
regulations governing clinical GMP sorting which also need to be
addressed.
Congress
Overview
practices in data sharing, visualization, and representation, and a
practical list of tools and resources under each category. The
attendees will have the opportunity to share their own experiences,
exchange
ideas, and suggest other useful tools. Copies of the
agenda will be published on-line or available at the door for
everyone to pick up as they come in.
Special
Lectures
44
Instrument PMT Voltage Optimization for Maximum
Sensitivity
Wednesday
15 June
Tuesday
14 June
Monday
13 June
Sunday
12 June
Saturday
11 June
Anna Brooks1,2, Simon Monard3
School of Biological Sciences, University of Auckland,
Auckland, New Zealand, 2Maurice Wilkins Centre,
Auckland, New Zealand, 3Cytometry, Walter & Eliza Hall
Institute, Parkville, Australia
It is widely agreed that PMT voltage optimization is essential to get
the best performance from flow cytometers. Over the years a
number of flow cytometry experts have stressed the importance of
optimizing machine settings and have proposed some protocols to
achieve this. However, very few have demonstrated how to do this
practically, or indeed addressed any caveats. It’s a bewildering area
for newcomers and confusing even for veterans. We will present
data comparing the outcome of each of these the methods (multipeak beads, antibody stained beads, antibody stained cells, etc) to
determine which methods are appropriate for optimizing voltages
and whether any of these have cell type-specific caveats. In
addition, a survey will be circulated to the flow cytometry
community to determine what is currently practiced, and the results
discussed with workshop participants. Together with the data
presented, we hope to determine whether a pragmatic, consistent
and practical method for voltage optimization can be achieved and
discuss ways this can be effectively disseminated to the wider flow
cytometry community.
Issues to be discussed:
Most flow cytometers have adjustable PMT voltages, there is little
agreement on how PMT voltages should be set. Can a consensus be
made?
Poster
Session
Voltage balancing: Is there any point?
Are bead based methods an appropriate surrogate for all cell types?
Commercial
Tutorials &
Exhibits
Scholars &
Emerging
Leaders
How accurate are bead based methods for maximizing stain index
of dim markers across all channels?
Oral Session
Abstracts
The output from this workshop will be a starting point for
establishing a best practice database for minimal-impact cell sorting
across a range of cell and instrument types.
46
Systems for Simplifying Core Management
Robert Salomon1, Rui Gardner2, Michael Thomson3
1
1
Why is it necessary to use optimized voltages?
Index
Abstracts
Association of Biomolecular Resource Facilities (ABRF) Flow
Cytometry Research Group, whose participants have been
investigating this question using a number of approaches.
Can the same approaches be used with autofluorescent cells as for
lymphocytes?
Garvan Institute of Medical Research, Sydney, Australia,
Inst Gulbenkian Ciencia, Oeiras, Portugal, 3St Vincent's
Institute of Medical Research, Fittzroy, Australia
2
Effective management of an SRL is a complex task that can be
simplified by designing and implementing systems to organize and
automate routine processes. These systems should be designed
around the overall goals of the SRL and the needs of its user. Welldesigned systems will not only simplify management, they will
allow identification of future needs of the SRL while providing for
the needs of the users and assist in facilitating high quality science.
During the workshop we will discuss key areas of an overall SRL
management system that may include but not limited to:
1) Scheduling (including rules for managing resources and booking
systems)
2) Cost recovery and finance tracking
3) Reporting (including ways of projecting future needs)
4) Data integrity (including system for automatic data QC)
5) User feedback and benchmarking
6) Facilitating collaboration and encouraging new users/projects
The workshop will 1) Identify what systems and respective key
features are most required by a) SRL staff and b) SRL users, 2)
Identify which systems are most commonly overlooked by the SRL
and 3) Explore solutions to address the limitations of current
solutions.
This will be an active workshop and audience participation is
encouraged.
47
Flow Cytometry for Cohort, Longitudinal and Multicenter Studies
Anis Larbi1, Attila Tarnok2
45
Are we Damaging the Cells we Sort?
1
SIgN, A*STAR, Singapore, Singapore, 2Univiversity of
Leipzig, Leipzig, Germany
Peter Lopez1, Andrew Box2
Flow Cytometry has been an important tool for the understanding of
immunological profiles, interactions and functions of immune cell
populations and subpopulations. Blood being the major source of
clinical studies many biomarkers have been developed and will be
developed to understand the biology/immunology in health and
diseases. Increasing numbers of clinical studies are longitudinal and
include increasing numbers of individuals to be tested. The main
reason for this is that studies aim at following health trajectories and
high numbers of participants are necessary for these studies to be
sufficiently powered. This workshop will discuss the findings from
such cohort studies and their translation into the biological/clinical
biomarker field. We will discuss with the attendees some of the
standardization processes that must be implemented in such studies
to ensure reliability of the data.
1
New York Univ, New York, NY, United States, 2Stowers
Institute for Medical Research, Kansas City, MO, United
States
This workshop will seek consensus from attendees on the question
of cell sorting potentially causing cellular alterations. Clearly there
are adverse cell sorting conditions that will result in outright cell
death, such as sorting cells using an undersized nozzle orifice and
excessive pressure. Using seemingly appropriate cell sorting
conditions, functional changes in a few cell types have been
described, although these observations remain undocumented in
peer-reviewed publications.
In this workshop we will seek attendee input to establish what types
of changes attributed to cell sorting have been observed by the
attendees, and if unobserved, would potentially be of concern.
Attendees will be asked to describe experiments that could be used
to evaluate these changes, either with broad application across
many cell types, or specific to one cell type. The workshop
organizers will briefly report on the ongoing study of the
124
CYTO attendees will hear experienced scientists in large-scale and
multi-center studies. Speakers will present some of their data and
what flow cytometry added to their study. This platform will enable
to discuss problems when running such studies and solutions they
have developed to overcome these difficulties. The expert panel
discussion will be moderated by speakers and experts in the field.
Disseminate the concept of standardization
Enable attendees to identify the gaps of their own studies
Identify feasible strategies developed by cytometrist
Create a network of cytometrist involved in such studies
1
49
Integrated, Multi-scale and Spatial-Temporal Cell
Biology – The Allen Institute for Cell Science
51
Biophysical Cytometry
Rick Horwitz
UCLA, Los Angeles, CA, United States
Allen Institute for Cell Science, Seattle, WA, United States
My lab uses microtechnology to interface at the scale of biology to
aid in scientific investigation, develop new approaches to diagnose
and monitor disease, and engineer therapies. In this talk I will focus
on a subset of our work to assay and exploit physical properties of
cells in diagnostics and drug screening. I will first introduce a
microfluidic toolbox that we make use of to precisely align, order,
and transfer cells between solutions in microchannels that takes
advantage of fluid inertia. These inertial microfluidic approaches
allow passive and high-throughput manipulations compatible with
processing large quantities of cells to assay single-cell
heterogeneity. We have combined these techniques with other
microfluidic manipulation strategies to perform multiparameter
biophysical cytometry and physical phenotyping. Physical
properties of cells can provide integrative, rapid, and low-cost
information about disease. I will discuss instruments we have
developed that quantify single-cell physical phenotypes including
deformability, size, morphology, motility, adhesiveness, and
contractility. I believe these new tools will enable new diagnostic
and screening approaches that assay immune cell function at the
point-of-care, tumor cell malignancy with higher confidence than
molecular markers alone, and many other cell fate decisions
associated with disease.
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Index
125
Speaker/Author
Poster
Session
A major goal of cell science is to understand and predict cellular
behaviors, promising major insights into homeostasis, disease,
development and regeneration as well as new approaches to
manipulate them. Most cellular behaviors are determined by
localized activities, resulting from the organization of cellular
components into discrete functional units, i.e., molecular machines
and regulatory complexes, that operate locally and transiently. This
localized organization and activity, in turn, arise from the subsets of
genes expressed, posttranslational modifications, and the cellular
environment, often results from the repurposing and specialization
of canonical cellular components. The mission of the Allen Institute
for Cell Science is to understand and predict cellular behaviors,
taking an integrative approach. The initial project will develop
dynamic, visual data on cell organization and activities at multiple
spatial and temporal scales, beginning with the mesoscale, using
fluorescently tagged, genome-edited, human induced pluripotent
stem cells. We will quantify the relative locations and dynamics of
the major cellular functional units and activities as the stem cells
execute characteristic activities, including differentiation, and in
response to perturbations. The goal is a database of dynamic
cellular organization and activity at multiple spatial and temporal
scales. The focus is on the integrative changes across the cell, rather
than a deep focus on a single process. The organizational and
dynamic image data will be used with genomic and other
information to model cellular organization and its transitions, with
an initial goal of predicting cell state and pathology from
organization. The quantitative image data will be integrated a visual
database, an “animated cell”, which will also serve as an output for
computational modeling. The animated cell will be multi-scale,
Dino Di Carlo
Wednesday
15 June
The image analysis algorithms and data mining approaches we
develop are freely available through the biologist-friendly opensource software, CellProfiler (www.cellprofiler.org), for both smalland large-scale experiments.
Tuesday
14 June
Free-ranging discussion of key questions, supported by the
experiences of facilitatorsKey Questions: Types of cytometry-related
innovations/inventions/services? Novelty? Paths to
commercialization? Available support? Innovation plan?
Understanding your goal or the unmet need? Prior art/competing
solutions? Disrupter/Improver? Time to capture/develop your ideas?
Participants in your innovation plan/evaluation/preimplementation? Desired Outcomes: List of specific DIY tasks that
can be carried out while still at your “day job”. List of information
needed on the CytoInnovation page of the ISAC website? Feedback
on the direction of the Innovation program within ISAC. List of
mechanisms to enhance the general awareness of the potential for
commercialization of innovations/inventions.
Images from microscopy and image-based flow cytometry contain
rich information about the state of cells, tissues, and organisms. Yet
it is common to measure only a few morphological features per
cell. There has been tremendous interest in harvesting the rich
information in images by extracting patterns of morphological
changes (“profiles”) from cells in response to perturbation by small
molecules and/or genetic manipulation. Often, the morphological
profile resulting from a particular genetic or chemical perturbation
is remarkably specific. These profiles are being used to identify
similarities and differences between various chemical or genetic
treatments, with the ultimate goal to identify the causes and
potential cures of disease. Our own goals in these profiling
experiments include identifying drug targets and mechanisms of
action, determining the functional impact of disease-related alleles,
creating performance-diverse chemical libraries, categorizing
mechanisms of drug toxicity, and uncovering diagnostic markers for
disease. We hope to make perturbations in cell morphology as
computable as other large-scale functional genomics data.
Monday
13 June
SciGro, Inc., NorthEast Office, Cambridge, MA, United
States, 2OncoTherics Ltd., Leicestershire, United Kingdom,
3
The TEKMILL, University of Illinois at Urbana-Champaign,
Champaign, IL, United States, 4FlowMetric, Inc.,
Doylestown, PA, United States
Imaging Platform, Broad Institute, Cambridge, MA, United
States
Sunday
12 June
Betsy Ohlsson-Wilhelm1, Paul J. Smith2, Gary Durack3,
Renold Capocasale4
Anne Carpenter
Saturday
11 June
48
First Steps as an Innovator: DIY Assessment of the
Commercial Potential of Ideas and Inventions in
Cytometry
50
A Picture is Worth a Million Numbers: Targeting
Disease by Profiling Cells via Microscopy and Flow
Cytometry
Special
Lectures
Raise interest in this topic
including existing structural data, and useful for fine grained
models. It will serve to integrate spatial and temporal information
and predictive models of cellular cellular behavior. All data,
models, reagents, and tools generated by the Institute will be made
publically available.
Congress
Overview
An interactive format to enable attendees to participate more
actively:
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Edwin van der Pol1,2, Frank A. Coumans1,2, Leonie de
Rond1,2, Elmar L. Gool1,2, Anita N. Böing2, Auguste
Sturk2, Rienk Nieuwland2, Ton G. van Leeuwen1
1
Biomedical Engineering & Physics, Academic Medical
Center, University of Amsterdam, Amsterdam, Netherlands,
2
Laboratory Experimental Clinical Chemistry, Academic
Medical Center, University of Amsterdam, Amsterdam,
Netherlands
Introduction: Flow cytometers provide light scattering data in
arbitrary units, which hampers data interpretation and comparison
of results between instruments. Moreover, particle identification
requires fluorescent labeling, which is expensive, laborious and
prone to artifacts. Here we present a label-free method to obtain the
diameter and refractive index (RI) of submicrometer particles from
the light scattering signals of a flow cytometer. Besides the obvious
advantage of relating scattering signals in arbitrary units to
measurement units, we hypothesized that RI detection can
discriminate between extracellular vesicles (EV; 1.36<RI<1.42) and
lipoprotein particles (1.45<RI<1.60). This is clinically relevant,
because (1) both EV and lipoproteins are submicrometer particles
present in human blood plasma, and (2) EV change in composition
and concentration with disease.
Methods: A flow cytometer (A50-Micro; Apogee, UK) with a 70
mW 405 nm laser was used to detect forward scattering (FSC) and
side scattering (SSC). FSC and SSC of reference beads with known
diameter and RI were measured and modelled with Mie theory,
taking into account the optical properties of the beads and the
optical configuration of the flow cytometer. To determine the
diameter of unknown particles, we used the relation between the
optical scatter ratio (OSR=SSC/FSC) and the particle diameter. To
determine the RI of unknown particles, we used the measured
diameter and FSC or SSC to solve the inverse light scattering
problem by Mie theory. We validated the method by determining
the diameter and RI of a mixture with 250 and 390 nm silica beads
and 250, 310, 380 nm polystyrene beads. We applied RI detection
to differentiate between EV and lipoproteins from the plateletdepleted supernatant of an outdated platelet concentrate
(centrifuged 3-fold, 1,550 g, 20 min).
53
Extracellular Vesicle-Based Rare Event Analysis in
Body Fluids Using High Resolution Flow Cytometry:
Impact of Fluorescence Triggering and Swarm
Detection
Sten Libregts1, Ger Arkesteijn1, Andrea Németh2, Esther
Nolte-'t Hoen1, Marca Wauben1
1
Department of Biochemistry & Cell Biology, Utrecht
University, Utrecht, Netherlands, 2Department of Genetics,
Cell- and Immunobiology, Semmelweis University,
Budapest, Hungary
Background: Extracellular vesicles (EVs) are promising biomarkers,
since their molecular content may contain information regarding
disease state and efficiency of medical intervention. Due to their
size, heterogeneity, and the complexity of body fluids, detection
and characterization of EVs by conventional flow cytometry is
challenging and often involves detection of EV swarms. By adapting
a commercially available flow cytometer (BD Influx) and
developing a generic fluorescent staining protocol using PKH67, we
previously developed a high-resolution flow cytometric approach
(Hi-Res FCM) that allows multi-parameter analysis and sorting of
individual EVs. We here explored whether Hi-Res FCM can be used
to detect rare EV populations (0.1%) in body fluids.
Methods: We performed spike-in experiments using 100nm and
200nm Fluosphere beads or PKH67-stained EVs with or without
fluorochrome-conjugated antibody labeling. Beads or EVs were
spiked into either: 1) PKH67-stained EVs isolated from cell culture
supernatant or body fluids, 2) cell-depleted unstained body fluids
that were run over a size exclusion column (SEC) after spiking, or 3)
115nm and 200nm unstained polystyrene bead samples. After
spiking, samples were analyzed using Hi-Res FCM. To monitor
swarm-effects and coincidence, we performed serial dilutions of
samples, observed the event rate, and kept track of pulse shape and
baseline-restoration using an oscilloscope.
Results: The obtained relation between OSR and particle diameter
is independent of the RI and has a unique solution for particles
<500 nm. For each population in the bead mixture, the diameter
and RI were determined with measurement errors on the mean
<8% and <2%, respectively. For the platelet-depleted supernatant,
two populations with different RI were clearly discernable. More
than 86% of the population with RI<1.42 and less than 7% of the
population with RI>1.42 was positive for CD41-FITC, which
confirms the capability of RI detection to differentiate between EV
and lipoproteins.
Results: Based on fluorescence and light scattering, spiked 200nm
Fluosphere beads could readily be detected in PKH67-stained EV
samples up until a frequency of 0.03%. However, EVs stained with
PKH67 and fluorochrome-conjugated antibodies could not be
distinguished from other PKH67-positive EVs when spiked at low
percentages due to a low signal-to-noise ratio of light scatter and
fluorescence. Notably, when PKH67-stained EVs were spiked into
unstained body fluids and isolated using SEC, light scattering of EVs
changed significantly. While event rates were within a normal
range, the oscilloscope showed clear changes in baseline-levels.
Upon dilution baseline-levels went back to normal, indicating that
unstained particles in body fluids affect the detection of spiked
fluorescent EVs when fluorescence threshold triggering is applied.
After separation of EVs and soluble proteins/lipoprotein particles
from the body fluids using SEC, we found that unstained EVs induce
swarm effects and hamper detection of spiked fluorescently labeled
EVs or beads.
Conclusions: We have developed the first method that converts the
scattering signals of a commercial flow cytometer to the
comparable measurement units of dimension and RI. The method is
based on the relation between OSR and particle diameter, which is
independent of the RI and has a unique solution for particles <500
nm. We have demonstrated that this method is accurate and that it
allows label-free discrimination between EV and lipoprotein
particles in plasma.
Conclusion: We show that Hi-Res FCM technically allows for the
detection of rare populations of nano-sized particles when the
single-to-noise ratio of light scatter or fluorescence is high enough.
However, for Hi-Res FCM based rare EV-biomarker analysis
fluorescent staining strategies need further optimization. In
addition, we show that a generic fluorescent labeling of all EVs is
essential to be able to monitor and control swarm and coincidence
phenomena when fluorescence threshold triggering is applied.
Index
Abstracts
Oral Session
Abstracts
52
Size and Refractive Index Determination of
Submicrometer
Particles by Flow Cytometry
126
Regulatory T (Treg) cells play an important role in maintaining
immune homeostasis, they suppress auto reactive lymphocytes, and
control innate and adaptive immune responses. Treg cells express
the transcription factor FOXP3 which is necessary for the
development of this lineage and plays a critical role in coordinating
the expression of several genes involved in immune regulation.
Mutations in the FOXP3 gene lead to the inherited multi-organ
autoimmune disease IPEX (Immune dysregulation,
Polyendocrinopathy, Enteropathy, X-linked) syndrome, and a
similar disease in the Scurfy mouse.
Index
127
Speaker/Author
There is considerable interest in strategies to increase FOXP3+ Treg
cells in patients with chronic GVHD (graft-versus-host disease) and
other disorders. Most strategies are currently focused on the
adoptive transfer of Treg cells that have been purified and
expanded ex-vivo. These cellular therapies have been difficult to
standardize in humans, but measurable clinical benefit has been
Poster Session
Abstracts
Glaxo Smith Kline, Stevenage, United Kingdom
The increasing use of disease relevant human primary cells is
driving the requirement for more sensitive high throughput
technologies that derive maximum information from fewer and
fewer cells. It is widely recognised that heterogeneous primary cell
populations are more suited to high content single cell analysis
techniques such as flow cytometry.
Oral Session
Abstracts
Exosomes (30-150 nm) are lipid-bilayer-enclosed extracellular
vesicles (EVs) derived from all cells and circulating in the blood.
Because exosomes carry molecular contents of the cells from which
they originate, isolation and identification of cancer-specific
exosomes in body fluids have shown great potential for cancer
Rob Jepras
Commercial
Tutorials &
Exhibits
1
Department of Chemical Biology, Xiamen University,
Xiamen, China, 2The First Affiliated Hospital, Xiamen
University, Xiamen, China, 3NanoFCM, Inc., Xiamen, China
56
Identification of Small Molecule Inducers of FOXP3
in Human T Cells Using High-Throughput Flow
Cytometry
Scholars &
Emerging
Leaders
Leaders
Xiaomei Yan1, Ye Tian1, Ling Ma1, Guoqiang Su2,
Wenqiang Zhang1, Chaoxiang Chen1, Shaobin Zhu3
Poster
Session
55
Quantification of Cancer-Specific Exosomes by High
Sensitivity Flow Cytometry
Wednesday
15 June
Conclusion: Comprehensive SFC-based study of MVs in plateletrich plasma showed changes in ability of MVs to form aggregates
and the emergence of new MV subpopulations in response to
platelet activation induced by ADP and collagen. MV
characteristics, including size, refractive and relative count of single
MVs and their dimers, which cannot be obtained by any other
method, may serve as new indirect indicators of platelet
functioning.
10998-11006.
Tuesday
14 June
Results: As expected, ADP- and collagen-induced platelet
activation had a significant impact on MV characteristics. Changes
caused by platelet activation were observed in the increased
relative number of single MVs and their aggregates. We also
observed formation of MV fractions with sizes and refractive indices
different from that in control not-stimulated sample. Moreover, this
effect depended on inductor, i.e. ADP and collagen action led to
the emergence of different MV subpopulations.
[1] S. Zhu, L. Ma, S. Wang, C. Chen, W. Zhang, L. Yang, W. Hang,
J. P. Nolan, L. Wu, X. Yan, Light-Scattering Detection below the
Level of Single Fluorescent Molecules for High-Resolution
Characterization of Functional Nanoparticles. ACS Nano 2014, 8,
Monday
13 June
Methods: We studied MVs and platelets in platelet-rich plasma
(PRP) before and after addition of ADP and collagen. PRP was
obtained by precipitation to minimize the effect of sample
preparation on the state of platelets and MVs. Analysis of plasma
samples was performed using a Scanning Flow Cytometer (SFC), the
unique instrument developed in our laboratory, which measures
light-scattering profiles (LSPs) of individual particles in flow. We
utilized our previously developed method of label-free
identification of single platelets, spherical MVs, and their dimers in
PRP based on measured LSPs, and characterization of particle
morphology, including platelet volume and MV size and refractive
index. This approach provides observation of MVs under nearnative conditions and also simultaneous monitoring of platelet and
MV morphological changes.
Sunday
12 June
Background: Blood microvesicles (MVs) are small phospholipid
vesicles, released from cells during stress conditions, including
apoptosis and activation. MVs perform a variety of physiological
functions and are considered as potential pathological markers of
some diseases. MVs are believed to be involved in coagulation
processes as their presence contributes to the formation of platelet
aggregates. Further detailed studies of the dynamics of MV
characteristics in response to the factors associated with platelet
activation and aggregation may reveal MV potential as diagnostic
marker of coagulation disorders and diseases associated with
platelet dysfunction. In this work we studied changes in MV
morphology in response to in vitro platelet activation by ADP and
collagen.
By reducing the detection volume for background reduction and
extending the particle transit time through the laser beam for
enhanced photon generation, we have developed high-sensitivity
flow cytometry (HSFCM) that allows light scattering detection of
low refractive index particles as small as 24 nm in diameter [1].
Particularly, the significantly reduced sheath flow rate renders the
interference of impurity particles considerably diminished. Here we
report the HSFCM analysis of exosomes isolated from cell culture
supernatant of cancer cells, and a good discrimination of single
exosomes against the background was achieved. In order to
accurately measure the particle size of single exosomes,
monodisperse silica nanoparticles (refractive index of 1.46) with
diameter ranging from 40 to 100 nm were synthesized and
employed as the size reference standards. The standard curve of the
side scattering intensity versus particle diameter was calibrated with
the refractive index of exosomes (1.41). With a throughput up to
10,000 particles per minute, high-resolution size distribution of
exosomes can be acquired in 2-3 minutes. The detected size
distribution agrees well with Cryo-TEM measurement. As CD147
has been identified as a specific surface protein marker for
exosomes secreted by colorectal cancer cells, cancer-specific
exosomes from the cell culture supernatant were quantified via
immunofluorescent labeling and single particle enumeration.
Moreover, analysis of circulating CD147-positive exosomes in the
blood of colorectal cancer patients will be reported. HSFCM offers
an advanced platform for the quantitative multiparameter analysis
of disease-specific exosomes and may serve as a potential noninvasive diagnostic and screening tool to detect colorectal cancer to
facilitate possible curative therapy.
Saturday
11 June
1
Cytometry and Biokinetics laboratory, Voevodsky Institute
of Chemical Kinetics and Combustion SB RAS, Novosibirsk,
Russia, 2Department of Physics, Novosibirsk State
University, Novosibirsk, Russia
Special
Lectures
Daria Chernova1,2, Anastasiya Konokhova1, Maxim
Yurkin1,2, Dmitry Strokotov1, Valeri Maltsev1,2
diagnosis. The large intrinsic heterogeneity of exosomes in particle
size and molecular contents calls for advanced analytical tools to
probe exosomes at the single-particle level. Flow cytometry is a
well-established technique for the multiparameter analysis of single
cells or cell-sizes particles. However, conventional flow cytometry
is incompetent to analyze single exosomes due to the limited
sensitivity and the large background signals generated from the
impurity particles in the sheath and sample fluid.
Congress
Overview
54
Collagen-Induced Platelet Activation on
Microvesicles in Platelet-Rich Plasma Studied with
Scanning Flow Cytometry
Congress
Overview
Special
Lectures
A robust high flow cytometry assay was developed to identify
compounds that up-regulate FOXP3 in primary human T cells.
Pooled frozen PBMCs were cultured in media containing anti-CD3
and IL-2 for 6 days in the presence and absence of suitable controls
and compounds. Cells were then processed for flow cytometry
analysis. FOXP3+ cells were identified in populations of unstimulated and stimulated PBMCs using specific fluorescent
markers. Over 70,000 small molecule compounds were screened in
single shot and several thousand hits were followed up at full dose
response.
Several compounds were identified that had a profound effect on
FOXP3 expression in T cells, one in particular had an EC50 of <100
nM. These novel tool compounds can be used for dissecting
signaling pathways upstream of FOXP3 and provide a molecular
target that may lead to a therapeutic intervention for autoimmune
and inflammatory disease.
Index
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Scholars &
Emerging
Leaders
Poster
Session
Wednesday
15 June
Sunday
12 June
Monday
13 June
The goal of the program was to identify small molecule
pharmacological agents that can modulate the immune response by
increasing the frequency of FOXP3 positive T cells, thus leading to
an effective therapy for inflammatory diseases. Several signaling
pathways common to all T cells are involved in FOXP3 expression
and it is uncertain what pathways control FOXP3 turnover or halflife. This makes prioritizing a single ‘signaling pathway’ target
difficult. An alternative method was taken where a high throughput
phenotypic flow cytometry screen was established to identify
compounds that influence FOXP3 expression.
Tuesday
14 June
Saturday
11 June
seen in Phase I studies of GVHD. Recent evidence also indicates
that pharmacological control of Treg cells may be possible in
patients by the administration of low-dose IL-2, a growth factor
critical
for Treg cells. Data from these studies showed an increase
in Treg cell numbers and a concomitant clinical improvement in
patients with GVHD or the autoimmune condition HCV-induced
vasculitis.
57
A Novel Approach for the High Throughput Imaging
of 3D Spheroids Using Imaging Mass Cytometry
Vito Zanotelli, Fanny Georgi, Artur Yakimovich, Raúl
Catena, Hartland Jackson, Bernd Bodenmiller
Zurich, Switzerland
Background: In vitro studies using monolayer 2D cell cultures
inherently fail to model a physiological 3D setting, including 3D
cell-to-cell contacts as well as nutrient and oxygen gradients. 3D
tissue models such as spheroid cell cultures overcome this
drawback and resemble the in vivo situation more faithfully.
Consequently these tissue models are currently broadly adapted in
biomedical and pharmaceutical research.
Every cell senses its local 3D environment and adapts its phenotype
accordingly. This process is a major driver for the regulated
phenotypic heterogeneity in tissue development and homeostasis.
When deregulated it can drive diseases such as cancer. However,
how the interactions of cells with their environment collectively
shape cellular phenotypes in tumors and contribute to tumor
heterogeneity is largely unknown. In order to tackle this question
we set out to develop a high throughput setup to quantify the
influence of microenvironmental influences and cell-to-cell
communication on the phenotypic heterogeneity in an in vitro 3D
breast cancer cell culture system.
Method: Imaging mass cytometry (IMC) allows the simultaneous
quantification of more than 40 phenotypic and functional markers
at subcellular resolution in slices of 3D tissues. This makes this
technology suitable to study the spatial relationships of complex
phenotypes and how both, relationships and phenotypes, are
perturbed by small molecule inhibitors. To enable screening
approaches using 3D tissue models and imaging mass cytometry,
we have developed a workflow based on a metal label based
barcoding approach that allows pooling of spheroids treated with a
multitude of conditions before the embedding and cutting steps for
subsequent IMC. In the future the presented pipeline will allow for
the concurrent embedding and cutting of a 384 well sphere culture
128
plate with up to 384 different conditions, making a high throughput
screen with 3D spheroids coupled with an IMC readout feasible.
Results: We use this workflow to study the origins of phenotypic
heterogeneity of 3D cultured breast cancer cell lines.
Preliminary data assesses the feasibility and challenges a barcoding
based approach to combine 3D spheroid cell culture technology
efficiently with IMC. Further, based on data of unperturbed breast
cancer spheroids we show how IMC can capture phenotypic
heterogeneity and phenotypic coordination in the
microenvironment. We also explore how such data can be
integrated using mathematical modeling to gain quantitative
insights.
Conclusion: We present the development of a broadly applicable,
scalable screening approach efficiently combining high throughput
3D microtissue culture with IMC. The approach will also be
applicable to more complex cell culture settings, such as 3D cocultures and organoids and to advanced readouts such as 3D IMC.
Thus it will be a solid basis to study the phenotypic relationships of
single cells in 3D tissues in general.
58
Performing Systems Immunology Using Next
Generation High-Dimensional Single-Cell Cytometry
Systems: Bright Lights and Heavy Metals
Thomas Ashhurst1,2, Andrew Lim3, Larry Duckett4, Robert
Balderas4, Barbara Fazekas de St Groth2,5, Adrian
Smith2,6, Nicholas King1,2
1
Pathology, The University of Sydney, Sydney, NSW,
Australia, 2Ramaciotti Facility for Human Systems Biology
(RFHSB), Camperdown, NSW, Australia, 3BD Biosciences,
Macquarie Park, NSW, Australia, 4BD Biosciences, San Jose,
CA, United States, 5The Centenary Institute, Camperdown,
NSW, Australia, 6Sydney Cytometry Facility, The Centenary
Institute, Camperdown, NSW, Australia
The rise of mass cytometry has allowed for characterisation of the
immune system in unprecedented detail, enabling the detection of
more than 40 parameters simultaneously on single cells.
Additionally, the comparative lack of spillover between reagents,
when compared with fluorescence cytometry, has lead to the
development of assays capable of interrogating highly related
cellular subsets. However, the development of new fluorescent
dyes, and enhancement of acquisition electronics has lead the way
in the development of next generation fluorescence flow
cytometers. Here we sought to employ these new cytometry
techniques to move beyond bulk measurement of a system, and
towards interrogating the complex interaction of large numbers of
cells that define the behavior of the immune system. For mass
cytometry, we utilised a CyTOF2/Helios (Fluidigm) platform. For
fluorescence cytometry we equipped a 10-laser Becton Dickinson
(BD) LSR-II special order research product (SORP) platform with a
newly developed electronics system, increasing the number of
detectible signals to ~30. We incorporated a range of traditional
dyes into our panels, in addition to novel polymer dyes developed
by Sirigen. Here we also describe the development of a novel
optimisation program, combining the use of the existing cytometer
setup and tracking (CS&T) program, with newer methods to
generate optimised voltage target values for panels of up to 30colours. Using both systems, we conducted comprehensive
profiling of the murine haematopoeitic system in the bone marrow
during viral infection. Our investigation revealed cell cycle and
signaling modifications that resulted in enhanced monopoietic
outputs.
Daniel Kalb1, Frank Fencl1, Gian Maestas2, Alireza
Goudarzi3, Bruce Edwards4, Andrew Shreve5, Steven
Graves1
Ziv Porat1, Inbal Wortzel2, Roni Seger2
Poster
Session
1
Pharmaceutical Sciences, Texas Tech University Health
Sciences Center, Amarillo, TX, United States,
2
Immunotherapeutics and Biotechnology, Texas Tech
University Health Sciences Center, Abilene, TX, United
States
Oral Session
Abstracts
Poster Session
Abstracts
Index
Speaker/Author
129
Commercial
Tutorials &
Exhibits
T-cell receptor mimic monoclonal antibodies (TCRm) recognize
specific peptide-MHC class I complexes. They combine the
specificity of T-cell receptors with high binding affinities in the low
nanomolar to picomolar range characteristic of monoclonal
antibodies. Antibody affinity and internalization rate of antigenantibody immune complexes are often directly related and play
important roles to better understand functional properties of the
antibody. The TCRm RL6A targets YLLPAIVHI peptide-HLA-A2
complexes (YLL-A2), where the peptide is derived from the p68
RNA helicase protein. p68 protein is implicated in cell proliferation
and differentiation in tumor generation. Previous work showed that
YLL-A2 target is expressed by brain endothelial cells forming the
blood-brain barrier (BBB), and RL6A displayed significant antitumor efficacy by targeting specific YLL-A2 targets in vivo breast
cancer model. This findings further lead RL6A as a novel
immunotherapeutic agent against brain metastases of breast cancer.
Upon binding, RL6A crosses the BBB via a receptor-mediated
transcytosis mechanism. Confocal microscopy indicated that, after
internalization, RL6A co-localized with the early endosomal marker
(EEA1). Here we used conventional flow cytometry and imaging
flow cytometry to quantitatively analyze the internalization process.
Endothelial cell monolayers formed by hCMEC/D3 cells were
incubated with unlabeled RL6A for time periods up to 120 min.
Surface bound RL6A was detected by a secondary, PE-labeled
antibody. RL6A disappeared in a biphasic pattern from the surface
of hCMEC/D3 cells, with an initial half-life (t1/2) of internalization of
about 3 min and a second, much slower phase (half-life about 3 hr)
Scholars &
Emerging
Leaders
Wednesday
15 June
Conclusions: Using multi-node acoustic standing waves to precisely
focus particles for analysis, we have demonstrated the ability to
analyze 17 streams in real time at high event rates (100k/s) and flow
rates (10mL/min). Without complex fluidic or optical designs, this
system allows for high throughput analysis in a robust, compact and
relatively inexpensive platform.
Jee Hyun Park 1, Oriana Hawkins2, Navin Chintala2, Jon
Weidanz2, Ulrich Bickel1
Tuesday
14 June
Results: Initial work has demonstrated the ability of the system to
run at event rates of 100k events/s and flow rates of 10mL/min, well
above the current limitations of most cytometers. At high
throughputs we have demonstrated good CV’s (5%), sensitivity
comparable to a commercial cytometer and full resolution of
commercial six peak beads.
61
Flow Cytometry Analysis of T-Cell Receptor Mimic
Antibody RL6A Internalization by Brain-Derived
Endothelial Cells
Monday
13 June
Methods: Multi-node acoustic flow cells have been made out of
glass capillaries and etched-through silicon wafers. The current
system employs an etched through SI wafer with channel dimesons
of 2mm wide by 250um high, driven by a 4.9MHz PZT there are 17
focusing nodes at the optimal resonance condition near 5.3 MHz.
Previous work has demonstrated the ability to efficiency excite up
to 300 focusing nodes in a Si etched device. A 150mW 488nm
laser is spread into a wide (2mm) but narrow height (10-20um)
excitation beam in an epifluorescence illumination that matches a
high speed camera’s field of view (2048 x 8 pixels) that is acquiring
25k frames/s. This camera data of these 17 focused streams is
analyzed on the fly and transformed into typical flow cytometry
parameters of interest, such as the peak and integrated intensity of
each particle.
The Golgi complex of mammalian cells is a dynamic organelle,
which regulates the vesicles trafficking in the cell. Although cellular
import and export of proteins require active changes of the Golgi
membranes these are not accompanied by changes in the general
Golgi’s structure. On the other hand, other cellular processes
require dramatic fragmentation of the Golgi complex. These
processes include mitosis, in which the Golgi complex is divided
between the two daughter cells, in apoptosis in which the Golgi
complex undergoes sequential degradation, and migration in which
partial Golgi fragmentation is required for a proper directionality of
the moving cells. Currently, quantification of changes in Golgi
structure is performed by counting 100-500 stain cells using basic
immunofluorescence. This is followed by, a manual classification of
the Golgi structure in these cells by subjective assessments. Here
we describe the use of the unique imaging and analysis abilities of
the ImageStreamX, a high-throughput imaging flow cytometer, for a
non-subjective quantification of Golgi fragmentation. This method
provides a way to analyze the changes in Golgi structure in an
automated, quantitative and non-biased manner. Furthermore, this
method enables rapid and accurate way to analyze more than
104cells per sample. All these above features are exemplified by
structural Golgi changes during different mitotic phases. Using
these parameters, we provide a robust, non-biased statistically
powerful analysis of the Golgi structure that can be used in future
cell cycle and apoptosis studies.
Sunday
12 June
Background: Flow cytometry analysis of a single hydrodynamic
focused core is typically limited to ~50k events/s and ~250ul/min.
While these event rates and flow rates are adequate for a variety of
systems, there are applications spaces where these volumetric and
event rates are insufficient. Meaningful flow cytometric analysis of
truly rare cells, such as circulating tumor Cells (CTCs), require
drastic increases in throughput. Beginning with a whole blood
analysis of ~5*109 cells/mL, the number of CTCs in this population
would be less than 10 cells/mL. The necessary dilution to avoid
coincidences on a statistically significant population quick yields
volumes on the order of 100mL. Even at a relatively high analysis
rate of 50k events/s and 100ul/min, a typical flow cytometer would
take days to perform a CTC analysis. Here we present the
development of a highly parallel acoustic focusing flow cytometer
that uses a high frequency acoustic standing wave to precisely
focus particles into multiple standing wave nodes for highly parallel
(~20 streams) analysis. The parallel interrogation of these streams
allows us to drastically increase the event rates and volumetric
throughput in a relatively compact, simple and cost effective
system.
Weizmann Institute of Science, Rehovot, Israel, 2Biological
Regulation, Weizmann Institute of Science, Rehovot, Israel
Saturday
11 June
1
University of New Mexico, Albuquerque, NM, United
States, 2Albuquerque, 3Center for Biomedical Engineering,
States, 4Pathology, Univ of New Mexico, Albuquerque, NM,
5
Department of Chemical and Biological Engineering,
Center for Biomedical Engineering, University of New
Mexico, Albuquerque, NM, United States
1
Special
Lectures
60
Quantitative Analysis of Golgi Structure by Imaging
Flow Cytometry
Congress
Overview
59
Highly Parallel Acoustic Flow Cytometer for High
Event Rates and Volumetric Throughput
Congress
Overview
Special
Lectures
Saturday
11 June
likely reflecting recycling to the membrane. In a complementary
approach using imaging flow cytometry (Amnis ImagestreamX) and
IDEAS analysis software, the internalization of AlexaFluor488-RL6A
could
be directly visualized and evaluated. The results confirmed
that RL6A internalizes rapidly into hCMEC/D3 cells. In conclusion,
flow cytometry is a suitable tool to analyze trafficking of specific
peptide-HLA complexes in endothelial cells using TCRm.
62
The Cellular Fate and Transport of Silver
Nanoparticles: Hyperspectral Imaging, Flow
Cytometry and Morphology
Robert Zucker1, Laura Degn1, Jayna NR Ortenzio2, Will
Boyes1
Toxicology Assessment Divison, ORD- USEPA- NHEERL,
Research Triangle Park, NC, United States, 2Oak Ridge
Institute of Science and Education Appointee, Research
Triangle Park, NC, United States
The detection of nanoparticles (NP) that enter cells is relevant to
biological and medical fields. The dosedependent uptake, fate and
transport of silver NP (AgNP) into cells has been studied using flow
cytometry, microscopic hyperspectral imaging and microscopic
morphology. ARPE-19 cells were treated with citrate-coated or
PVP-coated 75 nm AgNP (BioPure, NanoComposix) for 24 hours.
The migration of AgNP throughout the cell was visualized by
darkfield microscopy in addition to identifying the nuclei, Golgi,
endoplasmic reticulum, endosomes, lysosomes, actin, and cell
membranes using organelle stains and BacMan 2.0 (Molecular
Probes) organelle transfection products. AgNP that are incorporated
into the cell, migrate usually via endosomal transport, to the
endoplasmic reticulum (ER). Once in the ER, the AgNP aggregate,
which alters their darkfield spectral scatter properties. Higher
concentrations of AgNP and longer incubation times resulted in
more clumping. This physical state was detected with longer
wavelengths using the prism-based PARISS hyperspectral imaging.
Analysis of data: A library of unique spectra was created from
nanoparticles in different configurations within the cell, and
individual AgNP were classified according to their spectral
wavelength patterns. The following observations were made: 1) The
AgNP concentration useful for morphological and hyperspectral
imaging was between 0.1 ȝg/mL and 3 ȝg/mL, which is about 100fold less than values typically reported in the toxicology literature.
When using between 10 and 50 ȝg/mL, the concentration of
particles was usually too high to observe internal organelle cell
morphology. 2) AgNP enter cells in a dose-dependent manner,
which can be quantified by flow cytometry and spatially observed
by microscopy and PARISS hyperspectral imaging. 3) AgNP appear
to enter cells within 4 hours. Once internalized, AgNP travel to the
ER where they combine with other AgNP to form aggregates. 4) The
clumping in the ER is measurable by an increased amount of longer
wavelength light scatter observed by hyperspectral imaging. 5)
AgNP were observed at low concentrations in lysosomes (under 3
ȝg/ml). At high concentrations (between 10 ȝg/ml and 50 ȝg/ml) the
NP were not observed in lysosomes. These experiments shed light
on the uptake, fate, and transport of AgNP in cells. Flow cytometry
was used to quantify uptake while the microscopic morphology
showed the spatial distribution of the particles. Hyperspectral
imaging demonstrated the aggregation status of the particles within
the cell over time and concentration.
This abstract does not reflect EPA policy.
Xiongtao Ruan1, Kole T. Roybal2,3, Taráz E. Buck1, BaekHwan Cho1, Danielle J. Clark2, Rachel Ambler2, Helen
M. Tunbridge2, Jianwei Zhang1, Paul Verkade4, Christoph
Wülfing2,5, Robert F. Murphy6,7
1
Computational Biology Department, School of Computer
Science, Carnegie Mellon University, Pittsburgh, PA, United
States, 2School of Cellular and Molecular Medicine,
University of Bristol, City of Bristol, United Kingdom,
3
Department of Immunology, UT Southwestern Medical
Center, Dallas, TX, United States, 4School of Biochemistry,
University of Bristol, City of Bristol, United Kingdom,
5
Department of Immunology and Cell Biology, UT
Southwestern Medical Center, Dallas, TX, United States,
6
Department of Computational Biology, Biology,
Biomedical Engineering and Machine Learning, Carnegie
Mellon University, Pittsburgh, PA, United States, 7Freiburg
Institute for Advanced Studies and Faculty of Biology, Albert
Ludwig University of Freiburg, Freiburg im Breisgau,
Germany
Fluorescence microscopy is a critical tool for the study of cellular
processes. However, the number of different fluorescent probes that
can be simultaneously imaged in living cells limits our ability to
study the 3D dynamics of many proteins in the same process. Here
to aid study of the actin dynamics of T cells during antigen
presentation, we designed a large-scale computational image
analysis pipeline to produce maps of the spatiotemporal distribution
of actin regulators in a common frame of reference. Time series 3D
images of T cell-antigen-presenting cell couples of eight
fluorescently-tagged core actin regulators under full stimulus and
costimulation-blocked conditions were collected and analyzed with
the pipeline. The pipeline successfully addressed the problems of
low contrast in images (since fluorescently-tagged proteins were
expressed at low endogenous levels) and variation in cell shape.
Analysis of the maps revealed that WAVE2 and Cofilin showed the
greatest diminishment of interface recruitment upon costimulation
blockade; selectively activating WAVE2 and Cofilin under
costimulation–blocked conditions restored actin signaling
dynamics. Our approach provides an unbiased way to study large
scale time series 3D cell images of cellular processes for cases in
which protein distributions in different cells can be related to some
common spatiotemporal event (in our case, synapse formation).
They also provide a basis for spatially-accurate simulations of cell
signaling biochemistry, and for identifying spatiotemporal
relationships between different probes.
64
Ex Vivo Analysis of Multiple CD4+ T-Cell Responses
Using Combinatorial HLA-II Tetramer Staining to
Evaluate the Response to Flu Vaccine and to
Citrullinated Synovial Ag in RA
Jane Buckner, Hannes Uchtenhagen, Cliff Rims, Gabriele
Blahnik, I-Ting Chow, William Kwok, Peter Linsley,
Eddie James
Benaroya Research Institute at Virginia Mason, Seattle, WA,
United States
Index
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Scholars &
Emerging
Leaders
Poster
Session
Wednesday
15 June
Tuesday
14 June
Monday
13 June
Sunday
12 June
1
63
Computational Image Analysis Pipeline for Creating
Spatiotemporal Maps of Signaling Protein
Distributions during T Cell Stimulation
Background: MHC tetramers have become an essential tool for the
direct characterization of antigen-specific CD4+ T-cells in health
and disease. We have applied MHC class II tetramer technology to
the analysis of citrulline specific CD4 T-cells in RA, establishing a
panel of synovial antigen peptides that bind DR0401 upon
citrullination. Using these tetramers we have shown that the ex vivo
frequency of cit- specific CD4 CXCR3+ memory T-cells is increased
130
Oral Session
Abstracts
Poster Session
Abstracts
Index
Speaker/Author
131
Commercial
Tutorials &
Exhibits
Results: Both panels proved to work well based on data from HIVuninfected and -infected adults with known cytomegalovirus
responses. In qualification testing, the panels were at least as
sensitive as a previous 12-color validated panel for IL-2 and IFN-Ȗ.
When applied to the RTS,S pilot study, we found higher response
rates to CSP and HepB for CD4+ T cells in RTS,S vaccinated
children than comparators at one month post-vaccination. Almost
no positive responses were detected at baseline or in comparators.
Effector markers detected in CSP-specific CD4+ T cells included
CD154, IL-2 and TNF-Į. When analyzing the memory subsets,
responding CD4+ T cells had a central memory and effector
memory (EM) phenotype with IL-4-expressing CD4+ T cells in the
EM subset. Similar responses for HepB were detected, but
Scholars &
Emerging
Leaders
Methods: We optimized and qualified two 16-color ICS panels to
evaluate multiple T cell responses including memory and T
follicular helper phenotype, and Th1, Th2, Th17 and cytotoxic
markers in cryopreserved PBMC. The panels also allowed
assessment of natural killer cells (NK), NK-like T cells and Ȗǻ T
cells. Both panels were used in a pilot study of 179 children (105
RTS,S vaccinated and 74 controls receiving a Rabies vaccine) from
Mozambique and Tanzania participating in the RTS,S phase III trial.
Samples from baseline and one month post-vaccination were
evaluated with one or the other panel against peptide pools of
circumsporozoite protein and Hepatitis B surface antigen (HepB)
included in the vaccine. Proportion of positive responses between
RTS,S and comparators and frequencies of cell subsets expressing
effector markers were analyzed. Polyfunctionality analysis is
currently being performed by COMPASS.
Poster
Session
Antibody secreting cells (ASC) are differentiated B cells that play a
major role in humoral immunity as they actively secrete antibodies,
which identify and neutralize foreign antigens. Normally, ASC
develop in response to immune challenges such as infection or
vaccination. In autoimmune diseases, antibody secreting cells
develop which secrete anti-self antibodies that may play a role in
generation of disease flares as well as the chronic inflammatory
nature of the disease. CD19 is a molecule that is present on B cells
from the earliest stages of development through the memory and
plasma-blast stage. Thus, anti-CD19 mAbs, such as MEDI-551,
have the potential to deplete a broader range of the B-cell lineage
than other B cell targeting Abs. Understanding this differentiation
based on CD19 expression is important for both safety and efficacy
considerations. Here we show that CD19 is expressed on the
majority of normal ASC found in human blood, tonsil, spleen, and
bone marrow. Recent reports, and our previous data, have
identified a population of CD19- ASC in bone marrow which
contains cells contributing to long-lived humoral immunity.
However unlike previous reports we show that the function of these
two cell subsets appear to indicate that both have a capacity to
contribute to humoral immunity via antibody responses to previous
immunizations, such as tetanus/diphtheria toxin and influenza, or
infections. In addition, we found that the bone marrow CD19
positive and negative ASC share similar expression of caspase, bcl-
Background: High-level polychromatic panels to assess antigenspecific responses in a limited amount of sample are key in vaccine
development efforts. Characterization of these responses is needed
to understand the mechanisms of vaccine-induced protection and
identify correlates in complex infectious diseases to rationally
design more efficacious vaccines. We developed two 16-color
intracellular cytokine staining (ICS) panels to evaluate the responses
to different vaccine candidates in adults and children. Here, we
present the panels and their application in an immunogenicity
study of the RTS,S malaria vaccine.
Wednesday
15 June
MedImmune LLC, Gaithersburg, MD, United States
ISGlobal, Barcelona Institute for Global Health, Hospital
Clínic - Universitat de Barcelona, Barcelona, Spain, 2VIDD,
Fred Hutchinson Cancer Research Center, Seattle, WA,
United States, 3Bagamoyo Research and Training Centre of
Ifakara Health Institute, Bagamoyo, Tanzania, 4Centro de
Investigação em Saúde de Manhiça (CISM), Manhiça,
Mozambique, 5Antigen Discovery Inc., Irvine, CA, United
States, 6Indiana University Fairbanks School of Public
Health, Indianapolis, IN, United States, 7Michigan State
University, East Lansing, MI, United States, 8Swiss Tropical
and Public Health Institute, University of Basel, Basel,
Switzerland
Tuesday
14 June
Christopher J. Groves, Bhargavi Rajan, Jeff Carrell, Chris
Morehouse, Jinya Wang, Radhika Rayanki, Tomas
Mustelin, Roland Kolbeck, Yue Wang, Ronald Herbst
1
Monday
13 June
65
CD19 Is an Important Target for the Treatment of
Autoimmune Diseases with the Involvement of
Pathogenic Antibody Secreting Cells
Sunday
12 June
Conclusions: These results validate the utility of our combinatorial
approach to efficiently characterize the frequency, surface marker
expression, functional phenotype, and transcript profiles of antigenspecific T helper cells. This powerful multi-color approach has
broad applicability in a variety of settings including vaccine design,
infectious disease, tumor immunology, allergy and autoimmune
disease.
Gemma Moncunill1,2, Aintzane Ayestaran1, Maximilliam
Mpina3, Augusto J. Nhabomba4, Kristen W. Cohen2,
Chenjerai S. Jairoice4, Joseph J. Campo5, Jaroslaw
Harezlack6, Héctor Sanz1, Núria Dí ez-Padrisa1, Nana A.
Williams1, Daryl Morris2, Clarissa Valim7, John J.
Aponte1, Claudia Daubenberger8, Carlota Dobaño1, M.
Juliana McElrath2, Stephen C. De Rosa2
Saturday
11 June
Results: As proof-of-principle, we first analyzed in detail the
epitope hierarchies, memory and activation status, cross-reactivity,
lineage commitment and cytokine expression of six different
epitopes-specific CD4+ T-cell responses to the seasonal influenza
vaccine from very limited samples. We then adapted this approach
to analyze RA peripheral blood and synovial tissue to efficiently
screen novel candidate epitopes for ex vivo responses and to
perform cross-sectional studies that assessed the disease association
of these epitopes from limited samples. Preliminary experiments
furthermore demonstrated that flow sorting of antigen-specific Tcells facilitated RNAseq profiling of as few as ten ex vivo tetramerisolated autoreactive T-cells.
66
Two 16-Color Intracellular Cytokine Staining Panels
Identified Immunogenicity Markers in a Malaria
Vaccine Clinical Trial
Special
Lectures
Methods: We first compared the efficacy of tetramers conjugated
with eight different fluorophores. Of the tested fluorophores, we
found that PE, PE-CF594, PE-Cy5 and APC consistently delivered
comparable ex vivo tetramer results. These can be used together
and yield reliable double staining without significant bias or
spectral overlap and were therefore amenable for developing a
combinatorial staining protocol for the parallel detection of six
epitopes in a single staining tube. Applying this to the study of low
affinity autoreactive CD4+ T-cells we focused on the brightest
fluorophores (PE, PE-CF594 and PE-Cy5) and developed a staining
approach for the parallel staining of groups of tetramers in each
color to monitor a wide range of epitopes using a single sample.
2, and cyclin genes, which may indicate both are long-lived. We
show that CD19 is expressed on the majority of ASC in human
tissues as well as some long-lived cells. This important finding
which may provide an important new tool to selectively target and
treat the chronic nature of autoimmune disease while not
interfering completely with long-lasting acquired humoral
immunity.
Congress
Overview
in DR0401+ RA patients. Notably multiple synovial
proteins/peptides are recognized by RA subjects. Yet, the large
sample requirements of ex vivo analysis limit the number of
specificities that can be studied and restrict the applicability of this
technique, especially in clinical settings. To address this challenge,
we have developed a combinatorial staining approach that allows
characterization of multiple CD4+ T-cell specificities in a single
sample.
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Index
Abstracts
proportions of positive responses were higher than CSP. In addition,
for HepB, production of IFN-Ȗ and IL-13 from CD4+ T cells and IL21 from EM CD4+ T cells were detected.
Conclusions: Our panels allowed the assessment of a high number
of functions in several cell subsets in a small amount of sample and
provided significant insights into the cellular immunogenicity of the
pediatric RTS,S malaria vaccine. Based on the pilot results, a single
composite staining panel will be used in the case control study
from the phase III trial and will be valuable to identify cellular
correlates of protection to this and other vaccines.
67
Modeling Single-Cell Gene Expression Identifies a
Novel Correlate of Protection for an Experimental
Malaria Vaccine
Greg Finak1, Kailan Sierra-Davidson2, Andrew Mcdavid1,
Masanao Yajima1, Patricia Darrah2, Pratip
Chattopadhyay3, Mario Roederer3, Robert Seder2,
Raphael Gottardo1
1
Vaccine and Infectious Disease Division, Fred Hutchinson
Cancer Research Center, Seattle, WA, United States,
2
Cellular Immunology Section, National Institute of Allergy
and Infectious Diseases, Bethesda, MD, United States,
3
ImmunoTechnology Section, National Institute of Allergy
and Infectious Diseases, Bethesda, MD, United States
Single-cell transcriptomics is an incredibly powerful and promising
new technology that enables the interrogation of mRNA expression
levels in individual cells. It is increasingly used in immunological
studies since it can readily be paired with flow cytometry in order
to characterize the transcriptional heterogeneity of seemingly
homogeneous, sorted immune cell populations. However, single
cell gene expression data has unique characteristics, such as
bimodality, and zero-inflation which manifest as important sources
of technical and biological variation. We show that this variation
can be controlled for via the CDR (cellular detection rate), defined
as the proportion of genes detected in a single cell. If not properly
accounted for, the CDR can seriously bias inference leading to
serious overestimation of the number of differentially expressed
genes. We have developed a generalized linear modeling
framework for single-cell gene expression data, called MAST
(Model-based Analysis of Single-cell Transcriptomics), that models
these characteristics and allows us to control for the CDR and
nuisance variability induced by batch effects, enabling
experimentalists to assess differential expression associated with
specific experimental factors in complex designs, rather than just
pairwise comparisons. We apply it to a cohort of subjects from a
malaria vaccine trial in order to characterize flow sorted,
Plasmodium falciparum sporozoite (PfSPZ)-specific, CD4+ memory
T cells from protected and non-protected vaccinees as well as from
naturally infected subjects. This analysis is used it to identify a
novel biomarker of malaria vaccine protection, which we validate
on an independent study cohort.
68
Structure and Function of Tunneling Nanotubes –
Direct Cell-Cell Connections Formed between
Leukemia and Stroma Cells
Katarzyna Piwocka, Marta Walczak, Agata Kowalczyk,
Wioleta Dudka-Ruszkowska, Paulina PodszywalowBartnicka, Piotr Sunderland, Jakub Wlodarczyk
Nencki Institute of Experimental Biology, Warsaw, Poland
Introduction: Tunneling nanotubes (TNTs) are newly discovered Factin-based tubular connections that allow direct communication
between distant cells. The proposed functions include the cell-tocell transfer of large cellular structures such as membrane vesicles
and organelles, as well as signal transduction molecules. Although
TNTs have been described as novel mode of intercellular
communication, their presence and importance in the leukemic
132
niche are not fully recognized. In this study we investigated
structure and activity of TNTs assembled between HS-5 stroma and
K562 leukemia cells, and role of factors secreted by leukemia cells
in the TNTs formation.
Materials and Methods: HS-5 stroma or K562 leukemia cells were
cultured separately or co-cultured together. Alternatively,
supernatant from CML cells was added to HS-5 cells. To visualize
TNTs either immunostaining of actin and microtubules followed by
confocal microscopy or scanning electron microscopy observations
were performed. To investigate number of TNTs and to study cargo
transport in living cells, we used live cell brightfield imaging, timelapse analysis using confocal or spinning disc microscopy upon cell
tracking with CMAC or DiD lipophilic dye. To identify the cargo,
cells were stained with MitoTracker or Dil, analysed by confocal
microscopy followed by 3D image reconstructions. Function of
TNTs and exchange rate between cells were verified by flow
cytometry. For this, population of donors was stained with DiD and
mixed with GFP-positive or fluorescent WGA-tracked cells to study
both, homotypic or heterotypic exchange of cytoplasmic material
and quantify DiD-positive populations. To exclude possibility of
transfer via exo-/endocytosis or by free diffusion, co-cultures were
also performed in cell culture inserts, which unable cells to form
TNTs without influencing other types of transfer.
Results: We detected and characterized tunneling nanotubes
formed either between HS-5 cells or HS-5 and CML cells.
Cytoskeletal components, length and diameter of TNTs were
calculated. We observed that supernatant from CML cells
stimulated TNTs formation between HS-5 cells, however the direct
interaction was not necessary. We established flow cytometry
protocol for statistical analysis of cargo exchange and we estimated
TNTs activity as percentage of cells which obtained material via
TNTs. We also found that activity and direction of transport of
different cargos was not equal between different types of cells.
Conclusion: Collectively, we identified TNTs as the way to
communicate between stroma and leukemia cells. Formation of
TNTs in stroma was stimulated by factors secreted by leukemia
cells. Implementation of broad range of microscopy methods
allowed to characterize TNTs formation, structure and function.
Utilizing flow cytometry methods led to quantification of TNTs
activity and exchange of different cargos. TNTs are involved in
exchange of mitochondria as well as other components, which
might be involved in remodeling of leukemia and stroma cells
behavior to promote disease progression and resistance.
Supported by the National Science Centre grant
2013/10/E/NZ3/00673 to K.P.
K.P. is an ISAC Scholar Fellow 2012-2016
69
Unraveling the Tumor Microenvironment in Three
Dimensions using Imaging Mass Cytometry
Raul Catena, Alaz Oezcan, Vito Zanotelli, Hartland
Jackson, Bernd Bodenmiller
Zurich, Switzerland
Cancer is a complex group of diseases characterized by
uncontrolled growth and dispersion of cells from different origins.
During the last two decades, extensive data provides evidence that
cell types besides tumor cells have key functions in driving tumor
cell malignancy and tumor growth. The different cellular
components that make up this tumor milieu are known as the
“Tumor Microenvironment” (TME). Recently we presented Imaging
Mass Cytometry (IMC), a novel approach that currently allows the
simultaneous antibody-based detection of up to 52 proteins in
tissue sections. With IMC it is for the first time possible to
comprehensively study multiple cell types, alongside with signaling
pathways, extracellular matrix components, cell function
surrogates, and lineage markers, in the original tissue context and to
determine how these cells and their interactions drive tumor
development and metastasis.
1
Cardiothoracic Surgery, University of Pittsburgh School of
Medicine, Pittsburgh, PA, United States, 2Medicine, Xiangya
School of Medicine, Changsha City, China, 3University of
Norte Dame, South Bend, IN, United States, 4Immunology,
University of Pittsburgh School of Medicine, Pittsburgh, PA,
United States, 5Medicine, University of Pittsburgh School of
Medicine, Pittsburgh, PA, United States
Index
Speaker/Author
133
Poster Session
Abstracts
Human tumors are complex and heterogeneous: epithelial,
mesenchymal, stromal, endothelial and immune cells can be
observed in both metastatic lesions and primary tumors. The
interplay of these distinct cell types and their role in tumor growth
and invasion represents an area of active study. The epithelial to
mesenchymal transition (EMT) represents a key step necessary in
the transition from occult primary to aggressive invasive disease.
EMT is a phenotypic change in which cancer cells lose epithelial
characteristics and take on the properties of mesenchymal stromal
cells, including the ability to detach, invade and disseminate. EMT
also endows cancer cells with stem cell-like traits, including
increased resistance to various therapies and expression of key stem
cell-associated markers such as CD90 and CD44. We have shown
that CD90 is present on cancer stem cells with high invasive
potential, as well as on tumor supporting stromal fibroblasts and on
normal perivascular cells which are responsible for
microvasculature generation. We have also shown that tumorigenic
EMT cells, physically located at the invasive front of primary
cancer, have been linked to tumor metastasis. Further, we
demonstrated that these cells interact with tumor-associated
macrophages (TAM) to create a CSC-niche, amplify EMT and
promote an invasive tumor type. Here we show that TAM present in
metastatic and primary breast cancer are of M2 polarization as
Oral Session
Abstracts
Vera Donnenberg1, Jayce-Jieming Zhang1,2, Vincent
Riccelli1,3, Per Basse4, Albert D. Donnenberg5
Commercial
Tutorials &
Exhibits
Material and Methods: We enrolled a small cohort of patients
undergoing therapeutic liver resection upon chemotherapy; Syto 16
and fixable viability stain were used to correctly identify live
nucleated cells from dead cells and debris generated during the
tissue digestion procedure. The following monoclonal antibodies
were used to characterize tissue infiltrating leukocytes: HLADRBV510, CD11b-BV605, CD14-BV650, CD15-BV710, CD16-
71
Tumor-Associated Macrophages Exhibit M2a-Like
Phenotype and Drive the Epithelial-to-Mesenchymal
Transition, Tumorigenicity and Invasion in Breast
Cancer
Scholars &
Emerging
Leaders
recognized as a hallmark of tumors. Macrophages (M˳) and other
myeloid cells (i.e. monocytes and Myeloid-Derived Suppressor
Cells) are present in tumor and peritumor tissues and are key
promoters of both angiogenesis and suppression of adaptive
immune responses. Clinical and experimental evidence have
shown that high density of these leukocyte subsets is associated
with poor patient prognosis and resistance to therapies. Indeed the
targeting of myeloid cells in tumor microenvironment is considered
a promising therapeutic strategy: the depletion of macrophages or
their “re-education” as anti-tumor effectors is currently under
clinical investigation and hopefully will contribute to the efficacy of
conventional anti-cancer treatments.
Aim :The aim of our study was to set up a reproducible and
efficient multiparametric flow cytometric analysis for myelomonocytic cell detection in peritumoral liver biopsies of patients
with colon cancer liver metastases.
These M˳ are distinguishable either by both morphological
parameters and marker expression. Further studies are needed to
define polarization of these subpopulations and their effect on
tumor progression.
Poster
Session
Introduction: The last decade witnessed an ever-growing
awareness of the promoting role of chronic inflammation in cancer
initiation and progression. Cancer-related inflammation is now
existence of different types of M˳ in peritumoral liver samples.
Wednesday
15 June
Flow Cytometry Core, Humanitas Clinical and Research
Centre, Rozzano, Italy, 2Department of Hepatobiliary &
General Surgery, Humanitas Clinical and Research Centre,
Rozzano, Italy
Conclusion: To our knowledge this is the first report showing the
Tuesday
14 June
1
Monday
13 June
Federico Colombo1, Cristiana Soldani2, Barbara
Franceschini2, Francesca Clemente1, Matteo Maria
Cimino2, Guido Torzilli2, Matteo Donadon2, Achille
Anselmo1
identify 5 different M˳ subpopulations depending on their marker
expression as shown below:
Sunday
12 June
70
Multicolor Flow Cytometric Analysis of Peritumor
Infiltrating Myeloid Cells in Patients with Colon
Cancer Liver Metastases
(range 0.7 - 14.3) and a median of 0.9*10^6 CD68+ total M˳/g of
tissue (range 0.1 - 3.4). A median of 55.8*10^3 CD1a+/ HLADR+
DCs/g of tissue (range 4 - 206.3) and a median of 4.3*10^3 CD15/CD33+/HLADR-/CD11b+ monocytic MDSCs/g of tissue (range 0 30.9) was observed among samples. Interestingly, we were able to
Saturday
11 June
We show that IMC is an ideal tool for the three dimensional
reconstruction of tissues in order to provide an improved and
comprehensive understanding of the TME and the mechanisms
driving cancer, as well as for screening for novel biomarkers with
clinical relevance.
Results: We obtained a median of 6.7*10^6 CD45+ cells/g of tissue
Special
Lectures
Application of 3D-IMC to mouse models of breast cancer and
patient samples uncovered three dimensional morphological
features and patterns of TME interactions, including precise
localization of different intracellular signaling signatures alongside
concentric rings in metastatic lesions. Cytokines and target
receptors were measured as well, through which both known and
novel paracrine loops in the tumor milieu were identified.
Quantification of these features and relation to clinical data
provided prognostic and diagnostic markers for an improved patient
classification.
BV786, CD68-PE, CD33-PE-Cy7, CD1a-APC and CD45-APC-H7.
Samples were acquired using a LSRFortessa equipped with four
lasers.
Congress
Overview
Typically, two-dimensional tissue sections are analyzed by
pathologists. 2D-IMC expands greatly the information from each
section. However, to understand the interplay of the elements of the
tumor microenvironment, three-dimensional reconstructions of the
tumors are needed as information from 2D sections can e.g. miss
existing cell to cell contacts. Here we present a novel experimental
and computational workflow to produce three-dimensional
reconstructions of tumors and metastatic lesions by IMC (3D-IMC).
The workflow consists of first, a novel approach to generate
consistent serial tissue sections, second, IMC analysis using a panel
of 44 antibodies generated to study the TME and third, novel
algorithms developed to exploit IMC data for the alignment of
dozens of sections to reconstruct 3D tumor and tissue models.
Finally, we will show a multimodal imaging system that allows
overlay of the IMC images with confocal/fluorescence/light
microscopy images as well, thus increasing the dimensionality of
the 3D image stacks.
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
determined by the absence of HLA-DR and the expression of
CD163 and CD206. In metastatic breast cancer TAM are almost
exclusively CD14+CD11b/CD18+CD163+CD206+: 91.6 ± 8.5%
are
HLA-DR negative (Mean, SD, n=5). Further, M2-polarized TAM
associate with CD90+ CD44+ tumor cells, an association that
drives the EMT of patient-derived tumor cells. Co-injection of sorted
autologous M2-polarized TAMS with 100 CD44+/CD90+
metastatic breast cancer cells markedly enhanced tumorigenicity
(3-fold from 22.5% to 60%, 40 sites each treatment; 2 experiments)
and drove invasiveness in a xenograft model. Histological
examination of the leading edges of mammary fatpad xenografts in
NSG mice injected with TAMs + sorted tumor showed a marked
increase in intra-tumor blood vessels and sheets of tumor cells
invading the abdominal wall; these features were not seen in
xenografts from mesenchymal CD90+CD44+ tumor cells alone.
Taken together, the data indicate that M2 polarized macrophages
are capable of enhancing tumorigenicity and invasiveness.
Macrophage-tumor interactions may provide a novel and attractive
therapeutic target.
72
High-Dimensional Modeling of B and T Cells
Commercial
Tutorials &
Exhibits
Scholars &
Emerging
Leaders
Poster
Session
Wednesday
15 June
Tuesday
14 June
Monday
13 June
C. Bruce Bagwell
Verity Software House Inc, Topsham, ME, United States
Cellular development is normally a highly regulated process
characterized by the ordered, differential expression of numerous
cell-surface, cytoplasmic, and nuclear proteins. Driving these
measurable changes are the most advanced chemically-based
genetic programs known to science – the living system. Programs,
whether they are silicon or carbon-based, are characterized by
ordered sequences or stages. Though these steps can be complex,
it is unlikely they are chaotic in nature since they perform
numerous actions with extraordinary reproducibility.
The technology of cytometry was invented to provide a quantitative
and objective assessment of cells. Although much has evolved
within this technology, there is still too much subjectivity in its
interpretation. The gating paradigm has accomplished much, but
with the advent of cytometers capable of 40-50 correlated
measurements per cell, it needs to be replaced with more practical
solutions - solutions that are as reproducible as the systems they are
analyzing and that also serve to promote the understanding of the
true nature of biology.
This talk briefly chronicles efforts to model the developmental
stages of B cells in bone marrow and T cells in peripheral blood.
These are two very different systems yet they demonstrate that
cellular biology does not necessarily become geometrically more
complex with number of correlated measurements. The modeling
methods described are objective, reproducible, quantitative, and
scale well with number of measurements. Most importantly, model
results are easily interpreted by any scientist and directly confer
important information about the ordered phenotypic changes
associated with differentiating cellular systems.
73
Novel Insights into Human SLE Pathogenesis
Index
Abstracts
Oral Session
Abstracts
Virginia Pascual
Baylor Institute for Immunology Research, Baylor Research
Institute, Dallas, TX, United States
Systemic Lupus Erythematosus (SLE) is a systemic autoimmune
disease characterized by a breakdown of tolerance to nuclear
antigens and the development of DNA and RNA-containing
immune complexes (IC). Genomic approaches have shown that
>95% of children with SLE display unique blood signatures, that
include type I IFN- plasma cell- and neutrophil-related transcripts.
A connection between these signatures seems to exist, as activation
of SLE neutrophils with TLR7-agonistic autoantibodies from patients
induces the release of DNA that triggers massive IFN production by
plasmacytoid DCs (pDCs). To gain further insight into SLE
pathogenesis, we have established a longitudinal cohort of pediatric
SLE patients followed for up to 9 years. A combination of analytical
approaches applied to blood gene expression profiling, clinical and
134
demographic data permitted us to classify patients according to the
behavior of five major transcriptional pathways. These tools might
be useful to stratify patients in clinical trials and enable tailored
treatments targeting the immune networks that drive disease
activity.
This work is supported by NIH AR054083-01 and the Baylor Health
Care System
74
T Cell Trafficking in Tumor Vessels: Yet Another
Checkpoint for Cancer Immunotherapy
Sharon Evans
Immunology, Roswell Park Cancer Institute, Buffalo, NY,
United States
The success of T cell-based cancer immunotherapy hinges on
efficient trafficking of cytotoxic T cell effectors across tumor
vascular checkpoints. While there is strong interest in overcoming T
cell exclusion within tumors using various inflammatory stimuli,
surprisingly little is known about the mechanisms that positively or
negatively regulate delivery of cytotoxic effectors via vascular
conduits. Our laboratory has used live-imaging microscopy
coupled with multiparameter flow cytometric analysis to investigate
in real-time the vascular parameters that contribute to T cell
trafficking in aggressive murine tumor systems as well as in cancer
patients. These studies defined tumor vessels as a critical barrier to
adoptive T cell transfer immunotherapy in preclinical tumor
models. Thus, T cell trafficking across tumor vasculature must
exceed a critical threshold in order to observe tumor control during
adoptive T cell transfer immunotherapy. Despite expression of
multiple adhesion molecules (E/P-selectin ligand, VLA-4, LFA-1)
and chemokine receptors (CXCR3, CCR2, CCR5) by cytotoxic
murine and human CD8 effectors, only a restricted subset of these
molecules contribute to trafficking of cytotoxic CD8+ T cells within
the intravascular space during adoptive T cell therapy in preclinical
models. Intravital imaging further revealed that vessel geography
and high wall shear stress are major obstacles to trafficking of CD8
effectors in murine tumors but this unexpectedly was not the case
in patient tumors. These observations suggest that patient vessels
might be more permissive to T cell trafficking than anticipated, but
only if the appropriate trafficking molecules are available within
tumor vessel walls. Finally, we found that limited baseline
trafficking in tumor vessels can be overcome by preconditioning
regimens that involve thermal medicine or direct administration of
inflammatory mediators in some, but not all murine or human
tumors. Resistance to vascular preconditioning regimens is dictated
by the immune contexture within the local tumor
microenvironment. These findings support a model in which
therapeutic targeting of tumor vessels provides a window of
opportunity for improving the efficacy of T cell-based cancer
immunotherapy.
Supported by NIH R01 CA79765 and AI082039, T32 CA085183,
1F30 CA177210; and by the Jennifer Linscott Tietgen Family
Foundation.
75
The Key Role of Flow Cytometry in Developing
Antibodies for Immuno-oncology
Mark Selby
Discovery Biology, Bristol Myers Squibb, Redwood City,
CA, United States
Engagement of inhibitory receptors on T cells in the tumor
microenvironment reduces T cells functions required to mediate
antitumor responses. Recombinant monoclonal antibodies (mAbs)
to some of these checkpoint molecules have proven to promote
durable antitumor responses in cancer patients. While the
responses of patients to these current therapies is impressive, there
are many who are refractory. Additional mAbs and other agents
will need to be developed to provide more therapeutic options to
clinicians.
2
TAD, USEPA-ORD-NHEERL, RTP, NC, United States,
Biocytex, Marseille, France
What are the avenues for improvements?
Instrument modifications (exposure time, laser power, specific
optics, electronics, detectors, core-stream size, imaging capabilities,
spectral analysis).
135
Index
Brian Grimberg is an Assistant Professor of International Health and
Infections Disease and Immunology at the Centre for Global Health
and Diseases in the Case Western Reserve University School of
Medicine. He was an ISAC Scholar from 2011-2015 and has
received several awards for his work on malaria, conducted on
several continents.
Speaker/Author
Desired Outcomes: We expect that this workshop will help to
evaluate the current usage and future potential place of FCM in the
diverse technologies used for SMP analysis. The participants should
get a better understanding on how to achieve valid results in this
important and controversial field of research They should be able
to decide whether their objectives can be realized using
conventional FCM, and then with which care, or if they require
high-end specialized instruments. The necessary requirements to
Grace Chojnowski holds the position of facility manager at QIMR
Berghofer Medical Research Institute and is active in teaching and
education in malaria cytometry and facilitation of education in poor
resource countries.
Poster Session
Abstracts
Format: A two-side questionnaire will be provided to each
participant to answer questions both at session entry and at the end
of the workshop. This will help in evaluating the impact of
discussions and the acceptance of final
conclusions/recommendations.
Oral Session
Abstracts
Short presentations from leaders in this field will stimulate a lively
discussion that will help to either reach a consensus or at least
clarify the major trends in the field.
Howard Shapiro is one of the earliest advocates of cytometry,
author of the well known book Practical Flow Cytometry, now in its
4th edition, and of numerous publications. He recently formed the
nonprofit One World Cytometry to enable the dissemination of
affordable and sustainable cytometric technology to the resourcepoor countries that directly confront malaria and must also deal
with TB and HIV/AIDS.
Commercial
Tutorials &
Exhibits
Scattering vs fluorescence triggering. Pros and Cons of each.
Scholars &
Emerging
Leaders
QC controls to set-up and/or standardize the FCM? Advantages vs.
limitations of different controls. Already known artifacts and how
to overcome them e.g. “swarming”, counting biases, and sources of
interferences (nonspecific particulate matter, optics cleanliness,
sheath quality, un-bound fluorochromes, options and pitfalls for
wash/no-wash staining approaches).
Problem Focus and Key Questions: Malaria kills ~ 500,000 people
each year, >85% of them children under 5 and many of the rest
pregnant women. Most deaths occur in Africa and Asia and are due
to Plasmodium falciparum; other human malaria parasites (P. vivax,
P. malariae, P. ovale, and P. knowlesi) are less deadly. Prompt
diagnosis allows cure in a few days with inexpensive drugs,
although emergence of drug-resistant strains is a major concern.
Insecticide spraying and distribution of bed nets help control the
Anopheles mosquitoes that spread malaria, reducing its toll, and
vaccines and new drugs are under development.Since the first
malaria workshop, at CYTO 2012 in Leipzig, there has been some
progress in the use of cytometry of DNA content and base
composition and RNA content for malaria detection, species
identification, and determination of developmental stages and drug
effects. Measurement of hemozoin, the “malaria pigment” formed
by parasites’ digestion of haemoglobin in infected red cells, and
determination of parasites’ mitochondrial function and membrane
integrity, can also be informative. Problems with hemozoin
detection in flow cytometry using depolarized scatter signals may
be circumvented in imaging using simpler transmission
measurements. An overview of malaria cytometry that emerged
from the Leipzig workshop was published in Current Protocols in
Cytometry in 2013. Especially because today’s technology promises
to make cytometric apparatus substantially more affordable and
accessible, there is a need for communication between cytometrists
and scientists involved in malaria research and diagnosis. This will
speed newcomers’ progress up the learning curve and facilitate
optimal use of such resources as are available.The cytometry
community rose to the challenge of making CD4 counts for HIVinfected individuals easier and more accessible at the point of care
in both resource-poor and affluent countries; lessons learned from
that effort should enable us to achieve at least equivalent gains
against malaria.
Poster
Session
What is already achieved and accessible to standardization on
conventional FCM?
Intended Audience: Clinicians, malaria researchers, core managers
and flow cytometer users would all greatly benefit from attending
this workshop. Various probes can now be used with flow or
imaging cytometry to count and quantify various species of malaria
parasites in blood, permitting diagnosis, determination of efficacy of
drugs and vaccines, and monitoring progress of malaria
elimination. Measurement of DNA content and base composition
and RNA content, central in malaria cytometry, are also useful in
many other applications.
Wednesday
15 June
Limits of FCM for detection, counting and sizing.
One World Cytometry, Inc., West Newton, MA, United
States, 2QIMR Berghofer Medical Research Institute,
Brisbane, Australia, 3Case Western Reserve University,
Cleveland, OH, United States
Tuesday
14 June
Major Points to Discuss in the Workshop:
Techniques currently used to characterize and analyze SMPs (with
distinction of cell-derived vs synthetic SMPs). The place for FCM?
1
Monday
13 June
Problem Focus and Key Questions: Flow Cytometry ( FCM) is a key
technology that helps to investigate both cell-derived and synthetic
sub-micron particles (SMPs), The detection, characterization and
counting of SMPs, including ectosomes, exosomes and synthetic
nanoparticles, is a hotly debated topic due to the explosion of
knowledge on their biological and industrial potential as vectors
and biomarkers, as well as the associated toxicological risks of
nanomaterials There is a lack of agreement in the scientific
community on how to do an experiment that achieves useful data
without artifacts and how to apply FCM-based SMP analysis. In the
workshop, participants will discuss relevant issues to improve the
quality of fundamental as well as applied research in this field.
There will be an emphasis on the many sources of artifacts and
options on how to avoid them. This will extend/complete workshop
# 1 on sub-micron particles.
Howard Shapiro1, Grace Chojnowski2, Brian Grimberg3
Sunday
12 June
1
77
Malaria Workshop
Saturday
11 June
Robert Zucker1, Philippe Poncelet2
This abstract does not reflect EPA policy.
Special
Lectures
76
Sub-micron and Nano-particle Characterization,
Detection, and Analysis in Flow Cytometry
publish papers both for fundamental and applied/clinical research
on SMPs will be addressed.
Congress
Overview
Preclinical studies using mouse tumor syngeneic models have
helped to establish the utility of targeting checkpoint molecules. In
most cases, surrogate mAbs to mouse target proteins are generated
as clinical candidate mAbs often do bind to the mouse equivalent.
Flow cytometry has played a central role in the designation of
surrogate and human clinical mAb candidates. Flow-based binding
studies are used to identify mAbs with superior binding affinities
and identify those that block receptor-ligand interactions. FACS
analyses are also employed to help inform isotype selection in
engineering recombinant mAbs. Here I will summarize how flow
cytometry is crucial to the designation of mAbs with desirable
characteristics for use in immuno-oncology.
Congress
Overview
78
SRL Equipment Management: Future-Proof Your Lab
Jessica
Back1, Nina Lane2, Joanne Lannigan3
Sunday
12 June
Saturday
11 June
Special
Lectures
1
Oncology, Wayne State University/Karmanos Cancer
institute, Detroit, MI, United States, 2Cambridge Institute,
University of Cambridge, Cambridge, United Kingdom,
3
School of Medicine, University of Virginia, Charlottesville,
VA, United States
The goal of all Shared Resource Laboratories (SRLs) is to provide the
services that will most benefit the investigators they serve. No one
approach will serve all SRLs. The question becomes, how do you
know if your instrumentation options are the “best fit” for your users
at your institution and what do you do when what you have isn’t
fulfilling this promise? Manufacturers are eager to tell core directors
that you need all of the lasers, detectors, filter options, etc., and that
their instruments are the only ones that provide for all of your
needs, but how do you determine what is right and necessary for
your core? Key questions to be addressed are:
How do you decide what instruments to buy?
What is the most cost-effective plan to maintain your instruments?
Discussion will include needs assessments, user surveys,
marketplace research, and various equipment maintenance options.
The format will include slides with interactive participation and live
polling handsets. Attendees will have the opportunity to share their
own experiences and debate the pros and cons of different
approaches and come away with new ideas and tools on how to
select, acquire, maintain, and retire equipment.
79
Imaging Flow Cytometry: Applications and
Challenges
Dominic Jenner1, Andrew Filby2
1
CBR Division, Defence Science & Technology Laboratory,
Salisbury, United Kingdom, 2Centre for Life Flow Cytometry
Core Facility Laboratory, University of Newcastle, Salisbury,
United Kingdom
Imaging flow cytometry (IFC) is a growing field with a large and
multi-disciplinary user group. With the wealth of information out
there it can often be difficult for new IFC users to get a good and
accurate understanding of what the main challenges of working
with IFC are. The fact that Imaging Flow Cytometry is a hybrid
between conventional (non-imaging) flow cytometry and image
analysis has many benefits but also presents a number of significant
challenges to its successful adoption and utilization. This workshop
aims to bring new/experienced IFC users (as well as those with a
general interest) together to discuss the challenges of IFC method
development, data analysis ,data reporting and troubleshooting.
Discussion topics have been decided by the IFC community using a
pre-workshop survey thus giving an emphasis on the end IFC user.
Thus, this user driven workshop is a chance to give your views on
how as a community we can share, collaborate and enhance
working with IFC. The goal of this workshop is to focus on the
development of a IFC user interest group and/or IFC user forum
where people can share any information regarding IFC
development, issues or ask general questions.
Index
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Scholars &
Emerging
Leaders
Poster
Session
Wednesday
15 June
Tuesday
14 June
Monday
13 June
How do you decide it is time for new instrumentation?
136
80
Detective Workshop: How to Solve the Mystery of
Challenging Receptor Occupancy Assays
Virginia Litwin1, Cherie Green2, Jennifer Stewart3
1
Hematology/Flow Cytometry, Covance, Indianapolis, IN,
United States, 2Flow Cytometry Biomarker Development
Sciences, Genentech, Inc., a Member of the Roche Group,
South San Francisco, CA, United States, 3Flow Cytometry,
Flow Contract Site Laboratory, Kirkland, WA, United States
Receptor occupancy (RO) assays for biologics targeting cellular
antigens are critical in the drug development process. Data are
used to confirm physical target coverage and support dose selection
when combined with PK/PD modeling. RO assays are among the
most challenging flow cytometric methods to develop, validate, and
implement. The emerging importance of RO assays is highlighted
by the Special Issue of Cytometry Part B: Clinical Cytometry
dedicated to this topic. This workshop will be an in depth
discussion of the most challenging aspects of RO and is intended to
find concrete answers to the topics specified below. The final
outcome would be a detailed white paper.
Receptor (drug target expression): Dim receptor (drug target)
expression; Rare/infrequent target cell populations; Noncirculating/surrogate targets.
Receptor (drug target) internalization and/or shedding with or
without drug binding: How to assess and control receptor
internalization and shedding especially with activation antigen.
Lack of Critical Reagents: Strategy for the situation where suitable
competing and non-competing reagents are not available.
Variability: Day to day variability; Inter- and Intra-subject
variability; Receptor (drug target) stability during sample collection,
processing, and shipping.
Reagents: Requirements for qualification, monitoring practices, and
documentation of critical reagents such as non-commercial (inhouse, home brew) reagents (antibodies, cellular controls).
81
The Next Dimension in Single Cell Cytometry
Pratip Chattopadhyay1, Stephen Perfetto2, Julianne Hill2,
Mario Roederer2
1
ImmunoTechnology Section, Vaccine Research Center,
NIH, Bethesda, MD, United States, 2NIH / NIAID
This lecture will describe the development and application of 30+
parameter fluorescence flow cytometry technology. This
technology leverages new fluorescent dyes (the Brilliant family of
fluorophores), which are among the brightest dyes currently
available, and higher sensitivity electronics (with lower background
noise). We will examine the importance of quality control
methods, describe in brief the panel development process, and
demonstrate how panels perform in terms of sensitivity and
spillover. We will also explore informatic approaches to analyzing
30+ parameter data. Finally, we will describe the need for, and
power of, the technology for studying immunity, and explore how it
will interface with other high parameter technologies to provide a
deep, comprehensive, and complete profile of immune cells. An
important feature of 30+ parameter flow cytometry technology is
the potential for expansion to up to 50 parameters. We will explore
the path forward for that, and describe the dye and laser
considerations important to achieve this goal. In sum, we will
describe the leading technical and biological edge of this
technology, and illustrate the frontiers ahead that will allow for
even deeper cytometric analyses.
Jonathan Irish
Cardiology, Beth Israel Deaconess Med Ctr, Boston, MA,
United States, 2Flow Cytometry, Beth Israel Deaconess Med
Ctr, Boston, MA, United States
Introduction: Extracellular vesicles (EVs) and their microRNA
(miRNA) cargo can function as novel mediators of intercellular
communication. We have previously identified several microRNAs
that are associated with left ventricular (LV) remodeling in heart
failure (HF). We compare the characteristics and functional role of
cardiac-enriched EVs in HF patients compared to healthy controls.
Poster Session
Abstracts
Index
Speaker/Author
137
Oral Session
Abstracts
Results: SVT control patients (n=6) were younger (age 54±5.4 vs.
72±5.1, p=0.03) and had higher LVEF (63±5% vs. 25±3%,
p=0.0003) compared to HF patients (n=7). EV profiles in HF
patients demonstrated an increase in the geometric mean value of
side scatter compared to controls, indicative of increased EV size
(SVT, 1.02 ± 0.1943 gMFI; HF, 1.39 ± 0.06429 gMFI). Addition of
EVs isolated from heart failure patients to human embryonic stem
cell-derived cardiomyocytes produced an increase in APD at 24 hrs
(p=0.0649), with no change observed following addition of control
patient-derived EVs. Increases in several microRNAs previously
associated with heart failure were observed in the EV fraction from
HF patients compared to controls including miR-151a, miR-30d,
and miR-660 (2.6, 1.8, and 3.9 fold increase respectively). In silico
Commercial
Tutorials &
Exhibits
Methods: Coronary sinus blood was collected and processed from
patients either undergoing electrophysiology study for
supraventricular tachycardia (healthy controls) or implantation of
cardiac resynchronization therapy (CRT) for dyssynchronous HF.
Cell-free plasma was analyzed by small particle flow cytometry
(SPFC) (Beckman Coulter MoFlo Astrios EQ) to determine
differences in size and scatter properties of EVs. EVs were isolated
from plasma by Optiprep density gradient, and the expression of
miRNAs associated with LV remodeling assessed by qRT-PCR.
Finally, the functionality of these EVs was tested by addition to
human induced-pluripotent stem cells derived cardiomyocytes
infected with lentiviral vector carrying ArcLight, a genetically
engineered voltage indicator. Action potentials were obtained
through ArcLight fluorescence imaging and analyzed at 80%
repolarization.
Scholars &
Emerging
Leaders
Leaders
1
Poster
Session
Blood and urine were collected from twenty healthy volunteers and
twenty-two PAH patients. Echocardiography was performed on
Kirsty Danielson1, John Tigges2, Virginia Camacho2,
Vasilis Toxavidis2, Saumya Das1
Wednesday
15 June
Pulmonary arterial hypertension (PAH) is an insidious fatal disease
characterized by severe remodeling of the pulmonary artery and
right ventricular hypertrophy. There is an increasing need for novel
biomarkers to assess disease severity, and predict treatment
response in PAH. ȕ-adrenergic receptors (ȕAR) dysfunction in
lymphocytes has been found to correlate with cardiac function in
left heart failure. Endothelial cell-derived microparticles (Ec-MPs)
are established biomarkers for endothelial activation, injury, and
angiogenesis. We hypothesized that flow cytometric quantification
of these circulating cellular elements may provide novel biomarkers
of right ventricle function in PAH.
Tuesday
14 June
Cleveland Clinic, Cleveland, OH, United States
84
A Functional Role for Heart-Derived Extracellular
Vesicles in Heart Failure Associated Electrical
Remodeling
Monday
13 June
Kewal Asosingh, Jonathan Rose, Nicholas Wanner,
Kimberly Queisser, Patrick Barret, Margaret Park, Corrine
Hite, Sathyamangla V. Naga Prasad, Serpil Erzurum
Overall, flow cytometric quantification of peripheral blood cell ȕAR
density and urinary Ec-MPs may be utilized as routine techniques to
analyze clinical samples. These novel methods may be helpful to
further validate ȕAR density in circulating endothelial cells and EcMPs in urine as potential biomarkers of right ventricular function in
PAH.
Sunday
12 June
83
Flow Cytometric Quantification of White Blood Cell
ȕ-Adrenergic Receptor Density and Urinary
Endothelial-Derived Microparticles as Biomarkers of
Right Ventricle Failure in Pulmonary Arterial
Hypertension
The results demonstrated that flow cytometric detection of
alprenolol is a specific and fast method to quantify ȕAR density in
cells. ȕAR density was decreased in most WBC subsets in PAH
samples as compared to healthy controls (p=0.04 in total WBC
population), and there was a trend toward correlation between ȕAR
numbers in circulating endothelial cells and an echocardiographic
measure of right heart function, tricuspid annular plane systolic
excursion (TAPSE) (Spearman’s ȡ=0.49, p=0.05). Ec-MPs were
abundantly present in urine samples and were increased in PAH
compared to healthy controls (p=0.004).There was a strong
correlation between Ec-MPs and TAPSE (ȡ=0.49, p=0.04).
Saturday
11 June
In cancer, autoimmunity, and infectious disease, cells with novel or
unexpected phenotypes can be both therapy biomarkers and
clinical response mechanisms. However, revealing and
communicating the identity of new cell populations in complex
tissues is limited by two issues widespread in cytometry: 1) most
single cell analysis tools focus on reporting mean, median, or
percentages in a gate, and 2) complex populations are typically
described by metaphors intended to describe functions (e.g.
“cancer stem cells”, “central memory T cells”) or incomplete labels
based on a few features (e.g. “PD-1+ CD8 T cells”). While these
approaches have been useful, they can also be misleading when
cell subsets are heterogeneous or have additional functions that
contrast with their namesake. The lack of a quantitative framework
to describe feature enrichment on cell subsets is a key block to
clear communication of cell identity (cytotype). This problem has
become acute now that computational tools developed for machine
learning have been adapted to reveal and characterize cells in high
dimensional cytometry. To address these needs and help to teach
machines to recognize cytotype, we developed Marker Enrichment
Modeling (MEM) and applied it to characterize healthy and
malignant human tissue biopsies. MEM characterized and reported
cytotypes as context-specific feature enrichment scores for cell
subsets. MEM creates a simple way for machines and humans to
communicate cell identity quantitatively and with minimal bias.
This approach is complementary to expert analysis by humans,
traditional clinical analysis, and recent approaches to classify cell
subsets with computational tools. We implemented MEM as part of
a previously described workflow (Diggins et al., Methods 2015) that
includes viSNE based dimensionality reduction and SPADE
clustering. MEM was used after these steps to report cytotype
labels. We also developed a way to compare MEM labels and
score population similarity for cell subsets from contrasting studies.
This approach accurately described populations of T cells, B cells,
and cancer cells across a wide range of tissues, labs, and platforms,
including mass cytometry and fluorescence phospho-specific flow
cytometry (phospho-flow). In the long term, routine reporting of
MEM labels as population descriptors will facilitate machine
learning of cell identity in complex tissues, including human
tumors.
Special
Lectures
Cancer Biology, Vanderbilt University, Nashville, TN,
United States
PAH subjects. A flow cytometric technique was developed and
validated to quantify ȕAR density in white blood cells (WBC), using
biotinylated ȕ-blocker alprenolol as a probe. This method was
combined with immunophenotyping to quantify total (cell surface
and intracellular) ȕAR density in circulating WBC subsets, including
mononuclear cells, granulocytes, lymphocytes, progenitor cells and
circulating endothelial cells. To quantify Ec-MPs, vesicles were
isolated from spot urine samples and stained for annexin-V and
CD144, and analyzed on an Apogee A50-Micro flow cytometer.
Congress
Overview
82
Learning and Communicating Cell Identity in the
Context of Cancer Microenvironments
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Index
Abstracts
target prediction (TargetScan, MIRDB) produced several relevant
mRNA targets of these microRNAs including KCNJ10, MAPK11,
KCNJ12, and KCMB2 that may be involved in electrical
remodeling.
Suppression of these targets is a potential mechanism
for the observed prolongation of action potential within
cardiomyocytes, and will be addressed in future studies.
Conclusions: EV profiles and microRNA contents differ between
patients with HF and healthy controls, and EVs in HF patients
appear to play a functional role in electrical remodeling at the
cellular level. EVs and their contents may reflect a novel paracrine
signaling mechanism in HF that may be targeted for therapy.
85
Microparticles as a Novel Biomarker for Early
Prediction of Preeclampsia and Intrauterine Growth
Restriction
Anshul Jadli, Kanjaksha Ghosh, Shrimati Shetty
Dept. of Haemostasis and Thrombosis, National Institute of
Immunohaematology (NIIH), Mumbai, India
Background: Preeclampsia (PE) & intrauterine growth restriction
(IUGR) are major contributors to perinatal mortality & morbidity. PE
is the leading cause of indicated premature delivery & maternal
death affecting up to 5-8% of pregnancies & is responsible for over
50000 maternal deaths annually worldwide. The incidence of
IUGR is estimated to be approximately 5% to 7%. There is a strong
link between IUGR, chromosomal abnormalities, & congenital
malformations. The pathophysiology of PE & IUGR is poorly
understood even after decades of research. Even though the
symptoms of PE/IUGR generally manifest in the second to third
trimester of pregnancy, its underlying development largely takes
place in the first trimester. This finding has sparked great interest in
the search for biomarkers to predict PE & IUGR early in pregnancy
before these complications occur. Present study is aimed towards
utilization of cell derived circulating microparticles (cMPs) as early
predictive biomarker for prediction of PE & IUGR.
Methods: Blood samples were collected from primigravid pregnant
women attending for their antenatal examination. Plasma was
separated from whole blood & stored at -80°C until analysis. After
delivery, based on outcome, samples were divided into PE, IUGR &
control groups. 1:1 gestational age & storage time matched controls
were used for PE & IUGR samples. cMPs from different cell-origin
i.e. leukocyte, erythrocyte, tissue factor, endothelial & platelet,
were estimated by flow cytometry.
Results: Out of 587 primigravid women included in study, 97
women were excluded from study due to non-availability of
outcome data. 58 (11.83%) developed pregnancy related
complications. 40 (8.1%) women developed IUGR & 18 (3.67%)
developed PE. At 9-15 weeks, no significant change was observed
in cMP levels in PE patients compared to controls, while total
annexin (p-0.0217) & CD235a (p-0.029) showed significantly
elevated levels in IUGR group. In 16-25 weeks of gestation, all the
MPs, except CD41a, showed significantly elevated levels of MPs in
PE group compared to control group notably CD62e (AUC-0.8,
Sen.-90.5%, Spe.-69.2%), CD146 (AUC-0.82, Sen.-71.4%, Spe.76.9%) & CD142 (AUC-0.82, Sen.-71.%, Spe.-86.4%). In 16-25
weeks of gestation, total annexin (p-0.0052), CD41a (p-0.029), &
CD62e (p-0.035) showed significantly elevated levels in IUGR
group compared to controls. None of the circulating microparticles
showed significant change in PE group at 26-31 weeks of gestation.
In third trimester, only CD235a (AUC-0.889, Sen.-89%, Spe.-75%)
showed significantly elevated levels in IUGR group compared to
controls.
Conclusion: Our study reported circulating microparticles of
various cell origins in prediction of PE specifically in 16-25 weeks
of gestation. Similarly, study showed high predictive value of
CD235a in prediction of IUGR at 26-31 weeks of gestation. The
study for the first time shows the utility of circulating microparticles
for prediction of pregnancy related complications.
138
86
Label-Free Detection of Extracellular Vesicles in
Human Breast Milk Compared to Infant Formula
Leonie de Rond1,2, Edwin van der Pol1,2, Auguste Sturk1,
Rienk Nieuwland1, Frank Coumans1,2, T.G. van
Leeuwen2
1
Laboratory Experimental Clinical Chemistry, Academic
Medical Centre, Amsterdam, Netherlands, 2Biomedical
Engineering and Physics, Academic Medical Centre,
Amsterdam, Netherlands
Introduction:Extracellular vesicles (EV) in human breast milk carry
immune relevant structures (J Immunol.2007; 1;179(3)), and breast
milk has a beneficial long-term effect on infants’ health when
compared to infant formula. Therefore, we would like to study the
relation between EV and infants’ health. For this study the
concentration of EV has to be determined. Detection of EV in breast
milk and infant formula is, however, complicated by the abundance
of lipoprotein particles. We recently demonstrated that scatter
signals of a flow cytometer uniquely define the diameter (d) and
refractive index (RI) of particles below ~500 nm. RI detection can
discriminate EV (RI 1.37-1.42) from lipoproteins (RI 1.43-1.50).
Therefore, we determine the EV to lipoprotein ratio (EV:LP) of breast
milk, cow milk and (cow milk-derived) infant formula.
Methods: Forward and side scatter signals of particles between 200
and 500 nm are measured on an A50-Micro (Apogee, UK). To
validate the method, we compared the measured d and RI to the
specified d and RI for two silica and three polystyrene bead
populations. Breast milk (4 donors, 11-20 weeks postpartum) was
obtained with informed consent. The EV:LP was derived from the RI
distribution.
Results: Validation with beads showed that d and RI could be
determined within an measurement error between 1.5%-9.5% for d
and between 0.17%-2.7% for RI. The EV:LP of the breast milk
samples was between 1.3-3.9, which was much higher than the
EV:LP of infant formula (0.22). The EV:LP of infant formula was
comparable to cow milk (0.34).
Conclusion: Calibrated measurement of the forward and side scatter
signals on a flow cytometer enables label-free determination of the
number of EV and lipoproteins in milk. The average EV:LP of breast
milk is 9-fold higher than that of infant formula. The observed
difference indicates that EV may contribute to human breast milk’s
beneficial effect on the infants’ health.
87
High-Resolution Flow Cytometry of the Intestinal
Microbiota in Inflammatory Bowel Disease
Jakob Zimmermann1, Thomas Hübschmann2, Florian
Schattenberg2, Joachim Schumann2, Pawel Durek1,
Marie Friedrich3, Rainer Glauben3, Britta Siegmund3,
Andreas Radbruch1, Susann Müller2, Hyun-Dong Chang1
1
Deutsches Rheuma-Forschungszentrum Berlin (DRFZ),
Berlin, Germany, 2Umweltmikrobiologie, HelmholtzZentrum für Umweltforschung Leipzig, Leipzig, Germany,
3
Gastroenterologie, Charité Universitätsmedizin, Berlin,
Germany
The intestinal microbiota is a complex ecosystem of 1014 bacteria of
several hundred bacterial species. Besides nutrient metabolism, the
microbiota is vital for the proper development and functioning of
the mammalian immune system. Pathogenic changes to the
microbiota, known as dysbiosis, have been implied in immunemediated diseases like inflammatory bowel disease (IBD), arthritis,
and diabetes. However, microbiota analysis for the diagnosis and
prognosis of diseases is still rare, largely due to the lack of fast and
efficient preparative methods to dissect the heterogeneity of the
microbiota. Currently, most studies rely on next-generation
sequencing of the bacterial metagenome. This is rather slow,
88
Integration of Acoustic and Light Sensing for Marine
Bio-mining
Medical University of South Carolina, Charleston, SC,
United States
Ming Li1, Hector Enrique Munoz1, Jonathan Lin1, Keegan
Owsley1, Keisuke Goda2, Eric Diebold3, Dino Di Carlo1
1
Index
Speaker/Author
139
Poster Session
Abstracts
The capability to control the position of E. gracilis into a tightly
focused stream in a high-throughput and high-accuracy manner is
essential for various applications ranging from cell counting,
screening, and sorting to downstream biochemical assays and
genetic analysis. Although a variety of passive and active
approaches have been developed to focus particles in
microchannels, inertial microfluidics offer benefits in terms of highthroughput, simplicity, precise manipulation, and low cost in
comparison to other techniques.
Oral Session
Abstracts
Euglena gracilis (E. gracilis), a single-celled eukaryotic alga, has
been proposed as one of the most attractive and ideal feedstocks to
produce biodiesel [1]. This is because it can be grown through
photosynthesis without dependence on arable land, and both waste
carbon dioxide (CO2) and wastewater can be used. Moreover, it
has high volumetric biomass productivity of about 7.7 g/L/day, and
high wax ester content exceeding 20% of the dry weight biomass.
We apply inertial focusing to align and uniformize velocity for
these ellipsoidally-shaped algae enabling imaging flow cytometry to
characterize lipid content within populations of single E. gracilis
cells.
Commercial
Tutorials &
Exhibits
UCLA, Los Angeles, CA, United States, 2Univeristy of
Tokyo, Tokyo, Japan, 3Omega Biosystems Incorporated
Scholars &
Emerging
Leaders
Leaders
90
Inertial Focusing and Imaging Flow Cytometry of
Euglena gracilis for Biodiesel Applications
Poster
Session
Image cytometry has further improved the application of flow
cytometry to the analysis of palaeo-environmental deposits,
enabling rapid and accurate classification of the microfossils
identified within sediments. In particular the identification of
extremely rare, cereal type pollen grains from other, more abundant
pollen types that exist in the fossil record has allowed us to pinpoint
the earliest date that cereal crops were cultivated at a specific
locality, leading to a greater understanding of early human
agriculture, cultural expansion and migration.
Wednesday
15 June
Maximum diversity of life exists within the estuaries and coral reefs
of the Globe. The absence of vertebrate and other land dwelling
adaptations has resulted in an enormous range of complexity
among invertebrates and their symbiotic biome resulting in the
generation of compounds finding uses in anti-tumor and antibiotic
applications. It has been widely reported that the greatest factor
limiting progress in characterizing and processing new therapeutics
derived from invertebrates is the lack of adequate original material.
Symbiotic bacteria within specific tunicates often synthesize antitumor compounds as secondary metabolites. We describe a 3-stage
protocol that utilizes acoustic and photonic analysis of large areas
of marine ecosystem and life forms. We refer to this as Estuary
Assessment System (EAS), which includes a multi-frequency
acoustic transducer/sensing instrument mounted on our research
vessel. This generates a topological map of surveyed tracks of
marine locations known to be habitats of useful actinobacteria
laden invertebrates. Photonic devices are used to generate image
and pulse data leading to location, identification and isolation of
tunicates and actinobacteria. A description of human-mounted
search and survey apparatus was illustrated within a poster
presented at CYTO15. This included a contrast of electro-optic
methods developed to compile an image-gallery of bottom dwellers
and flow cytometric bacterial analysis. Three steps are utilized to
achieve our bio-mining objective. First, an aquatic or esturine
location is surveyed by acoustic analysis to determine a likely or
confirmed presence of invertebrates of interest. This is based on
known predator and grazing requirements of sponges, tunicates,
soft corals, etc. This bio profile, which most importantly includes
plants, amounts to an environmental assessment of marine location
accessible by our boat – laden bio-acoustic instrumentation. A
relatively low-resolution image of the sea bottom and its organisms
is obtained by a variety of sonar methods. ( Hummingbird ONIX
8ci) Adjustable sonar at 200/83/50 kHz is generated from the
transom mounted transducer. Two frequencies are received by a
DSP to produce maps. Adjustable CHIRP produces 360° imaging.
This complex sonar / image /GPS output is used to determine the
location for the diver to swim along a sinusoidal trajectory along a
plotted track. As a result of experiencing the enormousness of sea
tracks containing over 4000 tunicate species within the Philippine
seas and US eastern coast, we were compelled to widen our survey
system by implementing acoustic analysis as a preliminary step
towards locating, identifying and harvesting useful organisms.
Using FACS, we identified and purified representatives of a single
species of algae from lake sediment cores dating from 1,500 to
8,000 years ago. DNA was extracted from the purified algal cells
and sequenced by Illumina next generation sequencing. Analysis of
the ancient sequence enabled us to confirm that the algae that
existed in the lake at the retreat of the ice sheet are virtually
identical to those living there, today. Most importantly, that
conclusion could not have been reached without FACS.
Tuesday
14 June
Gordon Wiegand
Microscopic fossils are used routinely to date sedimentary deposits,
discover potential reserves of fossil energy and investigate
environmental change. Conventionally, microfossils are identified
by laborious and time-consuming methods that yield relatively few
representatives, thereby limiting analytical precision. We developed
new protocols for rapid analysis and purification of microfossils as
diverse as pollen grains, diatoms and unicellular chlorophytes from
ancient sediments using fluorescent activated cell sorting (FACS).
Not only has FACS dramatically reduced the time to characterise
entire populations of microfossils from months to days, the highthroughput offered by FACS enabled us to acquire microfossil
samples of sufficient purity and concentration to enable more
refined, downstream analyses than possible by microscopy alone.
Monday
13 June
/ = equal contribution
Biosciences, University of Exeter, Exeter, United Kingdom
Sunday
12 June
* °
Richard Tennant, Ann Power, Rob Lee, Richard Jones,
John Love
Saturday
11 June
In summary, high-resolution flow cytometry provides unique
opportunities for the analysis of the microbiota and its role in
disease, at the level of single cells, phenotype, and proteome, and
with the option to isolate defined bacterial species for subsequent
functional and molecular analysis, but also for therapeutic
corrections of the microbiota in dysbiosis.
89
Tracking Ancient Global Change Using Flow and
Imaging Cytometry
Special
Lectures
Here we present a flow-cytometric approach to unravel the
complexity of the commensal microbiota. High-resolution
determination of the bacterial forward scatter and DNA content
allows for the discrimination of up to 80 different bacterial
subpopulations per sample from formaldehyde-fixed murine stool
samples. Using this method, we tracked the changes of the
microbiota in the course of several murine models for IBD. We
observed a significant loss of microbial complexity upon intestinal
inflammation. 16s rDNA sequencing of sorted sub-populations
revealed that their composition is phylogenetically homogenous
and highly reproducible. Intriguingly, 16s rDNA sequencing also
confirmed the relative IBD-associated changes in bacterial
abundance detected by flow-cytometry thus validating our
approach.
Similarly to understanding how discrete differences in cellular
refractive and diffractive properties effect the scattering of light in
flow cytometry; I employed sound waves to optimize the detection
of targeted invertebrates within the marine biosphere.
Congress
Overview
expensive, population-based, restricted to genome and
transcriptome, and does not allow to isolate defined bacteria for
further analysis.
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Here, we use a device consisting of a low-aspect-ratio straight
microchannel and a series of expansion regions in channel height
called steps. By making use of inertial focusing and local secondary
flows
induced by steps, E. gracilis having diverse shapes are
directed to a single equilibrium position in a single focal stream.
We study horizontal and vertical focusing, orientation, rotational,
and transport behaviors of E. gracilis as a function of Reynolds
number and cell concentration, and we achieve focusing efficiency
higher than 90% with throughput of around 8,000 cells/s. In
addition, we demonstrate an integration of the focusing system with
imaging flow cytometry using radiofrequency-tagged emission
(FIRE) [2]. The results show that this technique is able to quantify
the wax ester production in E. gracilis, identify subpopulations with
different shapes, size, chlorophyll, and wax ester contents, and
determine viability. We also show that wax ester content is
correlated with unique cell shapes in E. gracilis.
This approach opens venues to discover and select E. gracilis with
desirable properties such as high wax ester content, growth rate,
and environmental tolerance for biodiesel production.
References:
Poster
Session
Wednesday
15 June
Tuesday
14 June
Monday
13 June
[1] T. M. Mata et al. Renewable and Sustainable Energy Reviews
14, 217–232 (2010).
[2] E. D. Diebold et al. Nature Photonics 7, 806–810 (2013).
91
High Level of Activation and Altered Mitochondrial
Functionality in T Cells from Patients with
Progressive Forms of Multiple Sclerosis
1
2
1
Sara De Biasi , Elena Bianchini , Milena Nasi , Lara
Gibellini1, Simone Pecorini1, Anna Maria Simone3,
Diana Ferraro3, Francesca Vitetta3, Patrizia Sola3, Andrea
Cossarizza1, Marcello Pinti2
1
Department of Surgery, Medicine, Dentistry and
Morphological Sciences, University of Modena and Reggio
Emilia, Modena, Italy, 2Department of Life Sciences,
University of Modena and Reggio Emilia, Modena, Italy,
3
Neurology Unit, Department of Biomedical, Metabolic and
Neurosciences, University of Modena and Reggio Emilia,
Modena, Italy
Index
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Scholars &
Emerging
Leaders
High Level of Activation and Altered Mitochondrial Functionality in
T Cells from Patients with Progressive Forms of Multiple Sclerosis
Introduction: Different forms of multiple sclerosis (MS) are
characterized by a different degree of T cell activation and
differentiation. After stimulation, T lymphocytes undergo profound
metabolic changes which are crucial for the differentiation towards
memory or effector phenotype. Thus, despite such a role for
metabolic changes in deciding T cell fate, in MS patients there are
no data concerning mitochondrial (mt) functionality and metabolic
profiles of T cell subsets.
Methods: We enrolled a total of 23 patients affected by MS: 2 with
a relapsing-remitting (RR) form in an inactive phase, 15 with a
secondary progressive form (SP), 6 with a primary progressive form
(PP). Fourteen age- and sex-matched subjects were used as healthy
controls (CTR). To characterize the activation and differentiation
status of T cells, freshly isolated PBMC were stained with anti-CD3PE-CY5.5, -CD4-AF700, -CD8-AF770, -CD45RA-PE, -CCR7-BV421,
-HLA-DR-FITC, -CD38-BV605 mAbs. CD4 and CD8 T cells were
purified using immunomagnetic beads, stimulated for 16 hours with
anti-CD3 plus anti-CD28. Then, by using flow cytometry, we
measured in each T cell population mitochondrial mass, mt O2level, mt membrane potential (MMP), level of GLUT-1 receptor and
phosphorylation of S6 Ribosomal Protein. Samples were acquired
using Attune NxT flow cytometer (Thermofisher). In parallel, CD4
and CD8 TEM, TCM, Tnaï ve, and TEMRA were sorted using eS3 sorter
(Biorad), stimulated for 4 hours and analyzed for metabolic gene
expression profile.
140
Results: PP patients were characterized by high percentage of
CD4+ TEMRA cells and low percentage of CD8+ Tnaï ve cells if
compared to CTR. SP patients displayed high percentage of CD8+
TCM cells. SP and PP patients had high percentage of activated
CD4+ and CD8+ T cells. SP patients showed high percentage of
pS6+ cells among CD4 TCM cells, while PP patients showed high
percentage of pS6+ among CD4+ and CD8+ TEM cells. CD8+ TEM
cells from PP patients expressed low level of GLUT-1 receptor after
in vitro stimulation. Mt mass decreased in PP patients after in vitro
stimulation. mt O2- production was high in CD8+ TCM cells of
progressive patients while MMP was reduced in all subsets of cells
after in vitro stimulation. Transcription factors that orchestrate the
metabolic switch from respiration to glycolysis were modulated in
progressive patients after in vitro stimuli.
Conclusion: Patients affected by progressive form of MS showed an
altered distribution of T cells subsets and high level of activated T
cells. Moreover, TEM cells from PP patients were characterized by
higher mTOR activity, high mitochondrial mass and high level of
O2- production. These data support the hypothesis that T cells from
patients with PP MS tend to shift more rapidly and easily towards
effector cells, and mediate a rapid progression of the disease.
92
Modulation of HLA-DR on Monocyte Subpopulation
in ICU Patients
Alessandra Cattaneo1, Elena Trombetta1, Cecilia
Frugoni1, Paola Cetrangolo1, Alessandro Protti2, Jacopo
Colombo2, Laura Porretti1
1
Flow Cytometry Service, Laboratory of Clinical Chemistry
and Microbiology, Fondazione IRCCS Ca' Granda
Policlinico, Milano, Italy, 2Department of Anesthesiology,
Intensive Care and Emergency, Fondazione IRCCS Ca'
Granda Policlinico, Milano, Italy
Background: HLA-DR molecules play a central role in the specific
immune response as they are required for antigen presentation and
activation of helper T-lymphocytes. Many studies have shown that
the HLA-DR decrease on monocytes after major injury, surgery or
organ transplantation, could correlate with infectious complications
and the development of sepsis. Despite the extensively studied
major population of classical CD14bright monocytes (Cmonocytes), little is known about CD16+ monocytes, which is
further divided in “intermediate” CD16+CD14+ (I-monocytes) and
“non-classical” CD16+CD14low/- (NC-monocytes). The aim of our
study is to look at the modulation of HLA-DR expression on the
three monocyte subpopulations by flow cytometry in patients
admitted in intensive care unit (ICU).
Patients and Method: 30 consecutively ICU patients (20 males and
10 females, mean age 52,7±13,5) and 13 healthy controls (6 males
and 7 females; mean age 41,7±13,4) were included in the study.
Patients were admitted in ICU for septic shock (16) or other causes
(2 hemorrhagic shock; 3 lung transplant; 2 acute respiratory distress
syndrome; 2 cardiovascular causes; 1 cranial trauma; 1 cancer; 1
acute pancreatitis). All patients survived the ICU period. For
monocytes immune-phenotyping 150ul of whole blood were
stained in a five color single tube containing CD14 FITC/CD16
PE/HLA-DR Per-CP/CD4 APC/CD45 APC-H7. Monocytes were
gated as CD4dim events in a SSCvsCD4 dot plot; then they were
subdivided in C, NC and I subpopulations in a CD14vsCD16 dot
plot and for all of them the percentage and the Median Channel of
Fluorescence (MCF) of HLA-DR expression were also evaluated.
Results: ICU patients had more I-monocytes (22,4±15,1% vs
10±4,5%, p=0,0004) and less NC-monocytes (3,5±2% vs
5,9±2,9%, p= 0,005) compared to controls. No significant
differences were found between ICU patients and controls in HLADR expression on NC subsets, both as percentage and MCF,
(92,9±11,3% vs 98,5±1,7% and 2180±1429 vs 2621±923,6,
respectively). Conversely, on C and I-monocytes the % and MCF
HLA-DR expression were significantly lower in patients as
compared to controls (79,2±11,3% vs 97,4±2% and 90,5±10,2%
vs 99,1±0,6%; MCF 602±468,8 vs 1379±531,8 and 2501±1942 vs
5811±2743 for C and I, respectively, p=0,0001). Interestingly, MCF
Congress
Overview
93
Biomarkers for Predicting Tolerance in Living Donor
Liver Transplantation
94
Parallel Monitoring of Human Rhinovirus- and
Allergen-Specific CD4+ T Cells during Experimental
Infection in Allergic Asthmatics
Special
Lectures
Kriti Jain1, Sangeeta Choudhury2, Naimish Mehta3,
Samiran Nundy3, Manoj Gupta3
Lyndsey Muehling1, Peter Heymann1, Paul Wright1, Duy
Mai2, William Kwok2, Judith Woodfolk1
Conclusions: HLA-DR is differently modulated on monocyte
subpopulations. In particular I- monocytes seem to be an interesting
cell population to monitor for the evaluation of monocyte immunerecovery in ICU patients.
1
Index
141
Speaker/Author
Conclusion: pDC:mDC ratio can serve as an index of clinical
unresponsiveness or rejection and may be used as a potential
biomarker to determine weaning-off immunosuppression in LDLT
Poster Session
Abstracts
Post-transplantation, pDC subsets expressed CD80,CD154
(CD40L); the mature DC marker CD83; apoptotic antigen CD205
(p<0.005) leading to low pro-inflammatory cytokine production
and T-cell dependent activation.
Oral Session
Abstracts
Conclusions: Longitudinal monitoring of antigen-specific cells
during viral infection reveals complex interactions between RVand Ag-specific cells. Our data confirm that T cell responses to RV
in atopic asthma comprise a mixture of RV- and Ag-specific cells
with heterogeneous phenotypes, and suggest a role for both
amplified Th1 and aberrant Th2 responses in virus-induced asthma.
Integrating these observations with clinical and other immune
parameters offers the promise of identifying new cellular and
molecular targets for the treatment and prevention of virus-induced
asthma exacerbations.
Commercial
Tutorials &
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Results: Both circulating RV- and Ag-specific memory T cells were
readily identified prior to RV inoculation in atopic asthmatics using
dual tetramer staining. In healthy controls, Ag-specific T cells were
rare and not modulated during RV infection. Both RV- and Agspecific T cell populations expanded during the effector phase of
infection in allergic asthmatics, with exaggerated RV-specific
expansion in allergic asthmatics as compared with controls.
Discrete molecular signatures were associated with each T cell
specificity. While Th1- and Th2-like signatures were associated
with RV- and Ag-specific cells respectively, we observed complex
antigen-specific populations with signatures that deviated from
“classical” Th1 and Th2 phenotypes. Both RV- and Ag-specific
phenotypes shifted during infection, consistent with cell activation
and re-direction to sites of infection. In healthy subjects, circulating
RV-specific T cells fit a Th1 profile, demonstrated cross-reactive
function, and included T follicular helper (Tfh) cells that are critical
for providing help to B cells for antibody production.
Scholars &
Emerging
Leaders
All patients showed CD80-pDC/mDC except HCV-induced
cirrhotic patients who had higher CD80+ expression in comparison
to normal. CD205 (endocytic receptor), crucial in induction of
immunity to intracellular viral pathogens was highly expressed
post-transplantation, before induction therapy. Higher expression
CD83 in mDC than pDCs when compared to pre-transplant status
were observed.
Methods: We utilized novel peptide/MHCII (pMHCII) tetramers in
conjunction with an experimental infection model to construct a
profile of T cell immunity to RV. Allergic asthmatic and otherwise
healthy control subjects were inoculated with RV-16. Venous blood
was collected prior to inoculation, as well as during acute (days 4
and 7) and convalescent (day 21) infection. RV- and Ag-specific T
cells were identified by staining cells from HLA-diverse subjects
with novel pMHCII tetramers, including both RV capsid proteinspecific and Ag-specific tetramers, and then analyzing by multicolor flow cytometry.
Poster
Session
Two acute rejection cases very clearly depicted significant gradual
decline in pDC:mDC ratio (upto 50%) correlating with allograft
rejection.
Background: Rhinovirus (RV) is a major cause of the common cold,
as well as a major trigger of allergic asthma exacerbations. These
episodes typically occur during peak exposure to inhalant allergens,
in the Spring and Fall. It is thought that virus-induced asthma arises
from deficient anti-viral Th1 responses, or else the amplification of
Th2 responses. While not mutually exclusive, these theories are
difficult to reconcile using conventional experimental systems and
tools. To surmount these barriers, we have designed an
experimental approach that allows us to monitor both circulating
RV- and Allergen (Ag)-specific CD4+ T cells in asthmatics during
experimental RV infection.
Wednesday
15 June
Result: The incidence of pDC decreases significantly posttransplantation, before induction therapy (28%-46%) as compared
to day 8th of induction therapy (24%-39%) with a significant higher
pDC:mDC ratios in normal donors (p<0.001) implicating an early
cell-mediated immune response, which reversed by day 8th of anticalcinurin and other DNA adduct cleavage drugs.
Benaroya Reserach Institute, Seattle, WA, United States
Tuesday
14 June
Peripheral blood mononuclear cell population were isolated to
identify Tregs (CD4+CD25+FoxP3+) and DCs (HLA-DR+lin–) to DC
subsets as CD11c+CD123–/lo(IL-3Ra–/lo (myeloid DC; mDC) and/or
CD11c– CD123hi(plasmacytoid DC; pDC) using a 8-color flow
cytometric analysis at 3 time points, pre-transplantation, before
induction of therapy (post-transplantation) and post-induction
therapy. Also delineated its association with the graft function
(CD80, CD83, CD205, CD154).
2
Monday
13 June
Introduction: Survival after living donor liver transplantation (LDLT)
has improved due to advances in surgical technique, perioperative
and post-transplant intensive care management and availability of
better immunosuppressive drugs. Thus, it is imperative to develop
robust biomarkers for detecting tolerance. Human dendritic cell
(DC) subsets play distinct roles in the induction and regulation of
immune responses. The present study aim to establish a set of
biomarker for early detection of rejection as per which immunosuppressive drug dosage can be optimized in LDLT patients
resulting in prevention of acute liver failure. In order to further
understand the status of maturation with reference to allogenic graft
acceptance, Tregs, CD28 and co-stimulatory (CD 80/83) molecules,
CD205,CD154 were evaluated.
Method: 50 LDLT patients (recipients) suffering from chronic viral
hepatitis (HBV/ HCV-induced) or HCC undergoing LDLT (based on
MELD score) and 50 healthy liver donors as controls were enrolled
in the study; comprising of both sexes, with a median age of 45
years (25-65 years). Exclusion criteria included those with active
viral infection and macrosteatosis/ liver attenuation index <5.
Immunosuppressive regime constitutes Methylprednisolone (pulse
therapy) / Tacrolimus / Prednison / Mycophenolate mofeti.
1
Sunday
12 June
NIMS University, New Delhi, India, 2Research, Sir Ganga
Ram Hospital, New Delhi, India, 3Surgical Gastroenterology
and Liver Transplantation, Sir Ganga Ram Hospital, New
Delhi, India
Saturday
11 June
patients. Anti- rejection therapy may be provided to those with
alteration in DC subset profile. Role of CD154 and CD83 correlated
with the clinical status of rejection and survival inducting the
maturation status and producing inflammatory cytokines. In future,
pDC may be a more appropriate cellular alternative for the
induction of clinically-applicable tolerance.
HLA-DR expression on I-monocytes evaluated in the same patient
at admission and dismission from ICU (n=13), showed a significant
increment (1337±802,9 vs 3263±1364, p<0,005).
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Index
Abstracts
95
Cell Painting Parametisation of Cellular Phenotypes
within
Heterogeneous Populations
Rachel A. Howard-Jones1,2, Karin Tuvstog1,2, Marie
Wiltshire1, Victoria Griesdoorn1, M. Rowan Brown3,
Alastair J. Sloan2, Rachel J. Errington1
1
School of Medicine, Cardiff University, Cardiff, United
Kingdom, 2School of Dentistry, Cardiff University, Cardiff,
United Kingdom, 3College of Engineering, Swansea
University, Swansea, United Kingdom
Background and Aims: U-2 OS cells are a human osteosarcoma
cell line derived from wild type p53 tumours; treatment with
Nutlin-3, a small molecule inhibitor of the MDM2/p53 interaction,
leads to non-genotoxic p53 stabilization and subsequently
accumulation of tetraploid cells through mitotic bypass. These
tetraploid cells can become stable in the tumour and are resistant to
many chemotherapy drugs, however, little is known about the
surviving cells and any specific features they have. The focus of this
study was to determine whether these polyploidy cells showed the
capacity for endoreduplication and cell cycle progress, whilst
profiling their biomarker expression for stem cell-ness. We also
sought to identify and characterise feature-based analysis of cellular
phenotypes of populations recovering from the perturbation. We
adopted an imaging process termed cell painting through the
labelling of cellular organelles with several fluorophores, to
determine numerous phenotypic features and patterns.
Methods: Cells were treated with Nutlin (5μM) for 24 hours,
washed and cultured for 2-9 days prior to extracting RNA for RTPCR; labelling with DAPI, MitoTracker Deep Red and Phalloidin
Alexa Fluor 488 or processing for cell cycle analysis. Images were
acquired using an automated wide field microscope and analysed
using CellProfiler pipelines.
Results: C-myc, Klf-4, Oct-4 and Sox-2 were expressed in both
treated and untreated cells, whilst Nanog and Lin-28 were not
detected. An increasing dose of Nutlin-3 led to the progressive
slowing of the cell cycle, evident from a depletion of the S-phase
compartment, which was complete at doses of 5ȝM Nutlin-3.
There was a significant increase in the tetraploid G1p subpopulation at the cost of G2 cells. When released after 24 hours,
the cells regained the ability to proceed through the cell cycle.
There was also an indication that the G1p sub-fraction had a
minimal capacity for replication. Next, cell painting was used to
detect these polyploid phenotypes in a 5-day recovering tumour
population. Morphological features were extracted from each cell
using CellProfiler. The polyploid phenotypes were detected
manually allowing optimisation of the pipelines using 696 (control)
and 477 (treated) cells. The established pipeline provided
morphological features that could be quantified by microscopy to
create image-based profiles of the polyploid cells. Total
fluorescence intensity (DAPI) enabled derivation of DNA content
and analysis of nuclear total intensity versus total cell size enabled
detection of the polyploid cells. Initial analysis found that macromorphological measures may not be suitable to differentiate treated
versus untreated populations. Further data mining will be
presented.
Conclusion: Our results showed that Nultin-3 alone could promote
mitotic bypass. The current work further demonstrated that cells
recovering from mitotic bypass were capable of endoreduplication.
This in turn imposed significant degrees of heterogeneity on cancer
cell populations while the cell cycle origins of such cellular cohorts
would not be apparent. Cell painting enabled us to process and
identify sub-populations of cells with specific features. This will be
useful for the development of new drug therapies and also for
application in regenerative medicine for identification of specific
cell populations within heterogeneous primary cultures.
96
Direct Learning of a Generative Model for
Endoplasmic Reticulum Distribution from
Fluorescence Microscopy Images
Kelvin Liu-Huang1, Tina Lee2, Gustavo Rohde3,4, Robert
Murphy1,2
1
Computational Biology, Carnegie Mellon University,
Pittsburgh, PA, United States, 2Biological Sciences, Carnegie
Mellon University, Pittsburgh, PA, United States,
3
Biomedical Engineering, Carnegie Mellon University,
Pittsburgh, PA, United States, 4Electrical & Computer
Engineering, Carnegie Mellon University, Pittsburgh, PA,
United States
The accuracy of purely quantitative biochemical models can be
improved by including a model of subcellular organization.
Generative models can capture subcellular relationships between
size, shape, and distribution of organelles. By applying statistical
machine learning to large cohorts of subcellular patterns, we have
developed generative models of these structures, primarily vesicular
organelles and microtubules (MT). However, generative models of
more complex structures such as reticular or cisternal organelles
have not been described. The endoplasmic reticulum (ER), the
primary biosynthetic and transport organelle, is a complex a
reticular distribution which interacts extensively with MT in
mammals.
Therefore we have developed a generative model of the
endoplasmic reticulum (ER) which captures the relationship
between the ER distribution and the cell shape, nuclear shape, and
MT distribution. In our model, ER is a graph whose vertices are
distributed according to MT intensity and fractional distance from
nucleus, and whose edges are distributed according to length. We
learn our model from three-dimensional two-channel fluorescent
microscopy images, in which ER and MT are fluorescently labeled
with atlastin and tubulin. These images are processed to construct
the ER graph. We then learn a probabilistic model of the
relationship between ER vertices and the fractional distance
between the nuclear membrane and cell membrane. Next we learn
the dependence of ER vertices on MT intensity. Finally we learn the
dependence of ER edges on length. With this generative ER model,
we can render images of synthetic cells belonging to the same
distribution as the training images. During synthesis, we sample
vertices of the ER from the product distribution of MT and distance,
treating them as independent, and then sample ER edges from the
length distribution. We render this ER graph as a three-dimensional
image.
We show that our method can learn and synthesize images of ER
which are visually similar to training images. The way in which
we've constructed this generative model uses easily interpretable
parameters, allowing us to compare models. We plan to use this
approach to compare ER distributions under various perturbations
and conditions, such as drugs, disease states, or cell cycle.
This work was supported in part by National Institutes of Health
grants GM090033, GM103712, and EB009403.
97
A Statistically Robust Automated Method for
Leukemia Detection Using Multi-parametric Analysis
on Imaging Flow Cytometry Data
Vidya Venkatachalam1, Yusuke Konishi2, Haley Pugsley1,
Phil Morrissey1, William Ortyn1, Ellinor Peerschke3, Peter
Maslak3, Tomohiro Tsuji2, Seiichiro Tabata2
1
Amnis, part of MilliporeSigma, Seattle, WA, United States,
Sysmex Corporation, Kobe, Japan, 3Department of
Laboratory Medicine, Memorial Sloan-Kettering Cancer
Center, New York, NY, United States
2
142
1
Miftek Corporation, West Lafayette, IN, United States,
Basic Medical Sciences, Purdue University, West Lafayette,
IN, United States, 3Bomedical Engineering, Purdue
University, West Lafayette, IN, United States
2
Oral Session
Abstracts
Cytometry
Index
143
Speaker/Author
Introduction and Background: We previously presented scanning
small laser beam cytometry titled Micro Imaging Flow Cytometry
(MIF). The principle of conventional flow cytometry is detection of
Mie scattering and spatially integrated fluorescence which is very
useful for quantitative cellular analysis. A fundamental difference of
the scanning small beam concept is time sequential detection from
any part of cellular structure on transmission, polarization,
diffraction and excited fluorescence. We now present a new
technology that is enhanced by using time domain analysis. This
Poster Session
Abstracts
Masanobu Yamamoto1,2, Keegan Hernandez1, J. Paul
Robinson1,3
Commercial
Tutorials &
Exhibits
Deep phenotypic profiling of the small number of cells that can be
acquired from low yield clinical samples is challenging using
current polychromatic flow methodologies. To facilitate acquisition
of detailed datasets from these important clinical samples, we have
developed MuSIC, a methodology that integrates nanowell arrays,
optimized microscope hardware, custom image processing software
and a novel bioinformatic analytic pipeline to enable integration of
high content cytometry and other proteomic and transcriptomic
measurements on sparse clinical samples. Cells are initially
captured in nanowell arrays enabling retention of live cells in
defined locations throughout the staining and imaging process.
After staining on the array with a polychromatic antibody panel,
spectral image stacks of the cells are acquired through a
combinatorial combination of excitation and emission filters on an
epifluorescent microscope driven by a custom software interface
optimized for efficient image acquisition. After image acquisition,
cells remain viable and can be further analyzed for protein
secretion or individually micromanipulated for single cell RNA
100
Time Domain Analysis by Scanning Small Laser Beam
Scholars &
Emerging
Leaders
Leaders
Koch Institute, Massachusetts Institute of Technology,
Cambridge, MA, United States, 2Chemical Engineering,
Massachusetts Institute of Technology, Cambridge, MA,
United States
Poster
Session
1
Keywords: imaging, flow cytometer, cell sorting, fluorescence, high
throughput, PMT, microfluidic, spatial-temporal transformation
Wednesday
15 June
Todd Gierahn , Denis Loginov , J. Christopher Love
Flow cytometry and cell sorters analyze and sort cells based on
multiple physical characteristics of a large population of single cells
as cells flow in a fluid stream through an excitation light beam.
Flow cytometers measure fluorescence and light scattering from
which information about the biological and physical properties of
individual cells are obtained. Although flow cytometers have
massive statistical power due to their single cell resolution and high
throughput, they produce no information about cell morphology or
spatial resolution offered by microscopy, which is a much wanted
feature missing in almost all flow cytometers. We invented a
method of spatial-temporal transformation to provide flow
cytometers with cell imaging capabilities. The method uses
mathematical algorithms and a specially designed spatial filter as
the only hardware needed to give flow cytometers imaging
capabilities. Instead of CCDs or any megapixel cameras found in
any imaging systems, we obtain high quality image of fast moving
cells in a flow cytometer using photomultiplier tube (PMT)
detectors, thus obtaining high throughput in manners fully
compatible with existing cytometers. Above all, the approach is
fully compatible with image-based cell sorting partly because the
high efficiency of image reconstruction algorithms support real-time
cell imaging and partly because the technology platform can be
readily integrated with existing cell sorting techniques. We will
demonstrate high throughput (1000 cells/s), multi-parameter cell
imaging and the feasibility of image-based cell sorting in a flow
cytometer platform.
Tuesday
14 June
1,2
1
Department of Electrical and Computer Engineering,
University of California, San Diego, San Diego, CA, United
States, 2Nanocellect Biomedical, Inc., San Diego, CA,
United States
Monday
13 June
1
Yuanyuan Han1, Sunghwan Cho2, Yi Gu1, Alex Zhang1,
Yu-Hwa Lo1
Sunday
12 June
1
Saturday
11 June
98
Deep Phenotypic Profiling of Small Clinical Samples
through Multispectral Imaging Cytometry
99
Imaging Flow Cytometer and Cell Sorter Using
Spatial-Temporal Transformation
Special
Lectures
We present the results of using this approach for leukemia detection
in a clinical study performed at Memorial Sloan-Kettering Cancer
Center in collaboration with Sysmex Corporation. For this study, we
evaluated approximately 150 samples from patients in various
stages of treatment for 6 leukemic conditions - ALL, CLL, AML,
CML, MDS and MPN; along with 120 normal samples and about
100 non-leukemia cancer samples. The incidence of leukemic cells
in the samples spanned a wide range from 0.02% – 97%. Every
sample was acquired in triplicate on 3 ImageStream® imaging flow
cytometers using 3 imaging modalities - Bright Field, Side Scatter
and Nuclear images. All data was analyzed using the IDEAS®
statistical image analysis software with a custom template that
contained the automated classification strategy with classifiers
trained to identify eosinophils, neutrophils, basophils, monocytes,
lymphocytes, blasts, immature myeloids and abnormal
lymphocytes. The efficacy of the classification output was
determined by comparing against the traditional manual differential
as well as flow cytometry analysis data and the sensitivity and
specificity of detection were computed. Our automated
classification method achieved a sensitivity of 98.5% across all
leukemia conditions and a specificity of 98.9% averaged over the 3
instruments with excellent concordance between the instruments.
Encouraged by these results, we are interested in extending this
automated classification method to a more detailed investigation of
leukemia while also evaluating the methodology for use in other
application domains.
analysis. For analyzing the images, fully automated image
processing software extracts both cell and well locations and cell
spectral intensities and then compensates the intensities for spectral
overlap using spillover matrices simultaneously generated from
images of singly stained beads. The resulting data can be
hierarchically gated using typical 2D plots or analyzed using
unsupervised nonlinear clustering algorithms. All data can be
traced back both to an individual cell and a well for integration
with further ‘omic measurements. Our current machine can image
up to 106 cells in 16 spectral channels in 25 minutes, enabling
scalable and robust phenotypic profiling of small clinical samples.
Congress
Overview
Leukemia is often associated with an abnormal manual blood cell
differential. This differential is typically performed by experienced
personnel and involves manually examining between 100-200
images to look for specific morphological characteristics of different
cell types. This manual process suffers from a lack of objectivity,
repeatability, reportability and scalability, making it difficult to
identify rare events or quantify subtle changes. We address these
limitations by designing an automated paradigm that can rapidly
classify several thousand cellular images into specific populations.
First, we extract over a thousand parameters from each cell image
that characterize the cell shape and texture. Next, we train our
classification engine using example images of populations of
interest to select the features that best discriminate between the
populations. We then combine these features to generate a
classifier for each population. Finally, we perform a statistical
evaluation of the classifier outputs to identify the prevalence of the
different populations in the sample. The analysis strategy can then
be applied to patient samples to identify specific populations that
are indicators of disease. This process removes the manual
subjectivity from the analysis, producing results that are objective,
repeatable, statistically significant and scalable with data size.
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Index
Abstracts
enables us to provide a new cellular analysis approach based on
time-sequential cytometry (TSC) with proof of concept.
Method
and Results: Time sequential observation of specific live
cells is not possible by conventional flow system. But because of
our high-speed beam-scanning with relatively slow flow velocity
(less than 1m/s), a flow chamber mounted on a high speed linear
motor slide can move faster than cell flow. The focused planar flow
has over 20mm of laminar flow, so it is possible to make initial
observation of a targeted cell, then observe after nǻt. If necessary,
separated 3 laser beams along the direction of flow can detect
localized flow velocity and shorter time interval observation. This
approach may contribute to analysis of rapid biochemical
responses in a cell (or multiple cells) under exposure of reagent,
temperature and ambient light, etc. Another new aspect is
fluorescence detection in the time-domain. The latest photodetector
like microPMT by SiMEMS process or SiPM as microcell APD
provides single photon resolution less than 1ns. The dark count at
room temperature is significantly improved comparing to previous
device. Combined with low noise and very high-speed electronics,
it is now possible to evaluate atto (10-18) watt level fluorescence
photons with picosecond resolution. Preliminary evaluation
suggests very promising frontier for cellular and intercellular
analysis- verylow level fluorescence detection, photon “plotting” on
cell morphology, photo-bleaching and resonance phenomena
analysis, etc. On the other hand, there are many hurdles to
overcome. One of the challenges is the significant autofluorescence
of detecting optics. We found many optical materials and coatings
exhibit auto fluorescence depending on illumination wavelength
and power, especially when using shorter wavelengths (<410nm).
The issues still have to be resolved and may require material
development and smarter design.
Conclusion: After the proof of concept, we demonstrated scanning
small laser beam concept in time domain has potential capability
and possibility for contributing cellular and intercellular analysis.
Single photon fluorescence detection is challenging and may open
new field by applying the latest device technology. We believe this
is a new opportunity using a technological paradigm change in
cellular analysis.
101
A Frequency-Multiplexed Parallel Inertial Flow
Cytometer for High-Throughput Screening and Drug
Discovery
Jonathan Lin1, Keegan Owsley1, Eric Diebold2, Dino Di
Carlo1
1
2
Bioengineering, UCLA, Los Angeles, CA, United States,
Omega Biosystems, Inc., Los Angeles, CA, United States
Conventional fluorescence flow cytometry is a ubiquitous and
invaluable tool in biology and drug screening, allowing multiparameter, single-cell-level measurements of biochemical markers.
Though conventional flow cytometers can be capable of high
throughputs (~10,000 cells/second), their sample throughput is low
due to the need for serial processing. In this work, we demonstrate
a parallel flow cytometer system that is capable of simultaneous
interrogation of eight samples through combination of Fluorescence
Imaging using Radiofrequency-tagged Emission (FIRE) and inertial
microfluidic focusing. FIRE, a high-speed optical technique, enables
the use of a single photomultiplier tube to measure the fluorescence
and scatter from multiple points in space. FIRE is used to interrogate
parallel streams of cells that are focused using inertial flow field
shaping, removing the need for a complex sheathing system.
Inertial focusing also allows for high cell densities by decreasing the
occurrence of multi-cell events.
FIRE microscopy enables fluorescent measurements from multiple
points in a sample utilizing a single laser for excitation and a single
photomultiplier tube (PMT) for signal detection. At its core, FIRE
works by simultaneously exciting multiple points in a field of view,
each at a unique beat radiofrequency created by two interfering,
frequency-shifted beams of light. In this application, eight points
coinciding with eight flow channels are excited and a PMT is used
to measure the scattered light and fluorescence emission of samples
passing through the flow channels. Due to the unique excitation
144
frequencies, fluorescence measurements from each channel can be
computed in real time by performing a Fourier transform on the
output signal.
In order to leverage the high-speed nature of FIRE microscopy and
to remove the need for complicated sheath flow systems, inertial
microfluidic focusing is used to organize cells into two ordered,
vertically-stacked streams. This is accomplished by taking
advantage of stable particle equilibrium positions caused by
opposing shear gradient lift forces and wall forces in high aspect
ratio flow channels and by disrupting undesirable equilibrium
positions using inertial flow shaping techniques. Additionally, we
exploit particle inertia in order to excite samples in a region of the
flow channel without bounding walls which would otherwise
decrease the sensitivity of the cytometer. It is worth noting that
inertial flow focusing is compatible with high velocity flows and
flow rates in excess of 6 ml per minute have been demonstrated in
this system.
This project represents a unique combination of microfluidic
inertial flow physics, radiofrequency photonics, and high speed
digital signal processing, which enables flow cytometry analysis at
unprecedented sample throughputs. This innovation opens the door
to the use of flow cytometry in truly high-throughput applications
including rare cell analysis and drug discovery using high-content
screening. In doing so, we bring a powerful biological research
technique to the high sample throughput screening world, allowing
for single-cell-level measurements which better represent
population drug-cell interactions compared to traditional aggregate
readout techniques.
102
Flow Cytometry Adds Imaging: Introducing a New
High Throughput Imaging Flow Cytometer from
Omega Biosystems
Eric Diebold, Jonathan Lin, Keegan Owsley, Matthew
Bahr, David Borlaug
Omega Biosystems Incorporated, Los Angeles, CA, United
States
The power of flow cytometry stems from the technique's ability to
enumerate the number of fluorescent probe molecules attached to
single cells at high throughput. This quantification can be
multiplexed such that many (currently up to ~50) probes for distinct
biological molecules can be analyzed at the same time. Modern
instruments can record data from cells at event rates of tens of
thousands per second, which provides incredible statistical
robustness in data collection. While flow cytometers can collect a
large number of parameters from individual cells, the measurement
of these parameters is fundamentally macroscopic and indirect in
nature, as flow cytometers lack the sub-cellular resolution to
visualize where probe signals originate within a cell. While
automated microscopy or plate imagers can provide high resolution
images of cells with relatively high sample throughput, the data
collected is typically limited to only 4 spectral bands, due to the
high cost and complexity of the optical systems and detectors
employed. Recently, imaging flow cytometry has emerged as a
commercial technology, which adds sub-cellular resolution to flowbased cell analysis. However, due to limitations in the detector
technology, the cellular throughput of these instruments is relatively
limited compared to flow cytometers, and like plate imagers, it is
cumbersome to scale their fluorescence parameters to large
numbers.
Here, we introduce a new class of flow cytometer that combines
the high throughput (>20,000 events/second) and multiparameter
detection capabilities of traditional flow cytometry with the subcellular imaging capabilities of fluorescence microscopy without
significant tradeoffs exhibited by other technologies. By utilizing a
novel engineered illumination and detection scheme employing
radiofrequency multiplexing techniques, conventional
photomultiplier tube detectors are rendered capable of functioning
as imaging detectors, allowing conventional flow cytometry
hardware to produce high resolution, high sensitivity, multiparametric image data in combination with conventional flow
cytometry data. There are 2 primary categories of advantages that
1
NYU Med Ctr, New York, NY, United States, 2Cincinnati
Children’s Hospital Medical Center, Cincinnati, OH, United
States
Index
145
Speaker/Author
Cell sorting in a Shared Resource Laboratory (SRL) is an often
complex task that requires multitasking, scientific understanding
and good customer service. In busy SRLs, where there are often
many users and extremely tight schedules, the efficiency,
Poster Session
Abstracts
Michael Gregory1, Monica DeLay2
Oral Session
Abstracts
Recent advances in quantitative neuroanatomy have called into
question whether neuron number is a more informative
measurement of computational capacity. This talk will focus on the
development of a highly novel method, which combines a
technique called isotropic fractionation with both standard and
imaging flow cytometry, to count neurons (versus non-neuronal
106
Best Practices for Providing Cell Sorting Services in a
SRL
Commercial
Tutorials &
Exhibits
Selection for cognitive ability has been proposed as a key factor
driving the evolution of larger brains and/or the brain structures
associated with problem solving, social behavior and other
cognitively demanding tasks. These brain structures are often
subject to different selection pressures, resulting in a significant
degree of variation in brain size and complexity across vertebrates.
Of particular interest are the cartilaginous fishes (sharks and their
relatives); although the most basal of the jawed vertebrates,
previously thought to have relatively small brains with a limited
behavioral repertoire, these fishes have brain/body ratios that are
actually comparable to birds and mammals. There is also high
variability in brain organization across this group that is associated
with habitat and or behavioral patterns, which allows us to be
predictive about sensory specialization and behavior, social
intelligence, and even survivorship.
It is a well-known fact that diagnosis of diseases, immunotherapies
and drug development is always made respectively to healthy
donor blood controls. It was demonstrated by Dr. Mario Roederer
that mean fluorescence intensity (MFI) is a critically important
parameter for differentiating autoimmune patients from healthy
controls in CYTO 2015. It is clear that after the linearity calibration
of flow cytometers by using calibration beads with assigned values
of equivalent number of reference fluorophore (ERF), a normal
blood reference control/material with known ranges of biomarker
expression, percentage of cell subtypes and/or certain genetic
contents would greatly help clinicians to make quick and robust
prognosis and diagnosis of diseases. In fact, Beckman Coulter
produces a freeze dried PBMC product, Cyto-Trol Control Cells, the
National Institute for Biological Standards and Control in UK
produces lyophilized PBMC pre-stained with anti-human CD4 FITC
antibody, UK NEQAS (National External Quality Assessment
Service) produces stabilized whole blood product, and BD
Biosciences is actively developing freeze dried PBMC pre-stained
with anti-human CD4 monoclonal antibody labeled with different
fluorophores. Additionally, NIST has been building the expertise
and capabilities in the characterization of blood cell preparations in
the last five years. Therefore, the time is ripe to best utilize all these
production resources and characterization capabilities for the
production of a normal blood reference material for the
advancement of cytometry. The key questions for the workshop are:
1) what biological reference materials are critically needed at
present and in near future? 2) who is interested and capable for the
production? 3) what quality matrix reference materials should fulfill
(properties, storage condition, and stability)? 4) what parameters
should be well characterized? 5) what evaluation and validation
study is needed for their use? The workshop will be formatted with
a few concise presentations followed by discussion and conclusion
with biological reference material recommendation.
Scholars &
Emerging
Leaders
Leaders
School of Animal Biology & UWA Oceans Institute,
University of Western Australia, Crawley, Australia
NIST, Gaithersburg, MD, United States, 2Consultant,
Poster
Session
Kara Yopak
3
Wednesday
15 June
104
Sending Sharks to School: Novel Methods For
Quantifying Cognitive Ability in Fishes
1
Tuesday
14 June
The ecosystems of the world’s oceans are supported by large
numbers of small phototrophic organisms. Flow cytometry is a good
tool for studying the composition and dynamics of these microbes.
Because of their diminutive size and dim fluorescence, optimal
measurement strategies for phytoplankton differ from the traditional
approaches in flow cytometry. I will discuss some of the technical
modifications that have been found useful in pico-plankton
analysis. Specifically, I will present the use of fluorescence action
spectra for the identification of phytoplankton forms. I will also
introduce high-resolution depth profiles obtained by flow
cytometry.I will illustrate the use of these techniques in the study of
the plankton communities at Station ALOHA and the Oxygen
Minimum Zone (OMZ)off the coast of Peru. The field studies show
that the phenotype distributions of populations of Prochlorococcus
and Synechococcus are highly stable. Both species display photoacclimation in response to deminishing light intensities with depth.
Physical disturbances of the water column rearrange the structure of
the microbial communities. The equilibrium state is restored within
a few days.
Lili Wang1, Robert A. Hoffman2, John P. Nolan3
Monday
13 June
Center for Marine Cytometry, Concrete, WA, United States
Sunday
12 June
Ger van den Engh
105
Biological Reference Materials for Enabling
Quantitative Flow Cytometry Measurements in
Antibodies Bound per Cell
Saturday
11 June
103
Phytoplankton Analysis with Flow Cytometry
This talk will present data on total brain cell counts in
representative fish species in the context of currently available
mammalian data, and discuss the potential for conservation of
neuronal scaling across vertebrates. These data will pave the way
for future work to assess whether the number of neurons within the
major brain regions show a linear relationship or reveals differential
rates of addition in relation to predicting more complex behavioral
repertoires in fishes, with implications for how “intelligence” has
evolved across vertebrates.
Special
Lectures
In this presentation, we will unveil the science and engineering
concepts that enable this revolutionary instrument, as well as
discuss some of the advanced data handling and processing
features of the system that enable new parameters, such as
fluorescence lifetime, to be extracted from samples. We will show
data from conventional experiments verifying the instrument
performance, as well as data from experiments such as rare cell
detection, which highlight the immense value that the ability to
image cells at high throughput adds to a conventional flow
cytometer.
glial cells) in the brain. To date, brain size-neuron number scaling
relationships have only been established for mammals, with no
validated or comparable data sets available for non-mammalian
vertebrates. Determining the degree of variation in brain size,
neuron number and cognition across fishes is an essential
component of advancing our understanding of fundamental aspects
of brain design that both constrain and enable behavioral
complexity.
Congress
Overview
such an instrument provides for the user. First, image-based assays
can be performed on samples quickly and with high statistical
significance, using a large number of image-based parameters
measured directly from large populations of cells. Secondly, imagebased analysis can be mapped directly onto flow cytometry data for
data confirmation with high sensitivity and specificity. This point is
particularly important for analysis of rare cell populations, but is
also generally important to any experiment in which the user
wishes to confirm assumptions made on flow data.
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Index
Abstracts
knowledge and skill of sort operators directly impact the success of
the SRL. Important factors that determine best sorting outcomes
include gathering relevant information, scheduling efficiently,
selecting
proper sort conditions, tracking experimental outcomes
and communicating effectively. Best practices for cell sorting aimed
at improving sort quality, time management, and investigator
engagement would be beneficial for those new to sorting as well as
those hoping to improve the success of their cell sorting services.
This workshop will be targeted toward anyone who sorts in a
Shared Resource Laboratory. We will identify the challenges
specific to providing cell sorting services in a SRL and the skills
necessary to meet them. We hope to identify and come to
consensus on best practices for the following: 1) effective ways to
gather information from the client that is needed to perform the sort
correctly 2) factors that should be considered and methods for
completing scheduled sorts in a timely manner and despite
obstacles 3) determining whether a sort was successful. Ultimately,
the workshop will formulate best practices for SRL sort operators as
well as a list of questions sort operators should ask users to ensure
they have all the necessary information for a successful sort.
107
Workshop on Perivascular Cell Subsets
Albert Donneberg1, Vera S. Donnenberg2, Julie
Phillippi3, Anna Brooks4, Mirko Corselli5
1
United States, 2Cardiothoracic Surgery, Univ of Pittsburgh,
SOM, Pittsburgh, PA, United States, 3Cardiothoracic
Surgery, University of Pittsburgh School of Medicine,
Pittsburgh, PA, United States, 4University of Auckland,
Auckland, New Zealand, 5BD Biosciences, San Jose, CA,
United States
Mesenchymal stem cells and lineage committed progenitor cells are
organized around small vessels in multiple tissues including
adipose, the dermis and the vasa vasorum of large blood vessels.
This workshop will consist of a survey of current and potential
research and clinical applications with emphasis on flow-based
quantification, quality assessment, and marker exploration. Experts
from the clinical world, the flow cytometry community and industry
will join the organizers in these presentations.
Desired Outcomes: Formal presentations will be minimized by
limiting presenters to 5 slides each and requesting that registered
participants e-mail data (up to 5 Power Point slides) to the organizer
([email protected]). Specific topics to be discussed
include preparing tissues for characterization and progenitor cell
isolation, in vitro expansion, in vivo graft implantation and
generation of validated SOPs which will be shared among the
participants. Participants will be asked to bring their own data
(BYOD, submitted at the time of registration). All data concerns
will be addressed by the organizers/facilitators off-line. Depending
on the response, selected data analysis problems will be presented
during the workshop.
108
Managing Operations in Multi-user, Multi-site, and
Consolidated SRLs
Christopher Groves1, Rachael Walker2, Claudia
Dumrese3, Derek Davies4
1
MedImmune, Gaithersburg, MD, United States, 2Babraham
Institute, Cambridge, United Kingdom, 3University of
Zurich, Zurich, Switzerland, 4The Francis Crick Institute,
London, United Kingdom
With an increase in the number of multi-user, multi-site SRLs, this
workshop will address different scenarios through group discussions
and expert panel experience.Although a majority of SRLs exist as a
single entity, there is a growing number of multi-site core facilities
which exist in different forms. These include cores that are
geographically dispersed, funded from multiple sources, or that are
being consolidated. With any core, but especially multi-site cores,
146
it is important for there to be standardisation in many areas of
operation including education of users, instrumentation and
instrument configurations, SOPs, and staff training. There are
challenges, which include the difficulty in managing users, ensuring
consistent results across sites, training staff, such that a high-level
service is provided equally to all sites.
During this workshop the audience will work through different SRL
scenarios:.
Single site SRL with users from multiple different locations
Multi-site SRLs working on the same project or study
Bringing together smaller cores to form a larger core
Running several small cores spread across a geographical region
The workshop facilitators will present how they have dealt with
each of these aspects and the workshop will draw to a close with
an audience discussion on ‘What all cores can learn from each of
these scenarios and how the standardisation that is needed in larger
cores can also be applied to SRLs of any size’.
Following the workshop, delegates will have a greater appreciation
of factors to be considered when managing several facilities and be
provided with potential solutions/approaches.
109
Organizing Local Events and Groups; Common Issues
to Overcome
Gelo dela Cruz1, Joel Sederstrom2, Karen Clise-Dwyer3,
Benjamin Daniel4
1
Danish Stem Cell Center, Copenhagen, Denmark,
Cytometry & Cell Sorting Core, Baylor College of
Medicine, Houston, TX, United States, 3M. D. Anderson
Cancer Center, Houston, 4Univ of Texas Health Science
Center, San Antonio, TX, United States
2
Organizing local cytometry user groups and events can be daunting
and overwhelming endeavors to the uninitiated. As Shared
Resource Laboratory professionals, we are expected to organize or
help organize these groups and events for educational, training, and
networking purposes. This workshop will address the numerous
issues that SRL professionals may encounter including funding,
meeting focus and scope, and event logistics. Organizers of several
succesful ISAC-related user groups will impart their expertise and
experience in a group discussion. Through this workshop, we hope
to gain insight and consensus to enable the improvement of existing
and the formation of new user groups, meetings and events.
110
The Hidden Layer of Regulatory RNA in Human
Development and Cognition
John Mattick
Garvan Institute of Medical Research, Sydney, Australia
It appears that the genomic programming of mammalian
development has been misunderstood for the past 50 years,
because of the apparently reasonable but ultimately incorrect
assumption that most genetic information is transacted by proteins.
The mammalian genome contains only ~20,000 protein-coding
genes, similar in number and with largely orthologous functions as
those in other animals, including simple nematodes. On the other
hand, the extent of non-protein-coding DNA increases with
increasing developmental and cognitive complexity, reaching
98.5% in humans. Moreover, high throughput analyses have shown
that the majority of the mammalian genome is differentially and
dynamically transcribed during development to produce tens if not
hundreds of thousands of short and long non-protein-coding RNAs
that show highly specific expression patterns and subcellular
locations, increasing numbers of which are being shown to play
important roles in cancer and other complex diseases. These RNAs
function at many different levels of gene expression, including
translational control and guidance of the epigenetic processes that
J. Huisken, Science (80-. ). 305, 1007 (2004).
8
P.J. Keller, A.D. Schmidt, J. Wittbrodt, and E.H.K. Stelzer, Science
322, 1065 (2008).
112
Rapid, High Dimensional Immunohistochemistry
with Next Generation Instrumentation for
Multiplexed Ion Beam Imaging
Michael Angelo
Pathology, Stanford University, Palo Alto, CA, United States
Multiplexed ion beam imaging (MIBI) is a novel approach to
immunohistochemistry (IHC) that uses secondary ion mass
spectrometry (SIMS) and antibodies labeled with elemental mass
tags to visualize dozens of proteins simultaneously in a single tissue
section. MIBI is compatible with standard formalin-fixed, paraffinembedded (FFPE) tissue specimens, the most common sample type
in clinical repositories worldwide, and can achieve single molecule
sensitivity across a five log dynamic range at resolutions equivalent
to brightfield microscopy. In recent work, MIBI was validated for
imaging breast tumor tissue sections stained with clinically relevant
metal-conjugated antibodies via side-by-side comparison with an
FDA-approved quantitative image analysis platform. Since that
time, my lab has validated over sixty antibodies and has
constructed a 45-plex MIBI panel for characterizing phenotypic and
epigenetic features of epithelial, stromal, and infiltrating immune
cells in clinical breast tumor biopsies. To permit broader use of this
method, we have designed and constructed novel instrumentation
optimized for MIBI that is capable of super resolution imaging and
one 100-fold faster sample throughput. These tools are being used
to comprehensively enumerate immune cell populations in normal
and neoplastic solid tissues, to construct classifiers for predicting
disease progression in pre-invasive cancer lesions, and to develop
quantitative IHC assays to be used in a clinical setting.
113
A Glorious Path of Plant Flow Cytometry
Jaroslav Dolezel
Institute of Experimental Botany, Olomouc, Czech Republic
Oral Session
Abstracts
Poster Session
Abstracts
Index
Speaker/Author
147
Commercial
Tutorials &
Exhibits
Probably the first report on flow cytometry in plants, by then called
impulse cytophotometry, was published more than forty years ago
in 1973. Since that time numerous applications of the method were
developed and used in basic and applied research as well as in
plant breeding and propagation. Interestingly, the spectrum of
applications differs considerably from that in biomedical research.
This reflects the nature of plant cells, which are not easy to isolate
from complex three-dimensional tissues, have rigid cell walls and
irregular shape that makes them unsuitable for flow-based analyses.
One option to make plant cells amendable to flow cytometry is to
strip them from cell walls and isolate protoplasts. Sorting intact
protoplasts provides an opportunity perform transcript and
metabolite profiling in specific tissues. Due to difficulties in
analyzing plant cells, a majority of applications involves the
analysis of subcellular organelles - isolated nuclei being by far the
most frequent target. Despite its relative simplicity, nuclear DNA
content analysis became vital for many areas of research and
practice. The most frequent use has been ploidy screening, which
literally revolutionized plant taxonomy, contributes significantly to
population biology and is irreplaceable in many crop breeding
programs. Estimation of genome size and the analysis of cell cycle
kinetics are other popular applications. While the analysis of other
subcellular organelles, such as chloroplasts, did not draw a
considerable attention, the analysis and sorting of mitotic
Scholars &
Emerging
Leaders
7
Poster
Session
F. Strobl, A. Schmitz, and E.H.K. Stelzer, Nat. Protoc. 10, 1486
(2015).
K. Greger, J. Swoger, and E.H.K. Stelzer, Rev. Sci. Instrum. 78,
23705 (2007).
Wednesday
15 June
E.H.K. Stelzer, Nat. Methods 12, 23 (2015).
2
E.H.K. Stelzer and S. Lindek, Opt. Commun. 111, 536 (1994).
6
Tuesday
14 June
www.researcherid.com/rid/A-7648-2011
1
5
Monday
13 June
In light sheet–based fluorescence microscopy (LSFM), optical
sectioning in the excitation process minimizes fluorophore
bleaching and phototoxic effects 1. Since biological specimens
survive long-term three-dimensional imaging at high spatiotemporal
resolution, light sheet-based microscopes (LSM) are an
indispensable tool in developmental 2, three-dimensional cell 3 and
plant biology 4. LSFM is based on two optical paths 5. The detection
path consists of a microscope objective, a filter, a tube lens and a
camera. The excitation path is perpendicular to the detection path
and directs a light sheet into the side of the specimen 6,7. The thin
light sheet and the focal plane of the detection overlap. LSFM
provides at least three important degrees of freedom, which are not
available in an epifluorescence microscope: a) the axial and b)
lateral locations of the light sheet and c) the axial location of the
focal plane. Further, probably less important, degrees of freedom
are the tilt and the incline of the light sheet. LSFM takes full
advantage of modern cameras, massively parallelizing the data
acquisition process 8 and recording ten to one hundred images per
second with a high dynamic range. LSFM does not rely on
traditional features that are required for ergonomic reasons. A
powerful multiple-sensors-based image processing pipeline is,
therefore, an inherent feature. Traditional fluorescence microscopy
enforces specimen preparation schemes that rely on hard and flat
surfaces. LSFM places the specimen in the center and arranges the
optics around it. Specimens can be prepared in new ways, their
three-dimensional integrity is maintained, and they can be used in
experiments hitherto regarded as impossible. Fluorescence
microscopy has several basic limitations. First, the excitation light is
absorbed not only by fluorophores but also by many endogenous
organic compounds, which are degraded much like fluorophores
and thus unavailable for vital metabolic processes. Second, the
number of fluorophores in any volume element at any given time is
finite, and fluorophores can degrade upon excitation. As a
consequence, the number of photons that are retrieved from a
fluorophore-labeled specimen is limited. Finally, life on Earth is
adapted to the solar flux, which is less than 1.4 kW/m2. This might
not be a hard limit, but it indicates that irradiance should not
exceed 1 nW/μm2 = 100 mW/cm2 when dynamic biological
processes are observed. When imaging living biological samples,
these challenges must be addressed. LSFM is perhaps the best
technology we have so far, which makes a sincere and honest effort
to address these challenges: 1) it provides optical sectioning, 2) a
true axial resolution, 3) reduces fluorophore bleaching and 4) photo
toxicity at almost any scale, 5) allows one to record millions of
pixels in parallel and 6) dramatically improves the viability of the
specimen.
A. Maizel, D. von Wangenheim, F. Federici, J. Haseloff, and
E.H.K. Stelzer, Plant J. 68, 377 (2011).
Sunday
12 June
Buchmann Institute for Molecular Life Sciences, Goethe
Universität Frankfurt am Main, Frankfurt am Main, Germany
4
Saturday
11 June
Ernst Stelzer
F. Pampaloni, E.G. Reynaud, and E.H.K. Stelzer, Nat. Rev. Mol.
Cell Biol. 8, 839 (2007).
Special
Lectures
111
Light Sheet-Based Illumination Provides for Highly
Corrected, Sensor-Based and Fully Automated
Microscopy
3
Congress
Overview
underpin development, physiological adaptation, brain function
and transgenerational communication, augmented by the
superimposition of plasticity via RNA editing, RNA modification
and retrotransposon mobilization. This suggests that there is a
massive hidden layer and network of RNA-based regulatory
information in mammalian genome biology and that the simple
protein-centric operator-repressor model of gene regulation derived
from studies of bacteria is incorrect in highly organized and
spatially specialized multicellular organisms. This in turn requires
reassessment of the nature, hierarchies and scaling of the regulatory
systems that control the evolution, 4-dimensional assembly and
cognitive capacities of humans and other animals. It also creates an
enlightened framework and new opportunities for therapeutic
development and genetic engineering.
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Index
Abstracts
chromosomes greatly facilitates plant genome sequencing and gene
cloning. Indeed, it is difficult to imagine current plant sciences
without flow cytometry, which became its vital part.
114
Deep Profiling of the Murine Myelopoietic System:
Signaling and Cell Cycle Responses to Neurotropic
Viral Infection Profiled by Flow and Mass Cytometry
(CyTOF)
Thomas Ashhurst1, Paula Niewold1, Darren Cox1, Adrian
Smith2,3, Nicholas King1,3
1
Pathology, The University of Sydney, Sydney, NSW,
Australia, 2Sydney Cytometry Facility, Centenary Institute,
Camperdown, NSW, Australia, 3Ramaciotti Facility for
Human Systems Biology (RFHSB), Camperdown, NSW,
Australia
Viral infection of the central nervous system (CNS) results in a rapid
influx of bone marrow (BM)-derived monocytes/macrophages, that
ultimately induce fatal pathology in the mouse. Whilst these cells
are derived from the BM, little is known about the kinetic and
migratory events that mobilise BM monocytes and their progenitors
in response to CNS infection. In this study we conducted
comprehensive mapping of the murine haemopoietic system in the
BM using high-dimensional single cell flow and mass cytometry
(CyTOF). Initially, we used this approach to refine the identification
of myeloid developmental intermediates involved in monopoiesis
and granulopoiesis, during steady state and inflammation.
Additionally we examined the expression of the transcription factor
IRF8 across the haematopoietic system and its role in instructing
myeloid developmental trajectories. During viral encephalitis, we
found a reorganisation of cellular outputs to favour monocyte
production, resulting in activation and expansion of monocytes and
monocyte progenitors, with increased proliferation of mature and
progenitor populations. In addition, we observed compensatory
downregulation of B-cell lymphopoiesis, and modification of
granulopoiesis in the BM, favouring monocyte expansion. The
upregulation of the interferon (IFN)-inducible marker SCA-1 on
specific lineages suggested a role for IFN signalling, and implicates
IFN gamma (IFN-g)-producing NK and T-cells in the BM in this
process. As such, antibody blockade of IFN-g resulted in a
reduction of monocyte proliferation, associated with improved
clinical outcomes. In this study we have used high-dimensional
cytometry approaches, and have characterized modifications to the
haematopoietic lineage during viral encephalitis that favour
production of pathogenic monocytes.
115
Dynamics of T-Lymphocyte Differentiation Revealed
by Tracing Single Cell Proliferative History
Zinaida Good1,2, Nora Vivanco-González1, Nikolay
Samusik2, Luciene Borges1, Garry Nolan2, Sean Bendall1
1
Pathology, Stanford University, Palo Alto, CA, United
States, 2Microbiology and Immunology, Stanford University,
Stanford, CA, United States
The variety of cell types comprising multicellular organisms are
derived from a single cell through progressive and coordinated
stages of proliferation, death, and differentiation. Still, the linkage of
cell division with lineage-specific differentiation, especially in
complex human cell systems remains elusive. We believe that
linking the fine details of proliferative status and history to cell fate
committement will lead to important applications in immunology
and regenerative medicine. A powerful technique for simultaneous,
high-throughput proteomic monitoring of single-cell phenotypes,
mass cytometry by time-of-flight (CyTOF), has recently enabled
deep analysis of differentiating cellular systems in primary human
tissues, overcoming the challenges related to stochasticity,
heterogeneity, and asynchrony of these systems. However, a
CyTOF-based method for tracking the number of cell divisions
(proliferative history) is not currently available. Here, we adapted a
148
well-established fluorescent dye dilution assay for tracking cell
proliferative history, to CyTOF. Specifically, we identified reagents
that can be tagged with monoisotopic mass reporters and used to
quantify carboxyfluorescein succinimidyl ester (CFSE) dilution in
our 30+ parameter single cell assays. Control experiment results
were quantitatively similar to CFSE dilution measured directly by
flow cytometry, where 5 cell divisions can be distinguished. As a
proof of concept, CyTOF CFSE dilution assays were then applied to
primary human T lymphocytes activated in a rapid expansion
protocol, currently applied in the clinic to expand T cells for
adoptive transfer cell therapies. Using a mathematical model to
extract proliferative history, we successfully profiled the temporal
expression of 23 clinically relevant markers across T-cell divisions
from the naï ve resting state. We found that expression of T-cell
activating receptors (e.g. ICOS, 4-1BB) or markers, which are linked
to activation (e.g. CD25, CD69) or immune cell fate selection (e.g.
CD45RA, CD45RO), is strongly linked to the number of divisions
for a given cell. Further, the relationship between the level of
marker expression and proliferative history could be
counterintuitive. For example, we show that T cells expressing the
lowest level of CD69, which would be normally considered as
poorly activated, have divided the most. In contrast, T-cell
inhibitory receptors (e.g. LAG3, PD1) had division-independent
expression. Finally, employing single-cell dimensionality reduction,
we have created a holistic view of early T-cell specification
dynamics as related to time and divisions number during immune
activation. These methods will be generally applicable across
investigations involving cellular proliferative processes and should
reveal how cell division can be harnessed in order to tune
outcomes.
116
Immuno-phenotyping and Functional Assessment of
Leukocyte Responses to M. tuberculosis Using
Cytometry by Time-of-Flight
Virginie Rozot1, Sheena Gupta2, Michelle van Royeen1,
Mark Hatherill1, Holden Maecker2, Thomas, J. Scriba1
1
South African Tuberculosis Initiative (SATVI), University of
Cape Town, Cape Town, South Africa, 2Institute for
Immunity, Transplantation and Infection, Stanford
University, Stanford, CA, United States
Our understanding of the human immune response to one of the
most successful pathogens, M. tuberculosis, remains incomplete.
Many studies have characterised components of the immune
response in isolation and revealed multiple immune cell subsets
that contribute to this pathogen-specific response. A highly
multiplexed approach that allows simultaneous measurement of
this multifaceted immune response to M. tuberculosis would
provide unprecedented knowledge about the diversity of immune
subsets, phenotypes and functions.
We have designed and optimised an assay to perform deep
immuno-phenotyping and functional assessment of peripheral
blood leukocyte responses by cytometry by time-of-flight (CyTOF).
We developed a PBMC stimulation assay, combined with cell
surface and intracellular cytokine staining with 44 metal-labeled
antibody probes, which allows simultaneous measurement of 40
cellular markers. Barcoding of cells stimulated with different
antigens allows seven stimulation conditions to be combined into a
single sample before antibody labeling and acquisition, minimising
inter-sample variability.
Our assay allows identification of CD4, CD8 and gd T cells, MAIT
cells, NKT cells, NK cells, monocytes and B cells, while measuring
expression of activation and inhibitory markers, chemokine
receptors and memory phenotype markers, such as PD-1, CTLA-4,
CD45RA, CD127, CD27, CCR7, CD38, CD7 and Ki67. It further
allows functional characterisation by measuring effector molecules
including perforin, IFN-Ȗ, IL-2, TNF-Į, IL-17, IL-6, IL-4, IL-22 and
MIP-1ȕ.
Novel analysis tools, such as tSNE or SPADE, allow clustering of
cells based on phenotypic and functional characteristics and may
117
Complex Modulation of the Immune Response to
Surgery by Immune Enhancing Nutrients
1
Stanford University, Palo Alto, CA, United States, 2Stanford
University
Poster Session
Abstracts
Index
Speaker/Author
149
Oral Session
Abstracts
Humans need to make 2 million RBCs every second to maintain a
steady state of 25 trillion circulating RBCs. Delineating
intermediates of erythropoiesis is a critical component of diagnosis
and study of diseases such as genetic anemias and myelodysplastic
Commercial
Tutorials &
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2. JRSS 67, 301-320 (2005)
University of Rochester, Rochester, NY, United States
Scholars &
Emerging
Leaders
1. SciTM 6, 255ra131 (2014)
Kathleen McGrath, Seana Catherman, Katherine Fegan,
James Palis
Poster
Session
References:
119
Combining Imaging and Flow Cytometry to Delineate
Erythropoietic Intermediates
Wednesday
15 June
Conclusion: The high dimensional mass cytometry analysis of perioperative AIN immune modulation revealed systems-wide
alterations in immune cell subsets of patients undergoing colorectal
surgery. While aspects of adaptive cell subsets were transiently
restored, AIN also induced a prolonged increase in MDSC
frequencies, a cell type associated with immune suppression in
sepsis and cancer. These findings have important implications for
the selection of patients that may benefit from peri-operative AIN.
Future studies will examine the utility of peri-operative immune
profiling to tailor pre-habilitation interventions to patient-specific
immune states.
Tuesday
14 June
Results: Consistent with speculations that AIN restores adaptive cell
function after surgery, a robust but transient increase in STAT1
signaling in memory CD8+ cytotoxic T cells was a prominent
feature of the immune network associated with AIN. However, AIN
also induced a prolonged increase in CD11b+CD14+HLA-DRlow
myeloid derived suppressor cell (MDSCs) frequencies, a cell type
intimately involved in Arginine-dependent suppression of adaptive
immune cells in the context of malignancy, sepsis and traumatic
injury.
Monday
13 June
Methods: 22 patients scheduled for elective colorectal surgery were
randomized to AIN treatment (IMPACTâ, 950mL/day for 5 days
prior to surgery) vs. no treatment. Whole blood samples were
collected before surgery (Baseline) then 4h, 24h, 72h and 7 days
after surgery and submitted to mass cytometry analysis of immune
cell subset frequencies and intracellular signaling responses. Plasma
cytokine levels were quantified on a Luminex 63-plex platform. An
Elastic Net analysis2 that integrated immune cell frequencies,
intracellular immune responses and plasma cytokine levels was
applied to identify groups of correlated immune features that best
predicted the effect of AIN on patients immune response to surgery.
Adipose SVF cells are prepared by enzymatic digestion, mechanical
disaggregation and centrifugation of whole adipose tissue or
lipoaspirate. We used the BDT FACSCAP Lyoplate system (BD
Biosciences) to determine the expression of 242 cell surface
markers on 10 populations defined by an 8-color backbone panel,
which included CD45, CD3, CD14, CD14 and CD34. 4 samples
were analyzed. Here we report on subsets of the CD45+
population. CD45+/CD3+ T cells represented 6.9 ± 3.5% (mean,
SD) of all nucleated single SVF cells. CD45+/CD14+ macrophages
(7.6 ± 5.5%) and CD45+/CD19+ B-cells (0.2 ± 0.1%) comprise the
remainder of immune cells in the SVF. The ratio of T cells to B cells
(30 to 1) and macrophages to B cells (32 to 1) differ from that of
peripheral blood (approximately 5:1 and 2:1, respectively),
suggesting that majority of T cells and macrophages are of tissue
origin with minimal peripheral blood contamination. Adipose T
cells and macrophage subsets are highly selected. T cells display a
unique phenotype partially resembling skin T cells, especially with
respect to expression of CD162, (cutaneous lymphocyte-associated
antigen). Like skin T cells, the majority (52.4 ± 16.3%) are CD4+.
Only 5.1 ± 4.0% of the T cells are CD8+, with the remainder
inferred to be double-negative. T-cell differentiation markers are
present in a unique combination, the majority of T cells being
CD45RO- and expressing CD45RB in the absence of CD45RA, or
CD62L. High expression of adhesion molecules (CD44, CD11a,
CD18), and markers associated with motility (CD162, CD151) are
consistent with extravasation into the adipose tissue. The overall
profile suggests a novel population of tissue resident memory or
effector-memory cells. The great majority of adipose macrophages
(92.2 ± 2.4%) express HLA-DR. They are mixed concerning M1
and M2 polarization markers. A sizable proportion express the Tcell costimulatory molecule CD86 and/or the FcȖ receptor CD64,
characteristic M1 macrophages, but an approximately equal
proportion express CD36, CD163 and/or CD206, receptors
associated with M2 macrophages. About a third express TLR-2.
Finally, the substantial population of CD45+/CD34+ cells (9.2 ±
4.4 of CD45+ cells), which are traditionally gated out in analyses of
mesenchymal, endothelial and adipose-committed populations,
revealed itself to be intermediate between endothelial progenitor
cells and pericytes by principal component analysis of all
informative markers. Although the CD45+/CD34+ phenotype is
associated with hematopoietic progenitor cells in bone marrow,
high expression of mesenchymal markers (CD90, CD73, CD44),
strong concordance with pericytes and EP (Spearman correlation
coefficient = 0.81 and 0.85, respectively), weak concordance with
T-cells, B-cells and macrophages (0.23, 0.33, 0.59), and absent or
very low ability of sorted CD45+/CD34+ cells to form
hematopoietic colonies, suggest a mesenchymal progenitor cell
despite the expression of CD45.
Sunday
12 June
Introduction: The recent emphasis on enhanced recovery after
surgery (ERAS) protocols in perioperative care highlights the
significance of efforts towards improving surgical recovery.
However the elements of these protocols that may improve
recovery are uncertain. A better understanding of the biological
mechanisms of ERAS protocols is critically needed.
Arginine-rich nutritional supplementation (Arginine ImmunoNutrition, AIN) is a component of ERAS protocols integral to the
perioperative management of patients undergoing colorectal
surgery. AIN has been the center of much debate, as AIN
consistently decreased post-operative infectious complications after
elective surgery, but increased morbidity and mortality in critically
ill patients. Thus, a mechanistic understanding of the immune
modulatory properties of AIN is essential to unify these apparent
discrepancies. In this study, we applied high-dimensional mass
cytometry1 to comprehensively characterize the effects of
perioperative AIN on major immune cell subsets and associated
intracellular signaling pathways of patients undergoing elective
colorectal surgery.
1
United States, 2Cardiothoracic Surgery, University of
Pittsburgh School of Medicine, Pittsburgh, PA, United
States, 3Univ of Pittsburgh, Pittsburgh, PA, United States,
4
BD Biosciences, San Jose, CA, United States
Saturday
11 June
Brice Gaudilliere1, Nima Aghaeepour2, Martin Angst1,
Garry Nolan2
Albert Donneberg1, Vera Donnenberg2, J. Peter Rubin1,
Ernest Michael M. Meyer3, Mirko Corselli4, Amitab Guar4
Special
Lectures
We will report results from ongoing analyses that characterise and
compare peripheral blood leukocyte responses in patients with
tuberculosis disease with those in controls with latent M.
tuberculosis infection.
118
Immune and Mesenchymal Cells in the CD45+
Compartment of the Adipose Stromal Vascular
Fraction
Congress
Overview
identify novel response subsets that contribute to the immune
response against M. tuberculosis.
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
120
CD Maps – Antigen Density Measurements of CD1CD100 on Human Lymphocytes
Tomas Kalina1, Daniela Kuæí lková 1, Marta Cuenca2,
Elena Blanco Álvarez3, Sophinus J. W. Bartol4, Martin
Andres-Perez3, Menno van Zelm5, Pablo Engel2
1
Charles University in Prague, Prague 5, Czech Republic,
Univ of Barcelona, Barcelona, Spain, 3Departamento de
Medicina y Servicio de Citometria, Universidad de
Salamanca, Salamanca, Spain, 4Department of Immunology,
Erasmus MC, Rotterdam, Netherlands, 5Department of
Immunology and Pathology, Monash University,
Melbourne, Australia
2
Over the past 4 decades, many human leucocyte receptors have
been characterised using antibodies validated by the human
leucocyte typing HLDA/HCDM organization (www.hcdm.org).
With rapid developments in immunology, a lot available
information is outdated or incomplete. Thus, HCDM has initiated a
CD Maps project that aims at quantitative mapping expression of all
CD molecules across the spectrum of leucocyte subsets.
Oral Session
Abstracts
Using the standardized approach developed by the EuroFlow
consortium, we measured PE conjugated CD1-CD100 antibodies in
the context of four 8-color panels on cells from three tissues (blood,
thymus and tonsil). We used Quantibrite PE beads to quantify the
number of PE molecules detected.
Initial analysis of 16 lymphocyte subsets in blood and 9 in thymus
from 8-12 donors revealed that the top expressed molecules are
CD45 (120,000 molecules in T-cells vs. 47,000 in B-cells) and
CD44 (78,000 vs. 47,000). In the thymus the highest expression is
that of CD99 (165 000 molecules in the DN stage).
Index
Abstracts
Commercial
Tutorials &
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syndromes, as well as the goal to create ex vivo sources of blood
sources to address the need for abundant safe transfusable blood.
In order to apply the power of flow cytometry to these studies, two
challenges
that are common to maturation of non-immune cell
types need to be addressed: limited immunophenotypic markers
and maturational changes that occur along a continuum instead of
distinct stepwise changes. We have developed a protocol that
delineates nine distinct erythropoietic intermediates in murine bone
marrow utilizing imaging flow cytometry (IFC) by integrating flow
cytometry characteristics of immunophenotype, cell cycle, and loss
of RNA (ribosomes), mitochondria and nuclei, with morphological
characteristics of cell and nuclear size, mitotic cell state and stages
of enucleation. Gating of specific stages of erythroblasts was
validated by correlation with known changes in cell cycle and
nuclear characteristics during enucleation. The overlap of flow
cytometric and microscopic measurements on the same cells was
used to evaluate approaches and markers used in traditional flow
cytometry and to correlate these erythropoietic intermediates with
erythroblast stages historically defined by morphology and
histologic staining. Finally, we utilized this classification system to
compare of modes of erythropoiesis. In the mouse, we compared
changes in cell morphology and organelle loss during adult bone
marrow steady state erythropoesis with the rapidly expanding fetal
liver erythropoiesis, an important and widely utilized model for
mechanistic studies of erythropoietic regulation. We also used a
similar scheme in human erythropoiesis to compare primary
erythropoietic intermediates with those arising from ES cell/iPS cell
derived erythropoiesis with particular focus on enucleation
intermediates, as efficient enucleation has been a barrier to efficient
ex vivo RBC production.
Comparison between naive and memory T lymphocytes showed
that the largest increase in expression (>20 fold) is observed in
CD45RO, CD95 and CD49e in both CD4 and CD8 T-cell subsets;
and CD11b and CD57 in CD8 T-cells. In contrast, CD62L,
CD45RA, and CD27 were significantly decreased. Amounts of
CD95, CD71, CD27, CD11b, CD54 and CD62L were significantly
higher on memory than naive B cells.
Quantitative information on receptor expression is important for
mechanistic studies as well as flowcytometric panel design and
design of novel biological therapeuticals. Therefore this database
will serve as a useful resource to widen and advance studies into
basic, translational and clinical immunology.
CD Maps project is supported by reagents from BD Biosciences,
BioLegend and Exbio. TK is supported by Ministry of Health Czech
Republic grant 15-26588A.
121
Genetically Encoded Fluorescent Proteins for
Tracking Circulating Tumor Cells with Flow
Cytometry In Vivo
Ekaterina Galanzha1, Dmitry Nedosekin1, Mazen
Juratli1,2, Vladislav Verkhusha3, Vladimir Zharov1
1
University of Arkansas for Medical Sciences, Little Rock,
AR, United States, 2Frankfurt University Hospitals,
Frankfurt/Main, Germany, 3Albert Einstein College of
Medicine, Bronx, NY, United States
The most important target of in vivo flow cytometry (FC) is
circulating tumor cells (CTCs). This is because approximately 90%
of all cancer deaths are caused by metastases produced by CTCs.
Studies performed by us and many other research groups have
demonstrated the great potential of CTCs as markers of metastasis
progression and therapeutic efficacy. However, despite enormous
research efforts, most metastatic cancers are still difficult to treat, if
not incurable, and the mechanisms of the metastasis development
through the multistep process of CTC dissemination is far from
being completely understood. The most promising approach for
CTC study involves using fluorescent proteins: photoswitchable
(called also photoconvertible) fluorescent proteins (PSFPs; e.g.,
Dendra2, Kaeda, and PSmOrange), green fluorescent protein (GFP)
and GFP-like proteins. Here we demonstrated how the integration
of Dendra2 and GFP with multicolor flow cytometry in vivo
provides new insights into the behavior of spontaneous CTCs (i.e.,
naturally shed into circulation over disease progression).
We used GFP to find new links between counts of spontaneous
CTCs, primary tumor size, and metastasis progression. We found
that in some mice with metastatic breast cancer and melanoma, the
highest CTC rate was detected when the primary tumor was small
and slowly growing. Later, when the primary tumor started
progressively growing, the CTC rate decreased. The occurrence of
this phenomenon varied from mouse to mouse, likely due to
specific features depending on the host environment. We also
showed that this phenomenon was attributed to aggressive tumors
developing from cancer stem cells. Furthermore, real-time
continuous monitoring of CTCs reveals that they occur at highly
variable rates in a detection point over a period of time (e.g., in the
range of 0–54 CTCs). In vivo dynamic fluctuation was observed in
epithelial and non-epithelial metastatic tumors, in different stages of
tumor progression, and in different vessels. These temporal CTC
fluctuations can explain false negative results of a one-time
snapshot test in humans.
We showed, for the first time, the unique capability of PAFPs to
define in vivo process by which tumor cells are released from a
primary tumor into blood circulation. Among many imaging agents,
PSFPs with controllable spectral shifts in excitation and emission in
response to light allow distinguishing light-treated (i.e.,
photoswitched) CTCs from nonphotoswitched CTCs by their
emitted color with the use of fluorescence microscopy and flow
cytometry in vivo. We inoculated carcinoma cells expressing the
PSFP Dendra2 into mice. Then we photoswitched a primary tumor
and performed real-time monitoring of CTCs in the blood
circulation before, during, and after photoswitching. Thus, we
monitored in real time the dynamic process of the appearance of
CTCs in circulation after their spontaneous shedding from primary
tumor.
The CDMaps project will generate a broad and updated online
database containing the expression profiles of all CD markers on
human leukocyte subsets present in blood, tonsil, and thymus.
150
1
States, 2Colorado School of Mines, Golden, CO, United
States, 3New Mexico Institute of Mining and Technology,
Socorro, NM, United States
Index
151
Speaker/Author
The home-built instrument incorporates an LSR II Fortessa flow cell
(BD) and off-the-shelf pumps and valves for cell handling. It utilizes
a flexible white-light supercontinuum laser light source (Fianium).
Waveshaping optics produces three spatially and spectrally distinct
excitation beams. Collected fluorescence emission light produced
by passing particles is routed through dichromatic mirrors and
emission bandpass filters on to three independent single-photon
photomultiplier detectors. Their electronic outputs are processed by
time-correlated single-photon counting (Becker&Hickl) and
analyzed using a custom Matlab script producing Flow Cytometry
Standard (FCS) files for subsequent interpretation. The instrument
offers 12 independent measurement channels by multiplexing three
Poster Session
Abstracts
Molecular interactions play a crucial role in cell signaling, drug
efficacy and disease pathology. They can be efficiently assessed by
measuring Förster resonance energy transfer (FRET) between two
fluorescent tags of interacting biomolecules separated by no more
than a few nanometers. Fluorescence lifetime readout of FRET is
advantageous compared to ratiometric and anisotropy-based
techniques by offering direct and quantitative measurement while
imposing the least experimental restrictions. Recently, we have
demonstrated the method and application of fluorescence lifetime
flow cytometry to studies of protein-protein interactions in living
cells1. The published instrument was limited to a single excitation
laser wavelength with a maximum of two emission channels. This
restricted its experimental application range, especially since FRET
acceptor presence could not be traced and no additional
fluorescent markers could be added. Here we report a 12-channel
fluorescence lifetime-measuring flow cytometer compatible with a
broad range of experimental assays and amenable to advanced flow
cytometry data processing and mining.
Oral Session
Abstracts
Background: Mononuclear cells (MNC) play an important role in
the immune system and are widely used in research as well as in
clinical applications. MNCs are usually collected from peripheral
blood in a batch process by density gradient centrifugation with
recoveries around 60 ± 20%. Here, we investigated the
performance of a continuous, microfluidic based, acoustophoresis
technique to enrich MNCs from peripheral blood. By applying an
acoustic standing wave field in a micro channel, cells passing
through the wave field can be separated depending on their
physical properties such as size and density in relation to the
surrounding medium. Despite the size difference between red
blood cells (RBC) and MNCs are these two cell types in blood
plasma or standard buffer systems too similar in their
acoustophoretic mobilities to allow for an effective separation in a
free flow acoustophoretic set-up. To address this bottleneck we
Cancer Studies, King's College London, London, United
Kingdom, 2Cardiovascular Division, King's College London,
London, United Kingdom
Commercial
Tutorials &
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Biomedical Engineering, Lund University, Lund, Sweden,
Laboratory Medicine, Lund University, Lund, Sweden,
3
Hematology, University Hospital Skåne, Lund, Sweden
2
1
Scholars &
Emerging
Leaders
1
Jakub Nedbal1, Izajur Rahman2, Alex Ivetic2, Simon
Ameer-Beg1
Poster
Session
Anke Urbansky1, Andreas Lenshof1, Pelle Ohlsson1,
Stefan Scheding2,3, Thomas Laurell1
Wednesday
15 June
123
Acoustophoretic Separation of Mononuclear Cells
from Red Blood Cells in Peripheral Blood
Tuesday
14 June
Conclusion: Observing the distinctive properties of high
concentration nanoparticles suggests that the primary acoustic force
is not the dominant effect. Notably the results are not consistent
with acoustic streaming. The observations are likely dependent on
secondary particle acoustic forces. We are developing a model to
illustrate that the secondary acoustic properties are a determining
factor of submicron particle positioning and that the exact position
and secondary nodal effects occur at predictable locations.
124
12-Channel Fluorescence Lifetime Flow Cytometry
for Investigation of Protein-Protein Interactions in
Intact Cells
Monday
13 June
Results: Our preliminary work has shown that at particle diameters
of 1ȝm and below, the predicted number of acoustically focused
streams is not obtained. In most cases we have observed three
focused streams instead of one. In addition to the predicted stream
at the center of the flow cell, two additional streams were observed
near to two sidewalls. In some cases we observed these additional
streams near to the center of the flow cells.
Conclusion: Acoustophoresis can be used to enrich MNCs from
peripheral blood with high separation efficiency and low RBC
contamination and offers the advantage of continuous separation as
compared to standard density gradient centrifugation.
Sunday
12 June
Methods: Acoustic focusing of silicon particles of diameter 1 μm
and below with carboxyfluorescein was performed in Micronit flow
cells with the dimensions of 500 μm wide, 1.1 mm deep, and 42
mm long. A lead zirconium titanate acoustic transducer with the
resonance frequency of 1.5 MHz was used to generate standing
acoustic waves across the flow cell. A Zeiss epifluorescence
microscope equipped with Andor Solis CCD camera was used to
obtain images of particle streams. Image analysis was performed on
collected images using a custom made Matlab program.
Results: Do to the similar acoustic properties of MNC and RBC it is
not possible to separate these two cell types from each other in
standard PBS buffer. At low amplitude the separation efficiency of
MNC was 99.9%, while no RBC where removed, whereas high
amplitudes moved the MNC (96%) as well as the RBC (98%) to the
waste fraction. By changing the buffer composition, and thereby
changing the acoustic forces acting on the cells, it was possible to
enable the separation of MNCs from red blood cells in diluted
whole blood. A log 2.2 ± 0.3 relative enrichment was achieved for
MNCs in the target fraction as compared to the starting sample. The
separation efficiency of the MNCs was 97.7 ± 0.3% while 99.2 ±
0.6% of the RBC were removed to the waste outlet.
Saturday
11 June
Background: Acoustic focusing is an efficient method of positioning
flowing microparticles and cells into tight streams for optical
analysis via flow cytometry. However, acoustic focusing does not
efficiently focus particles smaller than 1 μm in diameter. Here, we
explore the use of both the primary and the concentration
dependent secondary acoustic forces for the accurate and efficient
positioning of nanoparticles. Developing a model that enables
effective manipulation of nanoparticles via acoustic standing waves
will benefit several fields including nanoparticle self assembly and
nanoparticle analysis.
Study Design and Methods: Whole blood samples were obtained
from healthy volunteers (n=3). After labeling the blood cells with
fluorescent antibodies against CD45 and CD61, samples were
diluted 20 times and processed through an acoustophoresis
microchip consisting of a sample inlet, a pre-focusing zone, a
central buffer inlet, the main separation channel and a waste outlet
in the center of the channel as well as a target outlet at the side.
Different buffer compositions were tested at a set constant flow rate
of 100 μl/min sample flow and the amplitude of the acoustic field
was varied to obtain optimal separation conditions. Both, target and
waste outlet were analyzed for cell separation efficiency as well as
enrichment by flow cytometry.
Special
Lectures
Frank Fencl1, Jose Lopez2, Steven Graves1, Menake
Piyasena3
have therefore developed novel buffer protocols and optimized the
acoustophoretic conditions.
Congress
Overview
122
Concentration Dependent Acoustic Positioning of
Nanoparticles in Flow
Congress
Overview
Special
Lectures
Sunday
12 June
We
demonstrate the capabilities of the instrument and characterize
its performance using fluorescence microparticles. We will perform
and present the results of a FRET based assay investigating Lselectin cluster formation in human monocyte-derived THP-1 cells.
GFP and RFP-tagged L-selectin molecules will be coexpressed and
clustered with bivalent antibodies. The cluster-induced FRET should
cause measurable decrease in GFP fluorescence lifetime in cells
expressing wildtype but not signaling domain deficient L-selectin.
The flow cytometry results should mimic those previously reported
from single-color fluorescence lifetime imaging microscopy2. The
obtained multicolor fluorescence lifetime flow cytometry results
will complement the original images by providing unbiased
population data and expression levels of GFP and RFP variants of Lselectin in individual cells. Chiefly, the technology will enable
novel experiments requiring FRET readout combined with intensity
measurements of additional fluorescent stains within the same cells.
References:
Monday
13 June
Saturday
11 June
excitation and three emission wavelength bands with fluorescence
lifetime and intensity readouts.
[2] Rzeniewicz, K.; Newe, A.; Rey Gallardo, A.; Davies, J.; Holt, M.
R.; Patel, A.; Charras, G. T.; Stramer, B.; Molenaar, C.; Tedder, T.
F.; Parsons, M.; Ivetic, A. PNAS2015, 112, E1461–E1470.
[1] Nedbal, J.; Visitkul, V.; Ortiz-Zapater, E.; Weitsman, G.; Chana,
P.; Matthews, D. R.; Ng, T.; Ameer-Beg, S. M. Cytometry A2015,
87, 104–118.
Tuesday
14 June
125
Droplet-Based Microfluidics for Single-Cell Analysis
Dennis Eastburn1, Maurizio Pellegrino1, William Hyun2,
Adam Sciambi1, Jamie Yates1
Index
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Scholars &
Emerging
Leaders
Poster
Session
Wednesday
15 June
1
Mission Bio, Inc., San Francisco, CA, United States, 2Univ
of California San Francisco, San Francisco, CA, United
States
Identifying cellular subtypes based on the presence of RNA or
genomic DNA biomarkers constitutes a powerful approach for
examining heterogeneous cell populations. We have developed
novel microfluidic approaches that enable this capability. PCRactivated cell sorting (PACS) is a genomic cytometry method that
uses single-cell TaqMan PCR reactions performed in microfluidic
droplets to identify and isolate cell subtypes with high-throughput.
We extended this method and demonstrated that PACS allows highdimensional molecular profiling on TaqMan-targeted cells. Using a
random priming RNA-Seq strategy, we obtained high-fidelity
transcriptome measurements following PACS-sorting of prostate
cancer cells from a heterogeneous population. The sequencing data
revealed prostate cancer gene expression profiles that were
obscured in the unsorted populations. Single-cell expression
analysis with PACS was subsequently used to confirm a number of
the differentially expressed genes identified with RNA sequencing.
Our results demonstrate the applicability of PACS for transcriptional
profiling of rare cells from complex populations to obtain maximal
biological insight into cell states and behaviors. We will also report
the development of a high-throughput barcode-based single-cell
sequencing method. Unlike other single-cell droplet barcoding
methods, our approach can target both genomic DNA and
transcriptomes of individual cells for next-generation sequencing
analysis.
126
quantiFlash™-Based Method for Calibration,
Standardization and Optimization of High Parameter
Fluorescence Flow Cytometers
Stephen Perfetto1, James Wood2, Pratip Chattopadhyay1,
Julianne Hill1, Richard Nguyen1, David Ambrozak1,
Mario Roederer1
1
Vaccine Research Center, NIH / NIAID, Bethesda, MD,
United States, 2Wake Forest University, Winston-Salem, NC,
United States
152
Recent advances in fluorescence flow cytometry allow the
characterization of cell populations with an unprecedented depth.
Robust and sensitive analyses with these instruments requires
systematic procedures for calibration, standardization, and
optimization of biological measurements. We developed new and
improved tools for these purposes, that also provide insight into
instrument variation and begin to define the how QC measurements
affect immunophenotyping panel development.
Methods: We used quantiFlash™, previously known as an LED
pulser, to characterize PMT performance, voltage selection, and
background variation. The quantiFlash™ is used to define Q and B
values. Q values describe PMT efficiency, which in turn can be
used to compare staining resolution on different instruments; B
values describe PMT sensitivity. Using quantiFlash™ we could
standardize fluorescence measurement scales, such that the
instrument would directly report signal as photoelectrons measured
by the PMT. These standardized scales were used evaluate B and
Q-values across instruments. Next, we determined optimal voltages
for detectors by measuring autofluorescence (AF) and background
signals over a range of voltages; the ratio of robust standard
deviation (rSD) of autofluorescence to the rSD of quantiFlash™derived background measurements was used to define an optimal
PMT voltage range. We then examined compensation and
spillover-spreading matrices to refine our voltage selection. Finally,
we characterized performance using antibody capture beads or
PBMCs stained with CD4-conjugated antibodies.
Results: The optimal voltage range for each detector could be
defined by the linear portion of the AF/B vs. PMT voltage curve. We
found that channels where electronic or laser noise levels were
high had poor B-values, and, as predicted, detectors with higher Q
values gave better resolution of stained PBMC. Indeed, instrumentto-instrument variation was minimized by selecting PMTs for a
given detector that had similar Q & B values measured at the
desired detection wavelengths. Notably, we found that some PMTs
performed better detecting red (higher wavelength) photons than
green (lower wavelength photons), while others had the opposite
profile.
Conclusions: We have made considerable progress in developing
methods, centered on the use of quantiFlash™ for measurement of
Q and B values, for PMT voltage selection and characterizing
instruments. These procedures resulted in consistent, optimal
performance across multiple instruments.
Disclaimer: JW receives royalties from the sales of quantiFlash™
devices.
127
Synthetic, Cell-Like Particles with Optical,
Fluorescent, and Biochemical Multiplexing
Brandon Miller1, William Hyun2, Oliver Liu1, Jeffrey
Kim1
1
Slingshot Biosciences, Emeryville, CA, United States,
Laboratory Medicine, Univ of California San Francisco, San
Francisco, CA, United States
2
We have developed a new class of synthetic cell mimics for flow
cytometry and cell sorter calibration that match the optical and
biochemical properties of virtually any cell type. These particles are
shelf-stable and can be multiplexed along optical, fluorescent, and
biochemical parameters such as nucleic acid sequences and
antigens. These properties, such as forward (FSC) and side (SSC)
scattered light, can be tuned independently, generating a highly
flexible synthetic cell platform for a wide range of applications. Our
first target market addresses a major source of human error in
clinical immunophenotyping: the need to manually adjust gains
and gates between instrument setup/calibration and experimental
measurements. Existing polystyrene bead reagents possess different
optical and hydrodynamic properties when compared to cellular
material. These differences necessitate manual gating between
instrument setup, calibration, and experimentation. Our synthetic
cells exactly match the optical properties of blood cell
subpopulations, eliminating the need for manual intervention
between setup, calibration, and experimentation thereby removing
1
Index
Speaker/Author
153
Poster Session
Abstracts
Oral Session
Abstracts
Results: We used miCAT to process hundreds of IMC images. Our
main data set consist of ~200 breast cancer samples including more
than 200,000 single cells each with over 50 parameters and dozens
Commercial
Tutorials &
Exhibits
Methods: To process, segment, normalize and analyze these (large
scale) multiplexed imaging cytometry data, we developed a novel
open-source modular, multiplexed imaging cytometry analysis
toolbox (miCAT). This user-friendly Matlab based software enables
explorative data visualization, analysis modules to study cell types
and their marker expression, and several semi- and unsupervised
methods for neighborhood analysis within the TME. Finally all of
these image cytometry features can be linked with the
corresponding clinical data.
Scholars &
Emerging
Leaders
1
Zurich, Switzerland, 2Life Science Zurich Graduate School,
Zurich, Switzerland
Poster
Session
In this case study, we have assessed commonly used FACS
conditions, such as nozzle size, flow rates, laser power, etc. in
multiple cytometry machines and configurations using our
multiplexed cell sensors. With our established protocols we can
resolve differences in sensor signals as we fine tune sorting
conditions, highlighting the utility of the approach. We anticipate
these sensors will aid the flow cytometry and microfluidic device
communities to easily measure stress activation within their
platforms and consequently, design and operate systems in
configurations best suited maintaining optimal cell health.
Denis Schapiro1,2, Hartland Jackson1, Swetha
Raghuraman1, Vito Zanotelli1,2, Raúl Catena1, Bernd
Bodenmiller1
Wednesday
15 June
To achieve reporter multiplexing, we engineered each sensor to
constitutively express a specific fluorescence ‘code’ in the blue or
green spectra (using ypet or cerulean fluorescent proteins) for
convenient resolution of each cell sensor identity and its respective
stress level. This allows all sensor types to be used together for
simultaneous assessment of multiple stress pathways. We have
already disseminated these sensors to various laboratories who
have begun to adopt our approach to assess cell stresses in their
technological platforms.
130
Multiplexed Imaging Cytometry Analysis Toolbox
Coupled to Imaging Mass Cytometry Reveals Patterns of
Cell Interactions amongst the Heterogeneity of Breast
Cancer
Tuesday
14 June
Here we present cell-based sensors that emit fluorescence when
specific cell stress pathways are activated in microenvironments.
This live-cell assay now allows for quantitative monitoring of
specific cell stress pathway activations through the convenience of
fluorescence microscopy, or flow cytometry, and does not need
additional reagents. Using robust and commonly used NIH3T3
cells, we engineered three types of clonal cell-sensors: one to assess
DNA damage through the activation of the relevant p53-p21 DNA
damage pathway; one to assess stress due to electro-thermal effects
through the activation of the heat shock HSF1 pathway and a
sensor to assess fluid shear stresses through the transcriptional
activation of a mechanosensitive gene EGR1. We verified each
sensor functionality by chemical induction of their respective
pathways and noted population mean fold induction of RFP
fluorescence induction correlating to the ‘stress dosage’, and further
characterized sensor response with relevant physical stressors.
Monday
13 June
The use of engineered micro- and macroscale systems to study and
manipulate cells has increased markedly in the recent past.
However, there follows a growing concern such systems regarding
the impact of such systems on cell health and the potential artifacts
that can be induced via these technologies. Most existing methods
for assessing cell health suffer from either high cost and complexity
(e.g. genetic sequencing), or low specificity and information
content (e.g. viability dyes, morphology data). This necessitates a
metrology approach that is specific to cell stress markers, is easy to
apply and quantify, and remains inexpensive. Such methodology
could be applied to optimize flow cytometric, microfluidic and
other system designs and discover configurations that minimize cell
damage.
The consistency among a set of flow cytometry experiments hinges
on the existence of a constant ratio between the number of antigens
on a cell and the number of tagged antibodies detected by the
cytometer. Conventionally, this is ensured by staining the sample to
saturation where a sufficient quantity of tagged antibodies is added
so that each antigen is bound to an antibody. However in highdimensional mass cytometry using the CyTOF, saturation cannot
always be achieved, nor is it always desirable. Staining to saturation
may overload the detector leading to spill-over in other channels
and at high antibody concentrations, non-specific antibody binding
becomes significant. Without staining to saturation, changes in the
antibody-antigen binding ratio arising from variations in sample
preparation and shifts in the composition of each sample makes
each channel have a unique, unknown scaling coefficient that
varies between experiments. This makes quantitative comparisons
between multiple cytometry experiments impossible. Barcoding is
one approach to correct for this concern. By labeling each sample
with a unique tag and then pooling and staining the samples
simultaneously, this ensures consistency for each pooled sample,
but is limited to those samples that can be prepared simultaneously.
This is challenging for clinical trials where sample collection takes
place over a period of years requiring samples to be frozen, during
which time the sample may degrade. To enable quantitative
analysis across multiple mass cytometry experiments without the
need to run all samples simultaneously, we propose a new protocol
that adds in a barcoded reference population into each sample
prior to staining. Then our new algorithm Cell Level Expression
Abnormality Normalization (CLEAN) determines the optimal
correction to match the reference populations between experiments
and applies this correction to the sample cells. This corrects for
variations in the antibody-antigen binding ratio between
experiments and differences in sensitivity between instruments. This
technique could also correct for variations between instruments and
institutions thus enabling large scale, multi-site clinical trials using
the CyTOF.
Sunday
12 June
Electrical Engineering and Computer Science, MIT,
Cambridge, MA, United States, 2Cytometry Shared Resource
Laboratory, Stowers Institute for Medical Research, Kansas
City, MO, United States
1
Department of Leukemia, MD Anderson Cancer Center,
Houston, TX, United States, 2Department of Epigenetic and
Molecular Carcinogenesis, MD Anderson Cancer Center,
Houston, TX, United States
Saturday
11 June
Sarvesh Varma1, Andrew Box2, Joel Voldman1
Jeffrey Hokanson1, Ryan McCarthy2, Chris Benton1,
Ahmed AlRawi1, Michael Andreeff1
Special
Lectures
128
Multiplexed Cell-Based Sensors for Assessing the
Impact of Engineered Systems on Cell Health
129
CLEAN Corrects Variations in CyTOF Sample
Preparation
Congress
Overview
a major source of variability in clinical testing and site validation.
Here, we show how our cell mimics create assay-specific reference
templates for standard blood controls with consistent assay patterns
including scattered light. This new standard promises to increase
measurement accuracy across labs, instruments and decrease setup
costs, reduce the time for experimentation, and will save precious
sample material while improving detection limit controls for low
biomarker expression. These new particles represent the first
standards that will simultaneously verify instrument performance
with assay-specific parameter pass/fail criteria.
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Index
Abstracts
of spatial features. The data driven modeling modules implemented
in miCAT highlighted a surprising intra- and interpatient TME
heterogeneity in all breast cancer subtypes. Furthermore, marker
expression,
cell types and features of the TME correlated with
clinical outcomes. Finally, we found a link between infiltrating
CD68+ macrophages and metastatic carcinoma cells based on the
expression of markers of the epithelial mesenchymal transition.
Validation experiments are currently ongoing.
Conclusion: Highly multiplexed imaging approaches, including
IMC and serial immunofluorescence imaging, coupled to our
computational toolbox are a powerful tool to analyze highly
multiplexed single cell tissue data which can be linked to cell
phenotypes, morphological features and clinical data to identify
stratifying subpopulations and driving forces of metastasis and
disease outcome.
131
ggcyto: A New Flow Cytometry Data Visualization
Framework for BioConductor Based on ggplot
Greg Finak, Wenxin Jiang, Raphael Gottardo
Biostatistics, Bioinformatics and Epidemiology, Fred
Hutchinson Cancer Research Center, Seattle, WA, United
States
Open source flow cytometry data analysis tools are critical for
producing reproducible scientific results. In immune monitoring,
vaccine trials, and other immunological studies, large data sets
integrating multiple, complex assay types are common. Flow
cytometry is amongst the most complex and challenging of those
data types to analyze in a reproducible and open fashion. For the
past 10 years, via the Bioconductor project, we have been
developing open source tools for reproducible flow data analysis
that sophisticated statistical modeling, automated gating, and fast
data manipulation of single-cell event level data. Here, we present
an overview of those tools, and introduce a new data visualization
framework, called ggcyto, built upon the grammar of graphics
paradigm, that provides an intuitive and flexible FCM data
visualization platform and which is closely integrated with the core
Bioconductor flow cytometry tools. The framework allows for
plotting cell populations, gates, and associated data transformations
directly from Bioconductor’s GatingSet objects, which are created
upon import of FlowJo workspaces,Cytobank gatingML files or
automated gating of FCS files. Generating histograms, densities,
dotplots, or hexagonal 2D binned plots is straightforward in this
framework, as is adding population statistics and faceting of plots
by associated metadata related to subject-level or sample-level
annotations. We will demonstrate several examples of this
flexibility on real-world data sets used to generate publication
quality graphics. The ggcyto package is available on BioConductor.
132
Improving Marker Gene Discovery from HighDimensional Single-Cell Snapshot Data
Robrecht Cannoodt1,2, Wouter Saelens1,3, Katleen De
Preter2,4, Yvan Saeys1,3
1
Data Mining and Modelling for Biomedicine group, VIB
Inflammation Research Center, Ghent, Belgium, 2Center of
Medical Genetics, Ghent University, Ghent, Belgium,
3
Department of Internal Medicine, Ghent University, Ghent,
Belgium, 4Cancer Research Institute Ghent, Ghent, Belgium
The design of good panels of cell surface markers is a prerequisite
for successful flow cytometry experiments, and the current design
of such panels is mainly historically determined by previously
identified markers and availability of antibodies. However, recent
advances in RNA sequencing now allow to generate expression
data at the single cell level, allowing to get very detailed
transcriptional profiles of cells of a certain lineage of interest.
Computational methods that analyze single-cell RNA sequencing
data thus offer a new approach to look in an unbiased way for
novel and/or better marker panels, e.g. allowing to identify a certain
154
subset of cells in different tissues using a common panel, or to
identify novel, specific marker combinations that identify certain
precursor cells.
In this work we present SCORPIUS, a novel and fully unsupervised
technique to investigate transcriptional changes in dynamic cellular
processes. SCORPIUS starts from snapshot data that contains a
mixture of cells along different states in a differentiation process
and first reconstructs a developmental timeline model from single
cell observational data. Subsequently it predicts candidate marker
genes for stages of interest, making use of machine learning
techniques. SCORPIUS was shown to work accurately for a variety
of dynamic cellular processes, including cell differentiation, cell
cycle and cellular responses to external stimuli.
We evaluated the performance of reconstructing timeline models
using SCORPIUS with a new, quantitative evaluation pipeline on
10 publicly available single-cell RNA sequencing datasets, and
identified novel candidate marker genes for the different cellular
stages.
133
Reproducible Reduction: Deterministic tSNE Using
Regression Trees Enables Intra-sample Comparison
Michael Stadnisky, Shahid Siddiq, Jay Almarode, John
Quinn, Aaron Hart
Flowjo, LLC, Ashland, OR, United States
t-Stochastic Neighbor Embedding is a new and powerful technique
for visualizing high-dimensional data via dimensionality reduction,
and has been adapted to the model of 2-dimensional population
exploration in flow and mass cytometry as well as single cell
sequencing. However, two important limitations hold this method
back from wider use as a diagnostic and discovery tool.
First, it has relatively slow runtime that scales poorly (?(N2) for the
reference implementation or O(N log N) for Barnes-Hut ) and
second, there is no readily available method for out of sample
estimation, because tSNE learns a nonparametric mapping of the
data. These limitations taken together mean that in order to
successfully visualize large cytometry data sets with millions to tens
of millions of rows, spread across several samples, excessive
computation times, sample concatenation and down sampling are
all necessary. This limits opportunities for real-time interactive
analysis, sample comparison in the reduced space, and its use
outside of discovery research.
Herein, we combine two machine learning approaches to provide a
method for out of sample estimation of tSNE derived parameters
that is both fast (O(M(mN log N) for building the regression but
O(N) for out of sample estimation) and accurate as measured using
cross validation. We accomplish this using a Random Forest
regression model, an algorithm which has been shown be a
powerful tool for both classification and regression problems. We
provide several examples of this algorithm working in both high
parameter flow cytometry (>30 parameters) and single cell
sequencing (>1000 parameters) and propose a general workflow for
using this technique to deterministically examine high parameter
flow cytometry experiments.
Our regression model approach for out of sample estimation will
enable tSNE and other non-parametric dimensionality reduction
methods to be used extensively for visualizing and comparing
complex single cell data.
134
Minimal Residual Disease Detection in Acute
Leukemia
Brent Wood
Hematopathology Laboratory and SCCA Pathology,
University of Washington, Seattle, WA, United States
The detection of minimal residual disease (MRD) has emerged as
one of the most powerful prognostic factors in acute leukemia.
Flow cytometric methods of MRD detection rely on the recognition
Wednesday
15 June
Poster
Session
Oral Session
Abstracts
Poster Session
on
Abstracts
Index
Speaker/Author
155
Commercial
Tutorials &
Exhibits
1
Department of Internal Medicine, Division of Hematology,
The Ohio State University, Columbus, OH, United States,
2
Department of Microbiology and Immunology, Baxter
Laboratory for Stem Cell Biology, Stanford University,
Stanford, CA, United States, 3Division of Gynecologic
Oncology, Department of Obstetrics and Gynecologyicat,
Stanford University, Stanford, CA, United States,
4
Department of Internal Medicine, Division of Hematology,
Stanford University, Stanford, CA, United States
Scholars &
Emerging
Leaders
Gregory Behbehani1, Nikolay Samusik2, Zach Bjornson2,
Wendy Fantl3, Bruno Medeiros4, Garry P Nolan2
Acute myeloid leukemia (AML) is characterized by a high relapse
rate that has been attributed to the quiescence of leukemia stem
cells (LSCs), which renders them resistant to chemotherapy.
However, this hypothesis is largely supported by indirect evidence
and fails to explain the large differences in relapse rates across AML
subtypes. To address this, bone marrow aspirates from 41 AML
patients and five healthy donors were analyzed by highdimensional mass cytometry. All patients displayed
immunophenotypic and intracellular signaling abnormalities across
myeloid development and within CD34+CD38low populations and
several karyotype and genotype-specific surface marker patterns
were identified. The immunophenotypic stem and early progenitor
cell populations from patients with clinically favorable core-binding
factor AML demonstrated a five-fold higher fraction of cells in Sphase compared to other AML samples. Conversely, LSCs in less
Tuesday
14 June
The International Clinical Cytometry Society (ICCS) and the
European Society for Clinical Cell Analysis (ESCCA) have formed a
collaborative group to further consensus within myeloma MRD. As
a member of this joint ICCS/ESCCA effort, Dr de Tute will discuss
the practicalities and difficulties of developing platformindependent MRD approaches which are acceptable to a majority
of laboratories. Any such approach would need to be highly
sensitive and compatible with established assays used in previous
multi-centre trials. The challenge is to establish sufficient consensus
to generate inter-laboratory reproducibility whilst simultaneously
maintaining some flexibility to satisfy individual laboratory needs.
137
Mass Cytometric Functional Profiling of Acute
Myeloid Leukemia Defines Cell-Cycle and
Immunophenotypic Properties That Correlate with
Known Responses to Therapy
Monday
13 June
Minimal residual disease (MRD) has been demonstrated to be an
important prognostic tool in multiple myeloma (MM) and the huge
improvements in outcome resulting from recent therapeutic
advances have made it increasingly relevant. Treatment approaches
are now sufficiently effective that using progression-free and overall
survival as end-points in clinical trials is becoming untenable, with
trials of up-front therapy requiring 5-10 years of follow-up in order
to demonstrate survival differences. This is slowing down
identification of the most effective new treatments and novel
approaches to response assessment are required. As a consequence,
MRD assessment is currently been considered as a surrogate end
point for clinical trials and regulatory drug approval. A significant
body of evidence now exists demonstrating the clinical relevance of
MRD in multiple multi-centre studies. It has also been shown that
the prognostic relevance of MRD is independent of the treatment
received. There is acknowledgement, however, that acceptance of
flow cytometric MRD as an appropriate end point would require
consensus but there is no real clarity on the degree of
harmonisation or standardisation required.
Extracellular vesicles isolated from human ovarian tumor ascites
fluids and solid tumors have a size, lamellarity and composition
that are characteristic of exosomes. We report here that Cell Trace
Violet-stained tumor-associated exosomes bind to T-cells, and
inhibit signaling through the T-cell antigen receptor. The patient
derived immune suppressive exosomes significantly inhibit multiple
T cell functions including, the nuclear translocation of NFkB, the
upregulation of CD69, and CD107a, and cytokine production of T
cells following stimulation with immobilized antibodies to CD3 and
CD28. The exosome mediated arrest is dependent upon
phosphatidylserine (PS) that is expressed on the surface of these
vesicles, as a blockade of PS with Annexin V or anti-PS antibodies
significantly reverses their inhibitory activity. And the depletion of
PS-expressing exosomes using magnetic beads coupled to anti-PS
antibody significantly decreases the immune suppressive activity.
We conclude that exosomes are a novel T cell checkpoint in
ovarian tumors and represent a potentially viable target by which to
re-activate hypo-responsive T-cells in human tumor
microenvironments, and to enhance the therapeutic efficacy of
immune based therapeutic approaches to cancer.
Sunday
12 June
Haematological Malignancies Diagnostic Service, Leeds
Teaching Hospitals NHS Trust, Leeds, United Kingdom
1
Microbiology and Immunology, SUNY at Buffalo, Eden,
NY, United States, 2Microbiology and Immunology, SUNY
at Buffalo, Buffalo, NY, United States, 3Pharmaceutical
Sciences, SUNY at Buffalo, Buffalo, NY, United States,
4
Infectious Disease Section Veteran Affairs, SUNY at
Buffalo, Buffalo, NY, United States, 5Roswell Park Cancer
Institute
Saturday
11 June
Ruth de Tute
Richard Bankert1, Raymond Kelleher Jr. 2, sathy BaluIyer3, Jenni Loyall2, Gautam Shenoy2, Charles Berenson4,
Paul Wallace5, Joseph Tario, Jr. 5, Kunle Odunsi5
Special
Lectures
135
Consensus in Minimal Residual Disease Monitoring in
Multiple Myeloma: Development of PlatformIndependent Assays
136
Exosomes Present in Human Tumor
Microenvironments Arrest T- Cell Activation,
Suppress Proliferation, and Block Cytokine
Production
Congress
Overview
of differences in protein expression on neoplastic cells in
comparison to their closest normal counterpart. This approach is
well developed for the detection of B lymphoblastic leukemia at
early time points after therapy, and has demonstrated utility in T
lymphoblastic leukemia and acute myeloid leukemia. The
advantages of the method are its speed, reasonable cost, established
instrument base, and moderate sensitivity. However, subjective
interpretation and a lack of standardization between labs result in
significant variability that threatens the long-term viability of this
approach. By comparison, molecular methods rely on the
detection of mutated sequences specific for neoplasm, e.g.
translocations, point mutations or clonal sequences. The former are
only present in a subset of leukemias so have not served as a
general strategy for MRD detection, while the latter have required
laborious and expensive patient-specific primers. With the advent
of next-generation sequencing, highly multiplexed and highly
sensitive MRD assays have become possible and offer improved
standardization, but their performance characteristics have yet to be
firmly demonstrated. Each of these approaches and their potential
clinical significance will be discussed.
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
clinically favorable FLT3-ITD AML exhibited dramatic reductions in
S-phase fraction. Mass cytometry also allowed direct observation
of the in vivo effects of cytotoxic chemotherapy. This work
provides
a basis for using in vivo functional characterization of
AML cells to inform therapy selection.
Multimedia Abstracts
Dick had a very productive career, first leading the Instrumentation
Development Group in the Herzenberg Laboratory at Stanford, and
later at BD Biosciences, where he made significant contributions to
the Aria cell sorter.
Cell Sorting and Selection (B1)
This poster will highlight his contributions to Flow Cytometry, and
present some videos of talks he gave, some of which are in the
archives of the Smithsonian
138/B1
Pre-enrichment of ILC2 from Whole Blood Reduces
Sort Time and Results in Improved Purity
Hematological Disorders (B2 – B3)
1
1
1
Jennifer Wilshire , Yanet Valdez , Stephen Kyei , Grace
Poon1, Fumio Takei2, Carrie Peters1, Steve Woodside1,
Allen Eaves1, Terry Thomas1
1
R&D, STEMCELL Technologies, Vancouver, BC, Canada,
Pathology & Laboratory Medicine, Terry Fox Laboratory,
2
Group 2 innate lymphoid cells (ILC2) are a recently identified
subset of immune cells. Isolating these rare cells is challenging,
and currently multicolor flow cytometric analysis and cell sorting
are the only methods to identify and isolate ILC2s. However, the
scarcity of these cells makes cell sorting time-consuming, expensive
and often results in low purities and recoveries. Thus, better
approaches for effective identification and isolation are essential to
further understanding of ILC2 biology and function. We have
developed a rapid and efficient method for enrichment of human
ILC2 from whole blood. In brief, non-ILC2 cells in whole blood
were crosslinked to red blood cells (RBC) already present in the
sample using RosetteSep™. Cell surface markers on unwanted cells
are crosslinked to glycophorin A on RBC. After a 10 minute (min)
incubation, the sample was layered over Lymphoprep™ in a
SepMate™ tube, spun at 1200 x g for 10 min. The crosslinked cells
settled to the bottom of the tube, and the untouched, desired cells
at the plasma:Lymphoprep interface were simply poured off. Cells
were washed once and were then ready for cell sorting. Starting
with only 0.01 - 0.07% in whole blood, ILC2 were enriched 350
fold (90% range 104-718) in 35 min to 8.5% (median, n=21).
Subsequent cell sorting from these pre-enriched samples averaged 7
times faster and yielded higher purity ILC2 than sorting from nonenriched controls (n=3, p<0.05 paired t test). The post sort purity of
all pre-enriched samples was >95%, while the purities of nonenriched samples were < 70%. Low purity often necessitates a
second round of sorting, which adds additional sort time and
reduces total ILC2 recovery. Following culture and stimulation,
sorted ILC2, both pre-enriched and non-enriched controls, secreted
similar high levels of IL-13 as assessed by ELISA, indicating that
these cells are functional. In summary, pre-enriching ILC2s
reduces sorting time, increases ILC2 purity, and maintains ILC2
functionality.
Flow Cytometry Instrumentation (B312)
449/B312
In Memorium: Dick Sweet and His
Contributions to Flow Cytometry and Cell
Sorting
Marty Bigos1, David Houck2
1
Index
Abstracts
In the 1960s he and Mack Fulwyler discussed this technology and
how it could be applied to solve the problem Mack was engaged
with. The result was the first cell sorter.
Stanford University School of Medicine, Stanford, CA,
United States, 2BD Biosciences (retired), San Jose, CA,
United States
Richard Sweet died on November 28, 2015. In his life he had been
an avid fisherman and poker player, regularly playing Texas Hold
'Em at Bay 101,where he was known as 'Spider-Man', and most
notably he was the inventor of the ink jet printer.
156
139/B2
Automated Identification of Novel Cell Types Based
upon Signaling Characteristics in Chronic Myeloid
Leukemia
Jitakshi De1, Paul Kane2
1
Pathology, Deepath Medical Diagnostics, Palo Alto, CA,
United States, 2Computational Biology, Deepath Medical
Diagnostics, Palo Alto, CA, United States
Introduction: Chronic myeloid leukemia (CML) is a hematopoietic
stem cell neoplasm driven by BCR-ABL kinase and downstream
signaling cascades causing unabated proliferation of myeloid
lineage cells (neutrophils and precursors, basophils, eosinophils)
with suppression of lymphoid differentiation. CML cells are
generally indistinguishable from the non-CML cells arising from
healthy pluripotent cells. Mass spectrometry-based detection is
preferred over routine flow cytometry for massive multiplexing and
can potentially identify and characterize unknown cell types in
complex mixtures. In addition, assessing expression levels of certain
intracellular (IC) signaling markers alongside the lineage markers
can add a functional dimension to discern CML cell types with
different levels of lineage commitment (stem, progenitor, and
differentiated myeloid cells).
Methods and Results: Whole blood from a CML patient who
presented with molecular relapse was collected with informed
consent at UCSF Medical Center. A 100 ȝL aliquot (WBC: 7.9 x
10^3 cells/ȝL) was fixed followed by staining with 24 surface
markers associated with lineage and maturation, and 6 IC
antibodies towards phosphoproteins within the JAK-STAT, MAPK,
mTOR, and NF-kappa B survival pathways. Data from the single
tube assay were acquired on 63503 cell events by ICP-MS on a
CyTOF instrument (DVS Sciences, South San Francisco, CA), of
which 30579 passed the automated pre-gating. A reduced set of 17
markers allowed automated classification of all cells by EyelisTM
algorithms. Further dimensionality reduction and combining of
surface and IC markers improved the accuracy of classification of
CML cells. Novel p-STAT5hi cell subpopulations initially
differentiated based on surface markers by manual bivariate gating:
a) CD34+, CD45lo (0.04%), b) CD3+, CD45hi, and c) CD19+,
CD45lo (altogether <1% of the total cells analyzed) had substantial
overlap due to transition cells that expressed CD3 +/- CD19.
Combining additional surface and IC features in EyelisTM improved
the accuracy of classification, and allowed better separation of CML
and non-CML cell types in the blood.
Conclusions: We demonstrate further optimization of EyelisTM
statistical classification tool set by targeted feature selection,
facilitating automated distinction of CML and non-CML cell types in
a mass cytometry-based assay. By including IC signaling markers, at
least 3 CML cell subpopulations could be resolved in the setting of
relapse. The signaling activity profile and relative proportions of
rare CD34+ cells and p-STAT5hi cell types expressing lymphoid
markers could be of prognostic value in predicting response to
targeted inhibitors. Given the variability in response to BCR-ABL
targeted inhibitors, a cell-based prognostic test could provide a
basis for risk stratification in order to identify patients likely to
benefit from next-generation inhibitors. Further, the data provide
support for a clinical cytometry assay that includes measuring
signaling activities as a way to differentiate neoplastic clones from
non-neoplastic or reactive cell types.
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
ion
Abstracts
Conclusions: In additional to addressing the shortcoming of
existing tools, CyTOF Studio provides true computational
automation (normalization, clustering, classification, analysis),
while still allowing a high degree of interactivity to explore results.
It offers a robust analytical pipeline that is unbiased, reproducible
and yields higher quality results due to critical preprocessing steps
that generally go overlooked. Most importantly, it allows
researchers to more easily extract valuable information that has,
thus far, been elusive to many. The result is a robust, highlyautomated pipeline that can perform a far more comprehensive
analysis and allow a much more flexible visualization of the data in
a fraction of the time.
Poster
Session
Results: Calculations include measures of variability (if replicates
are defined), differential abundance, mean population shifts and
external variable correlations. Results can be immediately
visualized in customizable heat maps, differential scatter plots, and
a highly-interactive, rotatable 3D plot which allows for on-the-fly
filtering (by sample, cluster, phenotype) and color-coding.
Additionally, CyTOF Studio will automatically generate a properly
ordered and labeled phenotype lineage tree, with no further
manipulation or rearrangement required by end user.
Wednesday
15 June
Conclusion: Leukemic cells demonstrated a phosphorylation profile
that reflected survival of the intensively treated patients, but
surprisingly not correlated with mutational status of FLT3, NPM1,
cytogenetics or first course remission status. This suggests that
phosphoprotein determination in leukemic cells provide prognostic
information so far not available with current diagnostics. The robust
and relatively simple method of single cell signal profiling should
be tested in clinical trials to examine its feasibility in therapy
response prediction. More extensive mutational and epigenetic
analyses are needed in search for the molecular origin of the
low/high signal profiles.
Methods: The first step in the development of CyTOF Studio was
the codification of a standard nomenclature for expression. A 4level classification (with corresponding symbols) was chosen – Neg,
Low, High, Pos – with definitions that are clear yet still allow for
differences in magnitude between markers. Based on this standard,
an integrated (and modifiable) library of phenotype signatures is
used to drive the automatic classification of clusters downstream.
CyTOF Studio employs a number of novel algorithms and
techniques. Key among these new algorithms are: 1) a robust and
consistent means of translating numerical values into expression
levels; 2) a new peak-alignment normalization algorithm that
significantly outperforms the existing method; 3) a new (and
incredibly fast) density-based clustering algorithm capable of
handling millions of cells in seconds; and 4) a means of
automatically classifying unknown clusters, including the automatic
sub-typing if multiple flavors of a phenotype are detected. The
software has been implemented as a stand-alone Java desktop
application and requires no programming or scripting knowledge of
end users.
Tuesday
14 June
Results: Unsupervised clustering revealed two distinct signature
clusters based on low or elevated phosphorylation level among
AML cells. A similar cluster signature was absent in endogenous
non-leukemic lymphocytes from the same patients. No correlations
between baseline phosphorylation and prognostic mutations (FLT3
or NPM1), cytogenetics or response to first course of chemotherapy
were found. In AML patients treated with intensive chemotherapy
(n=45) the cluster with low phosphorylation level (n=19) correlated
with significant (p=0.007) shorter overall survival. Principal
component analysis guided a reduction to only three
phosphoproteins (STAT3, 4EBP1, ribosomal protein S6) with
statistically significant (p=0.014) stratification of survival. These
results were validated with a new set of 50 AML patients.
Introduction: Due to the relative infancy of CyTOF, there are
currently only a handful of tools available for analyzing mass
cytometry data. Unfortunately, these solutions all suffer from a
number of critical limitations and flaws which negatively affect
both the quality of results and the efficiency of analysis. Indeed, the
inherent high-dimensionality of mass cytometry data imposes a
number of challenges to the analysis. These challenges are further
compounded by technical variability across runs (misalignment of
peaks) and by the lack of a standard nomenclature for describing a
phenotype’s expression signature.
CyTOF Studio was developed as a solution that could overcome
these challenges in order to allow researchers to fully leverage the
potential of mass cytometry. This was accomplished by developing
and integrating several novel algorithms/techniques, wrapped in
graphical front end that is intended to facilitate ease of interaction
with the user.
Monday
13 June
Methods: Flow cytometric analysis of phosphoproteins central to
signal transduction pathways in myeloid cancer and analyzed
peripheral blood leukocytes from 133 (AML) patients. Cryo
preserved AML blood samples was thawed and equilibrated for one
hour in a defined serum-free medium which include insulin and
transferrin. Baseline phosphorylation was determined using 17
phosphorylation specific antibodies. All staining panels contain 4
surface antibodies and a live dead discriminator; CD33, CD38,
CD34, CD45 and phospho c-PARP. Two phospho specific
antibodies were added to each panel with the respective direct
conjugated AlexaFlour 488 and AlexaFlour 647; p38(pT180/pY182)
and ERK2(pT202/pY204), SRC(pY418) and Akt(pT308),
PDK1(pS241) and Akt(pS473), STAT1(pY701) and ribosomal
protein S6(pS235/36), STAT3(pY705) and STAT5(pY694),
CREB(pS133) and STAT3(pS727), ribosomal protein S6(pS240) and
NFkB(pS529), 4EBP(pT37/pT45) and STAT6(Y641), without pantibody and JNK(pT183/pY185). The lowest median signal for
each phosphoprotein in AML cells and lymphocytes, respectively,
were used as reference value for calculation of basal
phosphorylation. Hierarchical clustering were created using TM4,
and Principal Component Analysis was carried out using
UnscramblerX.
Immunoinflammation Therapy Area Unit, GlaxoSmithKline,
Cambridge, MA, United States
Sunday
12 June
Background: Dysregulation and mutations in signaling genes of
cancer cells characterize more than half of the acute myeloid
leukemia (AML) patients, and contribute to chemoresistance
through regulation of cellular processes. We investigated if
determination of single cell baseline phosphorylation of signaling
proteins reflected mutation of FLT3 and NPM1, cytogenetics,
response to first course of chemotherapy or overall survival.
Scott Davis, Mike Nolan, Jonathan Hill
Saturday
11 June
1
Centre of Cancer Biomarkers CCBIO, Department of
Clinical Science, University of Bergen, Bergen, Norway,
2
Department of Clinical Medicine, University of Bergen,
Bergen, Norway, 3Neuroimmunology Lab, Haukeland
University Hospital, 5021, Norway, 4Department of Internal
Medicine, Haukeland University Hospital, 5021, Norway
141/B4
CyTOF Studio: Automated Classification and Analysis
of High-Dimensional Mass Cytometry Data
Special
Lectures
Jø rn Skavland1, Stein-Erik Gullaksen1, Sonia Gavasso2,3,
Håkon Reikvam4, Øystein Bruserud4, Bjø rn Tore
Gjertsen4
New Software Development (B4)
Congress
Overview
140/B3
Survival Stratification in Acute Myeloid Leukemia by
Single Cell Signal Profiling
Index
157
Speaker/Author
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Index
Abstracts
Other
Biological Applications (B5)
142/B5
Flow and Image Cytometry Educational Outreach
Opportunities; Roswell Park Cancer Institute
Experience
Alexis Conway1, Adam Kisailus2, Orla Maguire1, Joseph
D. Tario1, Edward Podniesinski1, Hans Minderman1, Paul
K. Wallace1
1
Flow and Image Cytometry, Roswell Park Cancer Institute,
Buffalo, NY, United States, 2Educational Affairs, Roswell
Park Cancer Institute, Buffalo, NY, United States
The Flow andImage Cytometry shared resource (FICSR) at the
Roswell Park Cancer Institute(RPCI) was established in 1988 by
Carleton C. Stewart, PhD. Over the past 30years, technological
advancements in the fields of Flow and Image Cytometryhave been
progressing rapidly, and accordingly the importance of their role
andapplication in clinical diagnostics as well as basic research
settings. Educationaloutreach has been an important aspect of the
FICSR from its beginning. FICSR staffmembers have participated in
formal domestic and international educational coursesof Flow and
Image Cytometry and have also mentored high school, college,
andgraduate students participating in the RPCI summer research
program. The Departmenthas also developed introductory and
advanced courses to various aspects of Flowand Image Cytometry
which are regularly presented to our user base. FICSR hassince
expanded the educational outreach campaign to provide
educationalexperiences for teachers, students, patients, and
families. It has recentlyparticipated in the Interdisciplinary Science
and Engineering Partnership (ISEP)program from the University at
Buffalo. The ISEP project utilizes an innovativeapproach to the
professional development of teachers among a dozen ‘highneeds’urban schools. This approach incorporates educational
courses, interdisciplinaryresearch experiences, the development of
science and technology classroommaterials that are aligned with
state science learning standards, andinquiry-based curricula. The
project involved taking the principal componentsof a flow
cytometer, and making hands-on models that could be used to
explainin lay terms the principles behind the technology and how it
is used. Thiscurriculum has now also been developed into a
presentation which is piloted inthe RPCI Patient Resource center to
educate patients and visitors in order toprovide them with insight
into the supportive technology that may aid in thediagnosis or
treatment monitoring of cancer. Additionally, the FICSR has
developeda formal 14 week, 4 credit lecture/lab graduate course on
the Principles andApplications of Flow and Image Cytometry. In our
presentation we will summarizethe various educational outreach
initiatives that the FICSR is participating inwith the intent of
presenting the various opportunities that may be applicableto other
institutes as well. TheFICSR is supported by NCI Cancer Center
Support Grant P30CA16056.
Poster Abstracts
Antigen-Specific Immune Responses (B6 – B7)
143/B6
A Fluorescent Cell Barcoding Method to Measure
Human CTL Responses: Development of a “CytotoxFlow” Approach for Small Samples of Living Cells
Fatima-Ezzahra L'Faqihi, Manon Farcé, Sabina Mueller,
Delphine Lestrade, Salvatore Valitutti
INSERM U1043, Toulouse, France
The FCB technique encodes different cell samples with unique
fluorescent signatures and combines the samples together for
simultaneous antibody staining and data acquisition.
Our general aim is to understand the molecular and cellular
mechanisms regulating the CTL/tumor target cell encounter and
influencing both CTL fitness and tumor resistance.
In this work, our technical aim was to adapt this flow cytometry
multiplex approach to human disease. Our challenge was to apply
this technique to small cell samples (example: cells from biopsies)
and for living cells. To develop this method, we employed an in
vitro model of T cell activation based on B-EBV antigen presenting
cells and peptide specific cytotoxic T cell clones.
Cells corresponding to each condition of stimulation were fixed and
then labeled with different dye concentrations. Cells were labeled
with different fluorescent antibodies (anti-CD3, anti-CD8, antiCD20, anti-interferon Ȗ, anti-granzyme B, anti-perforin, antiactivated caspase 3) to analyze the activation of CTLs and the target
cell death. All of these conditions have been collected in one tube
and labeled with the same mix of CTL specific monoclonal
antibodies. The cells were then analyzed on a LSR-Fortessa
cytometer (BD Bisociences).
A limitation of this technique was that it can only apply to fixed
cells. This constrain does not allow to evaluate activation
parameters on live cells such as T Cell Receptor (TCR)
internalization, expression of CD107a on the CTL surface, or to
charaterize cell death using annexinV technique. All those
approaches require the use of living cells.
Then, the next step of our research was to adapt the technique of
cell barcoding to living cells. For this, we tested several fluorescent
probes for living cells. We observed that the best probes for our
experimental system were a combination of CFSE and CellTrace
Violet (CTV) (Invitrogen), probes that are compatible with many
others fluorochromes. This approach allowed us to expand our
conditions of barcoding to 12 samples and expand our panel of
specific antibodies to detect CTL activation and cell death.
We apply this method to study specific CTL activation against
targets cells in human health and disease.
144/B7
Recombinant BCG Over Expressing Ag85C Generates
Durable Central Memory T Cells and Showed
Enhanced Anti-mycobacterial Activity in Cattle
Anil Kumar, Hanumanthappa Krishna Prasad
Biotechnology, AIIMS, New Delhi, India
Tuberculosis (TB) is an infectious disease, caused by
Mycobacterium tuberculosis and or Mycobacterium bovis in
human and animals. These mycobacterial pathogens inflict huge
cost in financial terms and also pose a serious public health issue.
Six antigens, namely BCG (Danish 1331 Strain), M. indicus pranii
(Immuvac, Cadila), recombinant BCG over expressing Į-crystallin
(Rv2031c) & Ag85C (Rv0129c) and DNA vaccines with gene inserts
for Į-crystallin and SOD (Rv3846) were evaluated for their efficacy
in cattle. PBMCs from immunized and non immunized animals
(Control group) were isolated at 3, 7 & 30 months post
immunization (MPI) and polychromatic flowcytometry was adopted
to evaluate the T cell immune response. Comparative analysis of T
(CD4, CD8 & Ȗǻ) cells, showed that the highest induction of each T
cell subsets were seen in rBCG85C immunized cattle 3 MPI followed
by BCG immunized cattle. Central memory T cells (TCM) and
activated effector (AET) cells were also significantly induced in these
rBCG85C immunized cattle compared to BCG immunized cattle.
However the induction of effector memory (TEM) T cells was found
higher in BCG immunized cattle compared to rBCG85C immunized
cattle at 3MPI. TCM cells sustained up to 30MPI indicate the long
term immunological protection in rBCG85C immunized cattle.
Fluorescent cell barcoding (FCB) enables high troughput, high
content flow cytometry by multiplexing samples prior to staining
and acquisition on the cytometer. This technique minimizes reagent
consumption and maximizes data robustness (Krutzik and Nolan,
Nat. Methods. 2006).
158
Nicolas Bailly1, Damien Gheldof1, Sébastien Walbrecq1,
Louis Liu2, Michael Fere2, Cyril Gobinet2, Jacques
Clossat3, Edouard Cornet4, Xavier Troussard4, François
Mullier1, Bernard Chatelain1
At this time, the specificity and sensitivity of RM are 84.6% and
90.9% respectively when we targeted on region of interest of
spectrum. It allows us to make the difference between RM spectra
from Healthy B-Lymphocytes (HBL) and CLL B-Lymphocytes (CBL).
K Morton et al, Lab Chip, 2008.
2
D Inglis et al, J. Micromech. Microeng., 2011.
3
Y Chen et al, Biomicrofluidics, 2015.
Index
159
Speaker/Author
Further investigations must be conducted to define the exact
amount of lymphocytes that must be analyzed by RM to ensure the
(differential) diagnosis. Actually, one hundred B cells by sample are
inspected by RM. Decreasing the number of studied cells could
have a significant impact on the quickness and the comfort of the
CLL (differential) diagnosis. This could be done in a near future by
using quantitative phase-contrast microscopy.
1
Poster Session
Abstracts
Today, no predictive parameters for the clinical outcome
(independent prognosis factors) of the disease have been found but
immunoglobulin rearrangement of CBL could be detected by RM
keeping the hope for RM predictive parameters
With unlysed blood as the input at a rate of 10 microliter/min,
yielding 2 min on-chip incubation time, and R6G (mitochondrial
dye) as the label, the output leukocytes had fluorescence identical
to that of conventional preparation, with only 1% output
contamination by the R6G dye. Similar results were obtained with
SYTO13 dye.
Oral Sess on
Abstracts
RM offers a lot of advantages for a hematology laboratory including
automation, short time of acquisition (less than 30 minutes could be
expected), operator independent’s interpretation and informations
about molecular structure of the nucleus that both FC and
morphology are not able to give.
Commercial
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For those purposes, Classifier and Multi-Parameters Spectra
Analyzers (CMPSA) have been built to make automatically the
difference between HBL and CBL. To create the CMPSA, HBL, CBL,
T and NK lymphocytes have been sorted by Flow Cytometry( FC) to
perform analysis on purified cells from healthy subjects and CLL
patients.
Scholars &
Emerging
Leaders
These early results show that visual analysis and comparison of RM
spectrum are not easy and requires multi-dimensional and
mathematical analysis.
This paper integrates a long serpentine channel for extra on-chip
incubation time. Performing a series of sequential processing steps
in a continuous flow, we informally refer to the chip as a “Car
Wash.” A first DLD array of microposts of specific design1,2 harvests
and concentrates the target cells from the sample input and directs
them into a treatment chemical processing (e.g. labelling) stream.
The concentrated incubation mixture of label and cells feeds into
the serpentine channel for incubation. A final washing step is
performed in a second DLD array, which moves the cells from the
incubation mixture into a clean buffer for the output product. The
long serpentine channel enables long incubation time and
reasonably high flow rates to achieve low contamination of the
output (labelled and washed leukocytes) by the treatment chemical.
Wednesday
15 June
The aim of our research is first to evaluate the interest of the RM for
the diagnosis of B-Cell Chronic Lymphocytic Leukemia (CLL) and
the definition of easily accessible prognosis parameters and second,
to evaluate the practicability of RM in a routine hematology
laboratory.
Poster
Session
It has recently been showed that RM is able to discriminate
accurately benign from malignant lymphocytes 1 (in opposition to
haematological analyzers).
The labor-intensive steps used to prepare leukocytes for flow
cytometry (harvesting from blood, staining, fix/perm, washing, etc.)
can lead to variations in cell yield and quality. Morton et al.
presented a microfluidic cell-processing chip, in which
deterministic lateral displacement (DLD) arrays are used to move
target cells into and then out of a treatment chemical processing
stream (such as a label, etc.) in a continuous-flow process.1DLD
chips can harvest leukocytes from blood with 90% yield.2 In
Morton’s and later work,3 high flow rates were required to avoid the
diffusion of the treatment chemical into the washing (output) stream
and thus output contamination, limiting the maximum on-chip
chemical processing (incubation) time to a few seconds or less,
which is too short for this application. In this work, we demonstrate
an improved microfluidic approach which in single chip
sequentially (a) harvests and concentrates leukocytes from an input
blood stream, (b) moves them to a labelling/incubation region for 2
min, and (c) washes the cells to yield an output stream of the
leukocytes with low contamination (1%) of the labelling reagent.
Tuesday
14 June
Raman Microspectroscopy (RM) is a non-destructive method for the
observation and characterization of the molecular composition and
structure of materials and cells.
1
Electrical Engineering, Princeton University, Princeton, NJ,
United States, 2Physics, Princeton University, Princeton, NJ,
United States, 3Pediatrics and Physiology, University of
Maryland School of Medicine, Baltimore, MD, United
States, 4GPB Scientific, Richmond, VA, United States
Monday
13 June
The current strategy of B-Chronic Lymphocytic Leukemia(CLL)
diagnosis is mainly based on the immunophenotyping by FC. This
technique is operator dependent, time consuming (approximately
60 minutes for the diagnosis of CLL) and requires specific
knowledge’s and instrumentation. Since immunophenotypeis not
available in many laboratories, the diagnosis is often delayed and
this may impact the patient clinical outcome and always the
healthcare costs.
Yu Chen1, Joseph L. D’Silva1, Robert H. Austin2, James C.
Sturm1, Xiaochun Chen3, Kelly Reeder3, Curt Civin3,
Tony Ward4, Alison Skelley4, Khushroo Gandhi4, Lee
Aurich4, Zendra Peilun Lee4, Christopher Dosier4
Sunday
12 June
Hematology Laboratory, CHU UCL, Namur - Université
catholique de Louvain, Yvoir, Belgium, 22 MéDIAN
Biophotonique et Technologies pour la Santé, UMR CNRS
7369 MEDyC, Université de Reims Champagne-Ardenne,
Reims, France, 3Imagerie Médicale, TRIBVN, Chatillon,
France, 4Hematology Laboratory, CHU Caen, Caen, France
146/B9
“Car Wash": An Integrated Continuous-Flow
Microfluidic Device to Prepare Leukocytes for Flow
Cytometry by Deterministic Lateral Displacement
Saturday
11 June
1
Automated Sample Preparation (B9-11)
Special
Lectures
145/B8
Interest of Raman Microspectroscopy in the
Diagnosis and Prognosis of B-Chronic Lymphocytic
Leukemia
Reference:
Diagnosis approach of chronic lymphocytic leukemia on unstained
blood smears using Raman microspectroscopy and supervised
classification, T. Happillon et al., Analyst, 2015, 140, 4465
Congress
Overview
Automated Microscopy (B8)
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Index
Abstracts
147/B10
Automated Processing of Human Blood Leukocytes
by a Plastic, Disposable Microfluidic Deterministic
Lateral Displacement Device
148/B11
Preparation of Polychromatic Antibody Cocktails for
Immunophenotypic Whole Blood Assays Using a
Semi-automated Approach
Alison Skelley1, Tony Ward1, Khushroo Gandhi1, Zendra
Peilun Lee1, Christopher Dosier1, Joseph L. D’Silva2, Yu
Chen2, MinJung Kim3, Xiaochun Chen3, Lee Aurich1,
Diether Recktenwald4, Robert H. Austin5, Mike
Grisham1, Curt Civin3, James C. Sturm2
Iliana Gonzalez1, Tanisha Meeuwsen1, William Miller1,
Daniel Haley1,2, Valerie Conrad1, Tomasz Poplonski1,
Alice Tanibata-Branham3, Keith Bahjat1,4, Yoshinobu
Koguchi1
1
2
GPB Scientific, Richmond, VA, United States, Princeton
Institute for the Science and Technology of Materials,
Department of Electrical Engineerin, Princeton University,
Princeton, NJ, United States, 3Pediatrics and Physiology,
University of Maryland School of Medicine, Baltimore, MD,
United States, 4Desatoya, Reno, 5Princeton Institute for the
Science and Technology of Materials, Department of
Physics, Princeton University, Princeton, NJ, United States
As multi-parameter flow cytometry becomes increasingly more
valuable and widely used in research and clinical diagnostic
settings, the need for new methods to improve the efficiency of
sample preparation becomes more and more critical. Currently,
membrane and intracellular labeling processes can yield substantial
cell losses, especially using protocols that require multiple
centrifugation wash steps to maximize sample quality. Since each
wash step may lose ~10-15% of the cells, multiple washes during
processing for flow cytometry result in significant overall cell loss
and sometimes preferential loss of specific cell types. To counteract
cell loss, larger starting sample volumes are required, an especially
challenging problem in clinical testing of children and patients who
need many blood tests, and research using small animal studies. In
addition, the process is time intensive (requiring 30 min or more),
and the labor-intensive nature of these steps may also lead to
significant intra- and inter-operator variability.
Here we present a Deterministic Lateral Displacement (DLD)
microfluidic chip technology that harvests cells from blood purely
on the basis of cell size. The DLD approach involves pumping
blood through a microfluidic device the size of a microscope slide
containing a specially designed array of microposts. Cells above the
target size of the micropost array are gently deflected by the
microposts into a stream of run buffer, a process that is noninjurious to the cells. Our previous research utilized silicon-based
devices; this study demonstrates the reduction-to-practice of this
technology with a commercially produced, high precision plastic
microfluidic chip-based device. After a human blood sample was
incubated with fluorochrome-conjugated monoclonal antibodies
(mAbs), the mixture was input to a DLD microfluidic chip where it
was driven through a micropost array designed to deflect white
blood cells (WBCs) via DLD solely on the basis of size, thus
separating WBCs from smaller cells and particles and washing them
simultaneously. The DLD microchip processed 100 μL blood in
<18 min with >95% recovery of WBCs with no significant skew
across subpopulations. Under conditions optimized for WBC purity,
the WBCs outnumbered red blood cells (RBCs) by ~10:1 in the
microchip product, with >99.98% of the RBCs removed. Flow
cytometric evaluation of the microchip product, with no further
processing, lysis or centrifugation, revealed excellent quality WBC
forward and side light scattering characteristics and high signal-tonoise ratios for immunolabeled cells, similar to or better than those
of cell samples processed conventionally by centrifugation. These
results indicate that cost-effective plastic DLD microchips can be
used to replace multiple centrifugation steps to speed and automate
the processing of cells for high quality flow cytometry analysis, and
suggest their utility for a multitude of other applications.
1
Human Immune Monitoring Laboratory, Earle A. Chiles
Research Institute, Portland, OR, United States, 2Flow
Cytometry, Sony Biotechnology, San Jose, CA, United
States, 3Technical Support, Beckman Coulter, Inc. Life
Sciences, Brea, CA, United States, 4Discovery Research,
Bristol-Myers Squibb, Redwood City, CA, United States
Immunophenotyping of human peripheral blood determines
quantitative and qualitative changes in immune cell subsets. It
provides insight into mechanisms of action and resistance, aids
discovery of predictive biomarkers, and facilitates the development
of combination immunotherapies. Therefore, the validation and
standardization of immunophenotyping is an area of considerable
interest to academic researchers, clinical laboratories, and
industries.
Whole blood staining utilizes unmanipulated blood, which
minimizes artifacts that can occur during sample preparation (e.g.,
isolation and cryopreservation of peripheral blood mononuclear
cells). However, whole blood staining must also be done on freshly
collected blood to ensure the integrity of the sample. Additionally,
it is best to prepare antibody cocktails on the same day to avoid
potential instability of tandem-dyes and prevent reagent interaction
between brilliant violet dyes. Therefore, whole blood staining
requires careful standardization to control for intra and interexperimental variability.
Here, we report deployment of an automated liquid handler
equipped with a two-dimensional (2D) barcode reader into a
standard process of making antibody cocktails for flow cytometry.
Antibodies were transferred into 2D barcoded tubes arranged in a
96 well format and their contents compiled in a database. The
liquid handler could then locate the source antibody vials by
referencing antibody names within the database. Our method
eliminated tedious coordination for positioning of source antibody
tubes. It provided versatility allowing the user to easily change any
number of details in the antibody dispensing process such as
specific antibody to use, volume, and destination by modifying the
database without rewriting the scripting in the software method for
each assay. A proof of concept experiment achieved outstanding
inter and intra- assay precision, demonstrated by replicate
preparation of an 11-color, 17- antibody flow cytometry assay.
These methodologies increased overall throughput for flow
cytometry assays and facilitated daily preparation of the complex
antibody cocktails required for the detailed phenotypic
characterization of freshly collected anticoagulated peripheral
blood.
We will also show data from B-Cell, DC, NK and Helper T_Cell
Panels.
Biomarkers (B12 – B26)
149/B12
Analyses of Mass Cytometry Data Enables
Identification of Biomarkers That Associate with
Manufacturing Success for CAR-T Cell Therapies
Iulian Pruteanu-Malinici1, Piotr Pierog1, Arnaud
Colantonio1, Artur Veloso2, Sadik Kassim1
1
Cell and Gene Therapies, Novartis, Cambridge, MA,
United States, 2Oncology Bioinformatics, Novartis,
Cambridge, MA, United States
160
152/B15
Evaluation of the Oxidant Status of Patients with
Rheumatoid Arthritis as a Potential Biomarker for
Disease Progression
Poster Session
Abstracts
Mitali Chatterjee, Soumita De
161
Index
Pharmacology, Institute of Postgraduate Medical Education,
Kolkata, India
Speaker/Author
Conclusion: Our group has developed and validated a uEV assay
using high dimensional flow cytometry in healthy controls with a
coefficient of variation of less than 20%. Further validation of the
uEV assay in patients with CKD may provide a signature pattern of
uEVs that can serve as the basis for a clinical screen for KD.
Oral Session
Abstracts
Out of 7705 gene specific probes labelled on a micro-array chip,
669 genes were found to be up-regulated and 470 down regulated
in resistant strains. The cysteine protease-like protein of calpain
family [GenBank: CBZ34784] was found significantly over
expressed in resistant parasite strains both by DNA micro-array as
well as real time RT-PCR. Only sera from relapse patients showed
presence of anti calpain antibodies through western blotting.
Results: Spike-in of hPODs confirmed specificity of podocalyxin
antibody for podocyte EVs. uEVs were isolated from healthy human
urine and profiled with a coefficient of variation of less than 20%.
The following phenotypes were profiled: CD3+,
Podocalyxin+Annexin+, Podocalyxin+Podoplanin+,
CD105+Annexin+. A proof-of-concept study employing this uEV
assay in the urine of patients with CKD revealed a different uEV
pattern compared with healthy controls.
Commercial
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Splenic aspirates were collected and parasites cultured from
patients relapsed after initial cure (n=15) and successfully treated
(n=15) with miltefosine. Differentially expressed genes in cultured
parasite strains of miltefosine resistant strains, obtained by DNA
micro-array, were further validated by real time reverse
transcriptase polymerase chain reaction (RT-PCR) and western
blotting after preparing recombinant proteins.
Methods: Healthy controls and patients with Chronic Kidney
Disease (CKD) were recruited for this study. uEVs were assessed
using a modification of methodology previously developed to
measure plasma EVs. Briefly, fresh urine is centrifuged at 2500xg
and then treated with sodium chloride to remove Tamm-Horsfall
protein prior to staining with a panel of fluorochrome-labeled celltype specific antibodies detecting CD3, CD31, CD41, CD105,
Annexin-V, anti-Podoplanin, and anti-Podocalyxin. Cultured
human podocytes (hPODs) are used as a positive control. Triton is
added to selectively destroy EVs as a negative control. Data are
acquired on a modified Becton Dickinson FACSCanto A and the
pattern and number of uEVs is determined using FlowJo Software
(Treestar, Inc) and a computational algorithm called Cytometric
Fingerprinting.
Scholars &
Emerging
Leaders
Knowledge of bio markers for miltefosine resistance may be
beneficial for proper selection of treatment regimen.
This study developed methodology for high dimensional flow
cytometric profiling of various EVs in urine to discern a sensitive
and specific biomarker pattern for the early detection of
podocytopathies as precursors to KD before they progress to severe
disease.
Poster
Session
Miltefosine is the only orally administrable drug for the treatment of
leishmaniasis. It is used as a first line drug for the Kala-azar
Elimination Programme in the Indian subcontinent. But in recent
years, a decline in its efficacy points toward the emergence of
resistance to this drug.
Proteomic approaches to investigating KD have identified that
urinary EVs (uEVs) are involved in kidney damage or kidney repair
by affecting various aspects of KD, including, podocyte injury,
endothelial injury, tubulointerstitial damage, inflammation, and
repair. For example, proteomic studies have shown that uEVs
containing Podocalyxin correlate with disease severity in patients
with Diabetic Nephropathy while uEVs with Podocin are specific
markers of podocyte injury in KD.
Wednesday
15 June
Department of Medicine, Institute of Medical Sciences,
Banaras Hindu University, Varanasi, India
Background: A test that is minimally invasive and sensitive could
improve early diagnosis of kidney diseases (KD) and facilitate early
medical intervention, decreasing progression to end stage renal
disease and other serious complications.
Tuesday
14 June
Puja Tiwary, Dinesh Kumar, Shyam Sundar
Medicine, Perelman School of Medicine at the University
of Pennsylvania, Philadelphia, PA, United States, 2Pathology
and Laboratory Medicine, Perelman School of Medicine at
the University of Pennsylvania, Philadelphia, PA, United
States
Monday
13 June
150/B13
Gene Expression Profiling and Molecular
Characterization of Miltefosine Resistance in L.
donovani
1
Sunday
12 June
Conclusions: Using mass cytometry and automated analytical
workflows, we were able to identify several statistically robust
phenotypic signatures in the starting material of CLL patients that
associate with “manufacturing success” for CTL019 based
autologous cell therapies. Specifically, our results confirm that the
expansion of autologous T cells for CAR T cell therapy can be
highly variable and the variability is due at least in part to the
composition of the starting cell material.
Marisa C. Mizus1, Emile R. Mohler, III1, Richard D.
Schretzenmair2, Lifeng Zhang2, Wade T. Rogers2,
Lawrence B. Holzman1, Raymond R. Townsend1, Jonni
S. Moore2
Saturday
11 June
Results: Manual analysis of the D0 mass cytometry data failed to
identify a biomarker that predicts CTL019 “manufacturing success.”
By contrast, the automated analytical workflows identified several
signatures that are associated with CTL019 manufacturing
performance.
151/B14
Optimal Detection of Urinary Extracellular Vesicles
in Evaluating Chronic Kidney Disease
Special
Lectures
Methods: Peripheral blood samples were collected from a total of
15 chronic lymphocytic leukemia (CLL) patients. Cells were then
stained with a 37 parameter mass cytometry panel. Data were
subsequently acquired using a CyTOF instrument; this time-point
will subsequently be referred to herein as day 0 (D0). The 15 CLL
samples were then setup in small-scale cultures that mimic the 9
day CTL019 manufacturing process. Based on total cell expansion,
over the nine day period, samples were classified as either “high
proliferators” (NH = 7 samples) or “low proliferators” (NL = 8). In
order to identify a D0 biomarker signature that correlates with total
cell expansion in culture (i.e. “manufacturing success”), we utilized
the following automated analysis workflow. Subsequent to an
initial pre-gating step, the data were analyzed for biomarker
identification, using bio-statistical automated algorithms.
Calpain family cysteine protease-like protein can be useful as
potential bio marker of miltefosine unresponsiveness.
Congress
Overview
Introduction: CTL019 is a novel, investigational, cancer
immunotherapy wherein a patient’s own T cells are genetically
modified to express a chimeric antigen receptor (CAR) that targets
CD19 on the surface of malignant B cells. Early trial results of
CTL019 from multiple indications have shown tremendous clinical
efficacy. Yet, for some patients, CTL019 manufacturing represents
a logistical challenge; specifically T cells from some patients fail to
expand in culture; these lots are considered “manufacturing
failures” as the final product contains insufficient cell numbers for
full dosing of the patient. Here, we present a mass cytometry based
assay, that when combined with state of the art bioinformatics,
enables for the discovery of biomarkers that predict manufacturing
success of CTL019 therapeutic products.
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Index
Abstracts
Background: Rheumatoid arthritis (RA) is an autoimmune disease
whose etiology remains unknown, wherein studies have implicated
inflammatory mechanisms and importantly, includes a role for
oxidative stress. This study aimed to correlate the degree of
oxidative stress along with its downstream effects and its correlation
with disease activity score as a potential surrogate marker.
Methods: The redox status of neutrophils sourced from synovial
fluid (SF) and peripheral blood was measured by flow cytometry in
terms of total reactive oxygen species (ROS) and hydroxyl radical.
Among the molecular damage markers, protein carbonylation, lipid
peroxidation and advanced oxidation of protein products (AOPP)
was measured by spectrophotometry and S-nitrosothiols by
flurimetry. Additionally, the potential therapeutic role of an
established anti-oxidant Allylpyrocatechol (APC) was studied in
peripheral blood (PB) and synovial fluid (SF) along with its impact
on inflammation related signaling molecules.
Results: The basal levels of total ROS, superoxide and hydroxyl
radicals were significantly raised in neutrophils sourced from
peripheral blood and synovial infiltrate, as also they showed a
strong positive correlation. Furthermore, the raised generation of
ROS and hydroxyl radical correlated positively with markers of
oxidative damage and Disease Activity Score (DAS28). APC
effectively decreased basal levels of ROS, superoxide and hydroxyl
radicals in both PB and SF and mediated its anti-inflammatory/antioxidant activity by inhibiting the MAPKinase pathway.
Discussion: The enhanced generation of free radicals and oxidative
damage are a consistent feature of patients with RA. As the levels of
ROS and markers of oxidative damage sourced from peripheral
blood and synovial infiltrate correlated positively with the DAS28,
it suggested that (i) monitoring of oxidative stress could serve as a
marker of disease severity in RA and (ii) measurement of ROS in
peripheral blood can serve as an indirect measure of the degree of
inflammation in patients with RA. APC effectively attenuated the
oxidative stress in the emphasizing the importance of anti-oxidants
being included as an ‘add on’ therapy for improved management of
Rheumatoid Arthritis.
153/B16
Issues with Mesenchymal Stem Cell Markers in
Amniotic Cells
Wendy Weston1, Rachel Frisch2, Shabnam Namin1
1
Research and Development, UMTB/Vivex Biomedical,
Miami, FL, United States, 2Research and Development,
UMTB Biomedical, Miami, FL, United States
Flow cytometry is an invaluable method for the evaluation of cell
types within most tissues. Although we have become quite familiar
and comfortable with immunological markers, stem cell markers for
various tissues differ and combinations of markers are often
necessary to accurately determine the percentage of stem cells
present. In light of the variety of tissue and stem cell types, the most
varied in marker expression is the mesenchymal stem cell.
Unfortunately, many single markers and combinations of markers
are used to claim mesenchymal stem cell phenotype. Additionally,
there is a variety of levels of "stemness" that are claimed to be
discernable by specific marker expressions. In relation to amniotic
cells, we sought to determine some specific marker expression
levels utilized by biotechnology companies and their possible
misconceptions. We present here some issues with the use of most
single markers to establish mesenchymal percentages in amniotic
tissues and fluids. There are some specific, primitive mesenchymal
stem cell markers that have been shown to be reliable when used
alone. Most mesenchymal stem cells are not of this very primitive
phenotype and express some markers of other cell types. Although
the temptation exists to claim high percentages in a given tissue or
fluid, the extra research and diligence to rule out other cell types
that may also express these markers is crucial in the biotechnology
industry. Adoption of more stringent analysis ensures that the
claims of researchers and scientists are comparable to each other
and clinically reliable.
154/B17
Mass Cytometry: TNF Signaling in Patients with
Lucius Bader1,2, Stein-Erik Gullaksen3, Jø rn Skavland3,
Christian Vedeler4,5, Clara Gram Gjesdal1,2, Sonia
Gavasso4,5
1
Rheumatology, Haukeland University Hospital, Bergen,
Norway, 2Department of Clinical Science, University of
Bergen, Bergen, Norway, 3Hematology, University of
Bergen, Bergen, Norway, 4Neurology, Haukeland University
Hospital, Bergen, Norway, 5Department of Clinical
Medicine, University of Bergen, Bergen, Norway
Background:The pro-inflammatory cytokine tumor necrosis factor
(TNF) is a major driver of inflammation in patients with rheumatoid
arthritis (RA) and target of treatment with TNF inhibitors (TNFi).
Primary and secondary non-response to TNFi occurs in about one
third of RA-patients, exposing patients to treatment-related risks and
causing unnecessary drug-expenses. At present, treatment decisions
regarding TNFi are made empirically. Thus, there is a need for
reliable biomarkers to predict and monitor treatment effects.
Mass cytometry is a powerful tool in the search for such
biomarkers, allowing us to simultaneously look at a large number of
cell subsets, receptors, intracellular epitopes and cytokines at the
single cell level. The goal of this study is to contribute to the
cytometry characterization of TNF signaling in peripheral blood
from healthy donors and patients with newly diagnosed RA with the
purpose of identifying candidate biomarkers for TNFi treatment
responses.
Methods: We collected peripheral blood mononuclear cells
(PBMC) from 20 healthy donors and 20 newly diagnosed and
untreated RA-patients in an age- and sex-matched manner. RApatients are divided into one group with high disease activity
(disease activity score DAS28 >5.1, N=10) and one group with
moderate disease activity (DAS28 3.3-5.1, N=10).
Unstimulated PBMC, PBMC stimulated with TNF and PBMC treated
with a TNFi will be analyzed by mass cytometry (Helios). We will
apply a set of 17 standard surface markers, 2 receptor- and 8
phospho-antibodies. More specifically we will look at the
expression of TNF receptor 1 and 2 (CD120a and b) and at markers
of intracellular TNF signaling activation (p-p38, p-ERK1/2, p-IʃBĮ,
p-NFʃB) in common cell subsets (peripheral blood panel, Fluidigm).
We will additionally acquire data on the phosphorylation status of
STAT1, 3, 5 and pS6.
Results: This abstract describes work in progress; we expect results
from this project as described in May 2016.
Conclusions: TNF is central in RA-pathogenesis and treatment. We
aim to identify cell subpopulations and intracellular markers that
best reflect TNF activity as a basis for further study. Ultimately we
wish to translate results into flow cytometry based clinical assays.
We consider this study as a pilot project. As a part of our hunt for
diagnostic and therapeutic biomarkers in RA, we have collected
PBMC and clinical data from more than 120 patients with RA at
different time-points. We are aware of the fact that joints are the
primary site of pathology in RA, which is why we are planning to
expand this project by the collection of synovial fluid and synovia.
155/B18
New NIST Measurement Service for ERF Value
Assignments of Calibration Microspheres in Flow
Cytometry
Paul DeRose, Adolfas Gaigalas, Lili Wang
Bioassay Methods, NIST, Gaithersburg, MD, United States
Background: The accurate determination of antibodies bound per
cell (ABC) is still a great challenge in quantitative flow cytometry
(QFC). The comparability and accuracy of quantitative cytometry
162
served as the primary parameters of assay validation. Variability
between analysts, instruments, and labs was also assessed.
Specimen stability was characterized by holding the whole blood at
ambient temperature for 6, 24, 48 and 72 hours post sample
collection. Finally, assay performance was confirmed in whole
blood from patients with B-cell malignancies (CLL, NHL).
Congress
Overview
157/B20
Detection of PDL1 and PDL2 on Circulating Tumor
Cells Using Microfluidic Based Chipcytometry
Special
Lectures
Methods: Light obscuration and flow cytometer measurements were
used to determine the concentration of calibration microsphere
suspensions. SRM 1934, comprised of 4 fluorescent dye solutions
with known concentrations, was used to compare the fluorescence
intensities of dyes in solution to those in hard-dyed calibration
microspheres in the same spectral regions. A NIST-calibrated CCD
Fluorescence Spectrometer was used for this comparison to
measure fluorescence intensities.
Jinkai Teo1, Meihui Tan2, Anja Mirenska3, Janice Oh1,
Lewis Hong1, Ali Bhagat2, Richard Wnek4, Jan Detmers5,
Chih-Liang Chin1, David Skibinski1
David Lanham, Minesh Patel
Biomarkers, Eurofins Pharma Bioanalysis UK Ltd, Abingdon,
United Kingdom
Index
Speaker/Author
163
Poster Session
Abstracts
Materials and Methods: Peripheral blood samples were collected
into standard anticoagulant blood tubes, or sodium heparin CPT™
tubes for PBMC preparation. PBMCs were either assessed freshly
isolated and after resuscitation following cryopreservation.
Erythrocytes were removed from the whole blood by lysis and a
Oral Session
Abstracts
Introduction: Flow cytometry has been used in therapeutic drug
development for many years to inform scientific and clinical
decisions; from early discovery through to clinical sample analysis.
In this area the most common application is in pharmacodynamic
(PD) biomarker assessments. The growing reliance on this
biomarker data during drug development has led to an increased
scrutiny of the data being generated. There is now formal
regulatory guidance on assay validation that is being applied to
biomarker analyses by following a ‘fit for purpose’ approach. We
present data from a ‘fit for purpose’ validation of two flow flow
cytometry assays; a ‘TBNK panel’ to identify and a surface
phenotype 'T helper panel’ which identifies Th1, Th2, Th17 subsets
in whole blood and PBMCs.
Commercial
Tutorials &
Exhibits
Scholars &
Emerging
Leaders
T cell-recruiting bispecific modalities hold great promise in the field
of oncology as they direct a patient’s immune system, more
specifically the cytotoxic activity of T cells, against cancer cells.
Genentech has initiated an open-label, multicenter, Phase I trial
evaluating safety and pharmacokinetics of escalating doses of
BTCT4465A, an anti-CD20/CD3 TDB therapy, in patients with
relapsed or refractory B-cell non-Hodgkin's lymphoma and chronic
lymphocytic leukemia. The mechanism of action is T-cell mediated
B-cell killing following target-dependent T-cell activation. We have
developed, validated, and outsourced a comprehensive flow
cytometry assay to measure exploratory pharmacodynamic
response in patients with B-cell malignancies after treatment with
BTCT4465A. This 4-panel assay measures depletion and recovery
of circulating B cells, enumeration and kinetics of T-cell
populations, including activation (CD25, CD69), functional status
(Ki67, granzyme B), and checkpoint regulators (PDL1, PD1, TIGIT,
TIM3, OX40). Heparinized whole blood from healthy donors and
was used to develop and validate the assay at ICON Central
Laboratories in New York and Singapore. Inter- and Intra-assay
precision estimates of population percentage and antigen density
Poster
Session
Development Sciences, Genentech, South San Francisco,
CA, United States
Wednesday
15 June
Katie Dalpozzo, Cherie Green, Genevive Hernandez,
Chaitra Marathe, Jeffrey Wallin
158/B21
Performing a ‘Fit for Purpose’ Validation Exercise of
Flow Cytometry Assays for Lymphocyte Populations
Assessed as Pharmacodynamic Biomarkers during
Drug Development Clinical Trials
Tuesday
14 June
156/B19
Development, Validation, and Outsourcing of a Flow
Cytometry Assay to Measure Pharmacodynamic
Response in Patients with B-Cell Malignancies
Treated with BTCT4465A, an Anti-CD20/CD3 T-Cell
Dependent Bispecific Therapy
Recent advances in cancer therapy have demonstrated the potential
of the immune system in cancer control and rejection. Prominent
amongst these approaches has been the success of anti-PD-1
immunotherapy, to break the strong inhibitory signal, transmitted
by tumor specific ligands to the PD-1 immune modulatory receptor
expressed on T-cells. PD-ligand expression on the tumor is a
clinically validated biomarker of therapeutic response to anti-PD-1
immunotherapy. Obtaining tumor biopsies however for PD-ligand
interrogation is an invasive procedure not suited for frequent
longitudinal monitoring during cancer therapy. Furthermore, tumor
heterogeneity for PD-ligand expression may not accurately capture
the PD-ligand status of the whole tumor burden in a single biopsy.
An alternative approach is the analysis of blood samples for
circulating tumor cells which have broken away from the tumor
and entered the periphery. We describe the development of an
assay workflow to detect and characterize circulating tumor cells in
peripheral blood samples. Our approach uses a sized-based
microfluidic enrichment technique, and subsequent
characterization with microfluidic based cytometry
(Chipcytometry). Data measuring the expression of PDL1 and PDL2
on circulating tumor cells from breast cancer patients will be
presented.
Monday
13 June
Conclusions: NIST recently certified the concentration of four dye
solutions (Coumarin 30, APC, Fluorescein and Nile Red), which has
been released as SRM 1934. In order to assign ERF values to
microspheres, it is necessary to accurately measure both the
fluorescence intensities of free-dye solutions with known
concentrations (SRM 1934) and of calibration microsphere
suspensions, and the concentrations of microsphere suspensions.
The highly accurate particle counting measurements reported here
will improve the accuracy of ERF-based fluorescence intensity
assignments of reference microspheres for the calibration of the FC
channels centered from 425 nm to 800 nm. The improvements in
fluorescence calibration accuracy of flow cytometers will lead to
improved comparability of different flow cytometer platforms, and
set the stage for the application of biological standards to determine
ABC values.
Translational Biomarkers, MSD International (Singapore
Branch), Singapore, Singapore, 2-, ClearBridge Biomedics,
Singapore, Singapore, 3Zellkraftwerk GmbH, Hannover,
Germany, 4Translational Molecular Biomarkers, Merck &
Co. Inc., Rahway, NJ, United States, 5Central Lab Services,
Zellkraftwerk GmbH, Leipzig, Germany
Sunday
12 June
Results: The microsphere concentrations in suspension, determined
by two independent methods, were compared and systematic errors
related with each technique were explored. The concentrations of
the microspheres were determined with an uncertainty of less than
5%. Calibration curves of fluorescence intensity versus free-dye
concentration enabled ERF assignments of calibration microsphere
suspensions with uncertainties of about 5% or less.
1
Saturday
11 June
assays often depend on the accuracy and consistency of the
measured ABC values. Before ABC values of samples can be
determined the fluorescence channels being used in an assay must
be calibrated, typically using reference microspheres with known
fluorescence intensities. Fluorophore solutions of known
concentration are used to assign fluorescence intensities to the
calibration microspheres in units of equivalent number of
referencefluorophores (ERF). To make this assignment, the
microsphere concentrations of the reference microsphere
suspensions need to be known with high accuracy.
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Index
Abstracts
viability stain included along with the lineage-specific markers.
Samples were fixed in a 1% formaldehyde solution before analysis
using a 3-laser FACSCanto II™ flow cytometer.
Results: Viability Stain Comparison: A comparison of an amine
reactive fixable viability stain and a standard DNA intercalating
dye was performed. The DNA dye produced good discrimination
of live vs. dead cells for both freshly stained samples and stained
samples held for 24 hours at +4°C, without fixation. However, in
the presence of formaldehyde, there was a marked increase in
background staining for DNA dye in stained samples held for 24
hours, reduced live and dead discrimination.
Acquisition Events: Based on these results a minimum of 5,000
lymphocyte events collected for both TBNK and Th panels allows
application of reliable acceptance criteria These criteria are useful
where the disease state affects biology, such as lymphopenia.
Assay Precision: Within assay repeatability (intra-assay) for both the
whole blood analysis of TBNK and Th panels gave %CV <20%,
whilst isolated PBMC intra-assay precision for both the TBNK and
Th panels <10%. Reproducibility across assays (inter-assay
precision) for both TBNK and Th panels showed acceptable
precision (CV<20%), with exception to the monocyte populations,
where there was variability between donors and within runs. This
variation was most prominent in the whole blood assessment.
Variability seen within the Th17 population was also observed and
predicted since this is low frequency population. In general, the
PBMC samples were less affected. Between operator performance
for both panels using both PBMC and whole blood followed the
same pattern as the inter-assay performance.
Summary: In summary panels for TBNK and Th detection showed
generally suitable precision between donors and runs. Data from
blood samples collected in 3 different anticoagulants shows all 3
were suitable for use on freshly collected and analysed samples.
The number of lymphocyte events collected was confirmed to be
robust when only 5000 event acquired. The intra and inter- assay
and inter analyst precision for the TBNK and Th panels were found
to be generally acceptable on all parameters. Some inter-assay and
inter-operator variability was observed in monocytes and Th17
populations and these may require additional assessment to
determine the source of these variations.
159/B22
Development and Validation of a Clinical Flow
Cytometry Assay to Measure Intracellular Ki67
Expression in Memory T Subsets, NK and Treg Cells
in Human Whole Blood
Yongliang Sun, Terry Bridal, Katherine Yang, Lisa PattiDiaz, Michael Nathan Hedrick, Michele Cleary, Anka G.
Ehrhardt
Clinical Cytometry, Clinical Translational Technologies &
Operations, Bristol-Myers Squibb, Pennington, NJ, United
States
The cell proliferation marker Ki67 is a prognostic and/or predictive
biomarker in patients with breast cancer and other tumors.
Immunohistochemistry is commonly used to assess expression of
Ki67 in tumor tissues with the limitations of the assay
standardization and the sample access. Furthermore, Ki67 is a
continuous marker that needs to be monitored because of the
biology of tumor proliferation. Therefore, a quantitative flow
cytometry assay to measure intracellular Ki67 expression in human
whole blood (hWB) has been developed to support clinical trials in
immuno-oncology therapeutic area.
Intracellular multi-color flow cytometry is technically challenging
due to the fact that the permeabilization should be sufficient
enough to allow the exposure of intracytoplasmic/intranulear
proteins, and also be gentle enough to preserve the staining of
surface markers. We established a 9-color intracellular flow
cytometry assay to measure Ki67 expression in memory T cell
subsets, NK, NKT and Treg cells in hWB. Each marker’s clone and
its conjugated fluorochrome, as well as permeabilization conditions
were optimized. BD Cytofix/Cytoperm® buffer was selected for this
164
assay. To validate the assay for clinical use, Cyto-Chex® BCT blood
collection tubes were used to increase the duration of sample
stability. The Relative Error (RE) % was within 15% between the
data from fresh blood samples collected in the sodium heparin tube
and those in the BCT tube, indicating that BCT tubes were feasible
for this assay. Then, the sample stability was evaluated using blood
collected in the BCT tube. The RE % was within 20% for up to 72h
after blood collection, as compared to the fresh samples. Intra-run
precision was also determined to be within 15% (CV). Therefore,
the assay has been validated for clinical samples.
In conclusion, a multi-color intracellular Ki67 flow cytometry assay
using hWB has been developed and validated to support clinical
trials. The assay is quantitative, robust, and can be standardized
across labs and studies. Easy access to the peripheral blood enables
us to continuously monitor Ki67 expression in various immune cell
subsets from hWB as a biomarker for cancer patients.
160/B23
Detection of Durvalumab-Induced Changes in
Activated and Proliferating T Cells in Melanoma
Patients Using Flow Cytometry-Based
Immunophenotyping Assays
Nathan Standifer1, Jennifer Pearson1, Dominic Sinibaldi2,
Meina Liang1
1
Clinical Pharmacology & DMPK, MedImmune, Mountain
View, CA, United States, 2Research and Discovery
Informatics, MedImmune, Gaithersburg, MD, United States
Background: Durvalumab is a fully-human,IgG1 molecule that
binds to the Programmed Death Ligand-1 (PD-L1) protein.Blockade
of the interaction of PD-L1 with its receptor has been demonstrated
toresult in the activation of exhausted T cells. As such,
quantification ofcirculating activated or proliferating T cells is a
potentially highly-impactfulmeasure of durvalumab-induced
pharmacodynamic effects. Toward this end, wedeveloped and
validated a set of flow cytometry-based assays to quantify
thesepopulations. These assays were implemented in a trial of
durvalumab in melanomapatients.
Methods: The proliferating T cell assaywas designed to quantify T
cells expressing increased levels of Ki67. Toenhance its specificity,
we incorporated a negative control tube containingsaturating
quantities of non-fluorochrome labeled (cold) anti-Ki67.
Theactivated T cell assay was designed to quantify T cells with
increasedexpression levels of CD38, HLA-DR or CD278 (ICOS).
Each assay was validated forinter-assay and inter-operator
imprecision (acceptance criteria 20% CV ofmean values),
longitudinal stability and stability in the presence of drug
(acceptancecriteria 20% change from baseline values). In
addition, the sensitivity ofthe activated T cell assay was assessed by
spiking autologous, activated PBMCinto specimens. The assays
were implemented in a Phase I study of durvalumab (CD-ONMEDI4736-1108)administered to cutaneous and uveal melanoma
patients (n=46) at 10 mg/kg Q2W. Arange of variability for each
monitored population was calculated based on meanchanges
between two baseline samples.
Results: All monitored populations metthe acceptance criteria for
imprecision and stability for at least 48 hourspost-collection ± 300
μg/ml of durvalumab. We could reliably detect 5%increases in
activated T cell populations based on results from PBMC
spikingexperiments. Ranges of variability spanned from 30-45% of
baseline values. Analysisof percent changes in absolute counts of
proliferating CD4+ and CD8+ T cells inmelanoma patients
following durvalumab administration revealed 80 and 150 %
meanincreases, respectively, at d15. Additionally, we observed an
average increase of60% in CD8+ ICOS+ T cells. All increases
exceeded the calculated ranges ofvariability for each population.
Uveal melanoma patients exhibited a trend of increasedmagnitudes
of peak changes in proliferating or ICOS-expressing CD8+
compared tocutaneous melanoma patients. No additional
durvalumab-induced changes inactivated T cell subsets were
detectable outside of the ranges of variability.
162/B25
Development of a Dual mRNA (ISH) and Protein
(IHC) Biomarker Assay in Psoriasis Skin Biopsies
161/B24
Development and Validation of Immunophenotyping
Assays for Use on Cryopreserved Peripheral Blood
Mononuclear Cells Isolated in Trials of ImmuneMediated Therapies for Cancer Programs
Exploratory Biomarker Assays, GlaxoSmithKline,
Collegeville, PA, United States
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Gergely Toldi1, Péter Szerémy1, András Apjok1, Harjit
Bhattoa2, János Kappelmayer2, Zoltán Szekanecz3,
Sándor Szántó3, Gabriella Szûcs3, Szilvia Szamosi3,
Ágnes Horváth3, Andrea Domján3
1
MDQuest Ltd., MDQuest Ltd., Szeged, Hungary,
Department of Laboratory Medicine, University of
Debrecen, Debrecen, Hungary, 3Division of Rheumatology,
University of Debrecen, Debrecen, Hungary
Oral Session
Abstracts
2
Index
165
Speaker/Author
Background: Multi-Drug Resistance (MDR) protein function is an
independent prognostic marker in certain hematological
malignancies. Immune activation is also linked to the expression of
MDR proteins in autoimmune disorders. Furthermore, MDR protein
function may predict patient response to traditional DMARD, as
well as biological treatment helping the physician to tailor the
therapy. Switching the patient to biologicals is often challenging
Poster Session
Abstracts
Wednesday
15 June
Conclusion: We were able to overcome the inherent challenges of
validating flow cytometry-based immunophenotyping assays in
cryopreserved PBMC by adjusting the staining methodology
resulting in the majority of populations meeting validation
acceptance criteria. Validation of the flow-based assays in
cryopreserved PBMC was successfully completed, and assays are
ready for implementation in IMTC clinical trials.
163/B26
Multi-drug Resistance Protein Activity of T
Lymphocytes Assessed by Flow Cytometry Is a
Predictor of Biological Treatment Response in
Tuesday
14 June
Results: The majority of monitored populations met the acceptance
criteria for assay precision and ambient stability (1 to 3 days
depending on population). We observed substantial post-thaw,
time-dependent imprecision in the activated and memory T cell
populations that necessitated fixation of the samples following
staining. By implementing this step, all but two of the activated and
memory T cell populations met the precision and stability
acceptance criteria. Overall, the cryopreserved PBMC assays
demonstrate a wider range of imprecision and a shorter range of
ambient stability when compared to the same assays performed on
whole blood.
Monday
13 June
Methods: A set of four immunophenotyping assays, previously
developed and validated in whole blood, was validated in
cryopreserved PBMC. These assays were designed to enumerate the
following populations: 1) Activated and memory T cells; 2)
Proliferating T cells; 3) Regulatory T cells; and 4) T, B and NK
(natural killer) cells (TBNK). Prior to implementing validation
experiments, the optimum blood collection tube, cell
concentrations for staining, antibody clones and negative controls
(isotype vs FMO vs unlabeled antibody) were identified. Additional
assay development work focused on optimizing staining methods
and fixation buffers for the activated and memory T cell assay.
Validation parameters included assessments of inter- and intraassay imprecision and ambient stability. Inter- and intra-assay
imprecision acceptance criteria for the regulatory T cell and TBNK
assays were < 20% coefficients of variation (%CV) and ambient
stability acceptance criterion was < 20% change from baseline
values. The inter-and intra-assay acceptance criteria for the
activated and memory T cell and proliferating T cell assays were <
30% CV and ambient stability acceptance criterion was < 30%
change from baseline values.
Sunday
12 June
Introduction: Immune-mediated therapies for cancer (IMTC) are
therapeutics designed to enhance immune responses against
tumors. As such, evaluation of immune system changes in response
to IMTC may be useful for identifying biomarkers associated with
treatment. As part of MedImmune’s bioanalytical strategy for
developing IMTC therapeutics, we have designed and validated a
set of flow cytometry-based immunophenotyping assays for use on
cryopreserved peripheral blood mononuclear cells (PBMC). The
main advantage of using flow cytometry-based assays on
cryopreserved PBMC is the flexibility to retrospectively test specific
samples associated with clinical responses, potentially reducing
testing costs.
Assessment of biomarker expression and distribution in tissue
biopsies is increasing being added to clinical trial endpoints. These
histology biomarkers are used to measure drug effectiveness,
mechanism of action and drug safety. Immunohistochemistry is
used to label proteins of interest and multiplex labeling strategies
are often employed to study interactions, provide
immunophenotyping and maximize the amount of data that can be
obtained from a single tissue section. For novel targets, commercial
antibodies for IHC can be difficult to source and custom antibody
production can take an unacceptable amount of time. RNAscope®
is an in situ hybridization method to detect mRNA targets in tissue
sections. Custom probes can be provided in approximately three
weeks and they possess a high level of specificity and sensitivity.
This is due to the proprietary method that is used to create the
probes that result in strong signal with no nonspecific background
staining. In addition, this assay has been fully automated on the
Ventana Discovery Ultra® and is also available on the Leica Bond®
RX platforms. During the past year our lab has been developing
dual assays that combine IHC and RNAscope® mRNA detection.
The RNAscope® assay is performed first followed by the IHC
protocol. Part of the optimization process has involved the
empirical testing of a variety of colored chromogens to find the best
combination to demonstrate biomarker co-localization. Currently,
the automated RNAscope® assays are only available with brown
(HRP-DAB) or red (AP-Fast Red) chromogens. It was observed in
our hands and as well as widely reported in the literature, that DAB
should be avoided with performing dual IHC labeling. This is due
to the masking effect that the chromogen can have on epitopes
which result in blocking additional antibodies that co-localize with
proteins adjacent to the DAB from being detected by the antibody.
The second issue involves the masking of previous labeled proteins
when DAB is used as the second chromogen in the dual assay. The
density of the DAB tends to cover the previous chromogen making
it difficult to visualize the c0-localized events. For this reason, the
RNAscope® detection with AP-fast red was used to develop the
dual IHC/RNAscope protocol. This dual method was applied to
skin biopsies collected from psoriasis patients from lesional and
non-lesional areas. Non-proprietary gene and protein targets were
investigated to develop the assay and served as proof of concept
study. Semi-quantitative measurement of the mRNA and IHC was
performed using Definiens® Tissue Studio and iCyte® Laser
Scanning Cytometer.
Saturday
11 June
Translational Sciences, MedImmune, Mountain View, CA,
United States
David Krull
Special
Lectures
Jennifer Pearson, Meina Liang, Nathan Standifer
Congress
Overview
Conclusion: The use of sensitiveand specific immunophenotyping
assays to quantify activated or proliferating Tcells allows for the
detection of durvalumab-induced pharmacodynamic effectsthat are
consistent with the reported effects of PD-L1 blockade. These
assayshave been implemented in multiple durvalumab mono- and
combination therapytrials.
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Index
Abstracts
due to unpredictable drug susceptibility and high cost, especially in
patients with mildly elevated DAS28 scores. However, MDR
protein activity values can objectively reflect the actual disease
activity of RA patients.
Objectives: In this observational cohort trial, we wished to assess
lymphocyte MDR protein activity in RA patients on biological
therapy in comparison to healthy controls in order to predict
response to treatment.
Methods: We measured the activity of three clinically relevant
MDR proteins (MRP1/ABCC1, MDR/ABCB1, BCRP/ABCG2)
expressed in MDR activity factor (MAF) values in 23 responder and
8 primary non-responder RA patients before (at 0 week) as well as
at 2, 6 and 12 weeks after patients were switched to biological
therapy. For this purpose, a novel flow cytometry based, CE-IVD
certified diagnostic tool, the Solvo MDQ Kit™ was applied. In this
assay, fluorescent reporter substrates are trapped in the cytoplasm
and pumped out by MDR proteins depending on the presence of
specific inhibitors, allowing for quantitative, standardized
assessment. PBMCs were loaded with fluorescent MDR activity
reporter substrates (calcein-AM and mitoxantrone, respectively) and
treated with MDR protein specific inhibitors (verapamil,
indomethacin and KO134, respectively) to obtain MAF values. Cell
surface staining was applied to differentiate CD3+, CD4+ and
CD19+ cells. DAS28 scores, CRP, IL-6, aCCP and RF values were
also recorded. 35 age-matched, healthy controls were included.
165/B28
Leveraging Mass Cytometry for Deep Phenotyping
and Quantification of Target Proteins in a Macaca
fascicularis Model
Chad Stevens, Kondala Atkuri, Hendrik Neubert
Pfizer Worldwide R&D, PDM-NBE, Pfizer Inc, Andover,
MA, United States
Advanced single cell analysis technologies (e.g. Mass Cytometry)
for multiplexed cellular measurements in limited volumes of
primary cells are critical for biomarker discovery efforts. Mass
cytometry is the state-of-the-art technology in multi-parametric
single-cell analysis and provides powerful new capabilities for
multidimensional cellular biomarker investigations. Mass
cytometers (also known as CyTOF or Cytometry by Time-of-Flight)
combine the cellular analysis principles of traditional fluorescence
based flow cytometry with the selectivity and quantitative power of
Inductively Coupled Plasma-Mass Spectrometry (ICP-MS).
Conventional flow cytometry is limited in the number of parameters
that can be measured simultaneously due to the overlap in signal
when detecting the fluorescently labeled antibodies. In contrast,
mass cytometry uses antibodies tagged with stable isotopes of rare
earth metals, requiring minimal signal compensation.
Conclusions: Our results suggest that low BCRP/ABCG2 and
MRP1/ABCC1 MAF activities on CD3 cells may predict the need to
start biological therapy in RA patients whose symptoms do not
improve on classical DMARD treatment. Further decrease of CD3
BCRP/ABCG2 and increase in CD3 MRP1/ABCC1 MAF upon
follow-up may indicate a good therapeutic response to biological
therapy.
During development of biologics, quantification of target carrying
cell numbers, receptor numbers and determination of target cell
phenotypes are critical biomeasures that drive theoretical modeling
and simulation of the pharmacokinetic/pharmacodynamics
relationship for dose projections and determining safety margins.
Although the current standard procedures employ extensively
fluorescence based flow cytometric approaches, the ability to
multiplex to only 10-14 parameters and quantify only one target in
a given panel often requires large volumes of samples which can be
limiting in several preclinical models such a mouse or cynomolgus
monkey. Adoption of mass cytometry in preclinical drug
development enables researchers to seamlessly multiplex upwards
of 40 parameters and perform simultaneous quantification of
receptor numbers of multiple targets in limited sample volumes.
Here we cite examples of applications of mass cytometry in a preclinical model (Macaca fascicularis) including 1) deep phenotyping
of cynomolgus monkey blood and 2) multiplexed quantification of
a cellular receptors on cynomolgus blood cells.
Biopharmaceutical Applications (B27 - B28)
Cell Proliferation and Death (B29 – B33)
164/B27
Uber Your Analysis: Creating and Sharing a Custom
Analysis Platform Integrating Single Cell Data
166/B29
Development of a Multiplexing Imaging Flow
Cytometry Assay for Apoptosis Evaluation
Results: There was an inverse correlation between MAF of
BCRP/ABCG2 in CD3 and CD4 cells and DAS28 at 0 week in RA
patients. Multiple correlations between MAF values and
inflammatory parameters were also observed. While DAS28 and IL6 values decreased as the treatment progressed, MAF of
MRP1/ABCC1 on CD3 cells was increased at 12 weeks. A positive
correlation was noted between MAF of BCRP/ABCG2 on CD3 cells
at 0 week and IL-6 at 12 weeks (p = 0.0274, r = 0.4095) in RA
patients. MAF of CD3 BCRP/ABCG2 and CD3 MRP1/ABCC1 were
higher in non-responders compared to responders at 0 week.
Andreas Panopoulos, Shahid Siddiq, Jay Almarode,
Michael Stadnisky
FlowJo LLC, Ashland, OR, United States
With a budget of less than the commercial price of a dongle, we
leveraged professional data scientist algorithm development and
outsourcing via Experfy and UpWork. Using clinical trial data, we
developed a custom analysis algorithm which integrated single cell
sorting and genomic data and compared healthy and diseased
patient samples with a push of a button from FlowJo. We describe
the process of producing a novel plugin, the economics to produce
it from scratch with only biology-domain knowledge, and how to
share the code for re-use and citation using the FlowJo Exchange.
Thus, we address the challenges of constructing custom analysis
tools, data integration, and lower the barrier to development by
giving software engineers and biologists a user-friendly platform for
easy implementation and community for rating and sharing. By
uniting developers and biologists, the path to customized analysis
and discovery is readily accessible.
166
Diana Bonilla1, Kimberlyn Acklin1, Kathryn Ruissard1,
Martin Pichler1, Richard Jones1, Anna Sergueeva1, David
Dwyer1, Ryan Jewell1, Veena Papanna1, Shobana
Vaidyanathan2, Karen Ramirez1, Karen Clise-Dwyer1
1
2
MD Anderson Cancer Center, Houston, TX, United States,
EMD Millipore, Seattle, WA, United States
Apoptosis is a mechanism of programmed cell death implicated in
both physiological and pathological conditions, including cancer.
The study of defects in apoptotic cell death has given valuable
insights into cancer pathogenesis, tumor drug resistance and drug
discovery, the main translational research areas in our institution.
Since apoptosis cannot be defined by a single parameter, a
combination of techniques is recommended for reliable detection
of apoptotic events. Multicolor flow cytometry facilitates the
detection of specific biochemical steps throughout the apoptotic
pathway within the same sample. We developed a multiparametric
imaging flow cytometry assay that allows simultaneous
characterization of early and late stages of the apoptotic response,
giving a more complete picture of this particular cellular process;
and reduces the variability introduced by sample handling, timing
and changes across different tubes and staining protocols. We
evaluated the response of five cancer cell lines to different inducers
of apoptosis and quantified the most important apoptotic features:
mitochondrial membrane depolarization by using TMRE, caspase 3
169/B32
Real-Time Caspase 3/7 Measurement of Suspension
and Adherent Cells Using Celigo Image Cytometry
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Surekha Bonasu, Sarah Kessel, Leo Chan, Jean Qiu,
Dmitry Kuksin
Technology R&D, Nexcelom Bioscience, Lawrence, MA,
United States
Index
Speaker/Author
167
Poster Session
Abstracts
A novel assay has been developed for real-time staining and
detection of active caspases 3 and 7 in apoptotic cells using a platebased image cytometry method. Traditionally, this type of
apoptosis assay is an end point assay and is performed using
fluorescently labeled Annexin-V. Annexin-V labeling may increase
auto-fluorescence when used in imaging systems, thus can be
problematic when performing image analysis. Recently, a live-cell
caspase 3/7 dye was developed for directly staining apoptotic cells.
The caspase 3/7 live-cell dye can passively enter the cells, and
cleaved by the caspase 3/7 during apoptosis. Only when cleaved,
the molecules then enter the nucleus and bind to the DNA to
produce a strong fluorescent signal, which eliminates the possibility
of auto-fluorescence in the background and the need to wash the
cells. Since apoptotic cells are often fragile and can be easily
washed off the plate, performing an in-the-plate wash-free assay for
the detection of caspase activity is of significant value to the
Oral Session
Abstracts
Cell proliferation results between the two methods of Celigo and
Cell Titer-Glo were highly comparable with a correlation factor of
r2=0.998. The label-free image cytometry method is a simple,
rapid, and reagent-free approach to determine cell proliferation in
suspension cells. In addition, captured cell images can be used to
verify proliferation results obtained to eliminate uncertainties in the
traditional methods. Most importantly, the proposed method can
image and analyze cells in a time course study, so the same cells
can be assessed repeatedly over multiple time points throughout an
experiment rather than single, fixed end points, thereby reducing
time, supplies, and cost.
Poster
Session
Conclusions: To our knowledge, this is the first method
demonstrating that human serum can be used in a bioassay to
assess satellite cell proliferation in vitro. In the future, we will use
flow cytometry to analyze satellite cells from whole muscle samples
as it may provide more information on satellite cell behavior and
morphological features.
The Cell Titer-Glo method was used to determine cell proliferation
on the same plate imaged and analyzed by Celigo, which means
that the same wells were analyzed by both methods for a true
comparison. Dose-response curves and IC50 values were
calculated and compared between the two proliferation detection
methods.
Wednesday
15 June
Results: Satellite cells incubated with serum collected from male
subjects (but not female) following exercise resulted in a
significantly higher percent of satellite cells in later generations
then after incubation with control media.
Four suspension cell types, including Ba/F3 parental cell line (Cell
A), Ba/F3 expressing an oncogenic gene (Cell B), an oncogenic
gene mutant A or B (Cell C and D respectively) were plated at a
concentration of 5,000 cells/well and mixed in the presence of
various concentrations of four drugs (1-4) on Day 0. On Day 3, the
Celigo was used to image and analyze cell proliferation using the
bright-field application.
Tuesday
14 June
Methods: The purpose of this study was to develop a method
monitor and track satellite cell proliferation using image-based flow
cytometry. This method was established by culturing primary
human satellite cells with serum collected from human subjects
completed a heavy resistance exercise session (stimulated release of
hormones known to alter satellite cell proliferation). Cultures were
maintained in flat-bottom 96-well plates under sterile conditions.
Primary cells were loaded with profliferation dye (AffymetrixeBioscience) and then acquired on an image-based flow cytometry
(MilliporeSigma; Amnis FlowSight) at specific periods of time to
assess relative degree of proliferation.
Traditionally, cell proliferation has been assessed using detection
methods such as Cell Titer-Glo or MTT assay. However, these
methods require multiple steps, expensive reagents, and are fixed
end-point assays, prohibiting the same cells from being repeatedly
assessed over a time course. There has been a need for plate-based
label-free cell proliferation and cytotoxicity detection method to
directly determine the number of cells per well in order to eliminate
the issues with the traditional methods. In this work, we
demonstrated the label-free cell proliferation and cytotoxicity
detection method using the Celigo Image Cytometry by measuring
the effect of 4 proprietary compounds on the proliferation of 4
proprietary suspension cell lines from Ignyta. The same experiment
was performed simultaneously using the Cell Titer-Glo® Assay, and
the proliferation results were compared directly to Celigo.
Monday
13 June
Background: Postnatal muscle regeneration and hypertrophy is
dependent on a population of muscle stem cells, known as satellite
cells. Satellite cells are defined by their physical location between
the basal lamina and the sarcolemma and their morphological
features. Because of their remarkable ability to differentiate into
myonuclei (responsible for muscle protein synthesis), studies have
been devoted to many facets of satellite cell function. Previously,
satellite cell analysis has been limited to immunohistochemistry or
western blotting. Neither of these techniques provide information
about the propensity of satellite cells to proliferate. A better
understanding of the functional capacity of satellite cells will help
understand their role in normal developing, aging physiology, and
certain types of muscle wasting diseases.
Cell Biology, Ignyta, San Diego, CA, United States,
Technology R&D, Nexcelom Bioscience, Lawrence, MA,
United States
2
Sunday
12 June
Department of Biological Sciences and Kinesiology, Health
Promotion and Recreation, University of North Texas,
Denton, TX, United States
1
Saturday
11 June
Hui-Ying Luk, Danielle Levitt, Brian Mcfarlin, Jakob
Vingren
Roopal Patel1, Leo Chan2, Olivier Dery2, Gina Wei1
Special
Lectures
167/B30
Using Image-Based Flow Cytometry to Monitor and
Track Satellite Cells In Vitro
168/B31
Comparison of Label-Free Cell Cytotoxicity Image
Cytometric Detection Method to Cell Titer-Glo
Congress
Overview
and 7 activation by using Cell Event, phospatidylserine
translocation by using Alexa fluor 594-conjugated Annexin V,
changes in membrane permeability by using Sytox blue and nuclear
condensation and fragmentation by using DRAQ5, by both
conventional and imaging flow cytometry. We observed
comparable results in all parameters with both imaging flow
cytometry and conventional cytometry. Apoptotic features were
increased proportionally with concentration of the apoptotic
inducer, staurosporine. Analysis of cytarabine-treated cells showed
differential kinetics of TMRE, caspase activation and Annexin V,
Sytox blue and DRAQ5 staining, reflecting early and late stages of
apoptosis. Being this cellular process a common target for different
therapeutic strategies against cancer, the development of a
multiparametric approach to monitor apoptosis at different stages
and within heterogeneous populations becomes crucial for the
advance of our research and our attempts to developed apoptosisbased therapy. A study using p53 mutant myeloma cells
demonstrated differential responses to drug treatments. In summary,
this assay will improve our ability to monitor the activation and
progression of the apoptotic pathway simultaneously in specific cell
types, giving us the capability to identify differential responses to
treatment or stimulation between cell populations.
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
research community. In the recent years, the Celigo image
cytometer (Nexcelom Bioscience, LLC) has been used for highthroughput fluorescent cell-based assays. By coupling image
cytometry and the live-cell dye for caspase 3/7, we are able to
image and analyze adherent and suspension cell lines without cell
perturbation. Because the image cytometer is capable of capturing
bright-field and fluorescent images as well as perform gating
operations based on fluorescence intensity, it is possible to not only
carry out live continuous monitoring by detecting a green caspase
3/7 signal, but also perform an end point assay by counterstaining
the cells with Hoechst and thereby determine a percent nucleated
cells that are apoptotic. Here we successfully demonstrate realtime continuous monitoring and end-point caspase 3/7 assays using
adherent MDA-MB-231 and suspension Jurkat cells stained with the
ViaStainTM Caspase 3/7 reagent in a 96-well format and analyzed
on the Celigo image cytometer. This combination of live cell
staining and real-time image acquisition provides researchers a
unique tool for examining dynamic apoptotic events in a microwell environment.
Conclusions: Our novel probes, ROS Green, MitoROS 580,
Nitrixyte Orange and DAX-J2 PON Green, can serve as powerful
probes to selectively monitor total ROS, superoxide, nitric oxide
and peroxynitrite, respectively, in live cells. These new ROS and
RNS probes have been successfully used for investigating specific
inhibitory effects of curcumin on the production of superoxide and
hydrogen peroxide in cells.
170/B33
Flow Cytometric Analysis of Intracellular ROS and
RNS Production and Curcumin Inhibition
1
Department of Paediatric Haematology and Oncology, 2nd
Faculty od Medicine, Charles University Prague, Prague,
Czech Republic, 2IBA GmbH, Goettingen, Germany
Zhen Luo1, Qin Zhao1, Jixiang Liu2, Ruogu Peng2, Jinfang
Liao1, Zhenjun Diwu2
Background: Mass cytometry is a powerful new platform that
enables single-cell analysis of more than 100 different parameters
simultaneously. This unique feature is redeemed by its higher
sample demands. Notably, the instrument is processing cells at
“only” 500 events/s, so a preparation of cellular sample containing
only the cells of interest is preferable, to save time and detector
capacity.
1
Biology, AAT Bioquest Inc., Sunnyvale, CA, United States,
Chemistry, AAT Bioquest Inc., Sunnyvale, CA, United
States
Index
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Scholars &
Emerging
Leaders
Poster
Session
Wednesday
15 June
Tuesday
14 June
2
Background: Reactive oxygen species (ROS) and reactive nitrogen
species (NOS) are important biological regulators involved in cell
damages and health problems. Curcumin, a naturally occurring
phenolic compound, has long been recognized as a promising
anticancer medicine because it can effectively inhibit ROS
generation. However, ROS is a mixture of oxidative species, and
the effects of curcumin on different oxidative molecules are
unclear. Moreover, ROS analysis via flow cytometry is challenging
due to the lack of specific probes for selective detection of ROS and
RNS species. To address these issues, we have developed a few
novel ROS and RNS probes with high selectivity targeting different
ROS and RNS species.
Methods: Human immortalized T lymphocyte Jurkat cells were
monitored with: 1) ROS Green for measuring total ROS (mainly
H2O2); 2) MitoROS 580 for detecting superoxide; 3) Nitrixyte
Orange for targeting nitric oxide and 4) DAX-J2 PON Green for
probing peroxynitrite, respectively. After 1 hour incubation, cells
were treated with various oxidizing agents to trigger ROS or RNS
generations before flow cytometry analysis (ACEA NovoCyte 3000).
Results: In the absence of oxidizing reagents, all these probes had
negligible fluorescence. Upon reacting with H2O2, the fluorescence
intensity of ROS Green increased dramatically in a H2O2
concentration-dependent manner. Other oxidative species also
reacted with ROS Green, but generated lower fluorescence signal.
Nitrixyte Orange is a very selective dye exclusively reacting with
nitric oxide rather than other ROS and RNS species. Compared to
commonly used DAF-2 DA, Nitrixyte Orange showed much higher
signal-to-noise ratio and better cell permeability. MitoROS 580 is a
live-cell permeant dye and can rapidly and selectively target
superoxide in mitochondria, generating bright red fluorescence. In
terms of peroxynitrite detection, which is formed from the reaction
between superoxide radicals and nitric oxide in cells, our unique
novel DAX-J2 PON Green is a highly selective fluorescent probe. It
specifically reacted with intercellular peroxynitrite to generate a
bright green fluorescent product without interference of superoxide
or nitric oxide radicals.
We have used these novel probes to investigate the efficiency of
curcumin as a protective agent against ROS and RNS damage via
flow cytometry analysis. In absence of curcumin treatment,
fluorescence intensities of all probes were increased significantly in
cells. The addition of curcumin remarkably decreased intracellular
ROS level in a dose-dependent manner.
168
Cell Sorting and Selection (B34 – B54)
171/B34
CD81 T-Catch™ Cell Isolation Approach as an Ideal
Tool for an Advanced Cell Analysis on Mass
Cytometer
Ondrej Pelak1, Daniela Kuzilkova1, Jan Stuchly1, MarieLuise Kiene2, Kristian Stanar2, Martina Vaskova1, Herbert
Stadler2, Tomas Kalina1
To prepare highly purified cell suspensions of peripheral blood
mononuclear cell (lymphocytes and monocytes) for further analysis
on mass cytometer we used the new CD81+ T-Catch™ cell
isolation approach, which uses low affinity reversible capture of
target cells on an agarose beads matrix.
Methods: Cells from seven different donors were isolated through
CD81+ T-Catch™ (IBA GmbH, Göttingen, Germany) tips or
columns and Ficoll-Paque low speed (500g) density gradient
centrifugation in parallel for mass cytometry analysis. CD81 is
expressed on NK-, T-, B-cells and dendritic cells and less
abundantly on monocytes. According to this surface presentation
CD81 was selected as separation target to purge of unwanted cells,
such as granulocytes, platelets and erythrocytes. In order to
compare yields and purities, isolated products were subsequently
measured on mass cytometer (CyTOF®2) using Maxpar human
peripheral blood phenotyping panel kit enhanced by 4 more metal
conjugated antibodies and analyzed using supervised and
automated approaches.
Results: We reached 91.4% median purity of CD81+ cells of
CD45+ with T-Catch™ and 77.3% median purity with FicollPaque. However, we have noticed a lot of non-target, non-nuclear
events and also platelet to cell aggregates in Ficoll-Paque separated
cells. Using T-CatchTM, only 12.3% of CD81+ target cells were
stained positive with platelet antigen CD61, whereas we have
detected CD61 signal on 57.2% of PBMC by using the Ficoll-Paque
approach. While free erythrocytes were absent from cells separated
by either method (median 6.9% and 3.2% for T-catch™ and FicollPaque approach respectively), free CD61+ platelets were present in
Ficoll-Paque separated cells (median 93%) while low in T-CatchTM
(median 22.3%). No changes in proportions of NK-, T-, B-cells and
dendritic cells (p-values of 0.0004, 0.0004, 0.0067 and 0.0067
respectively) but monocytes (p-value 0.56) were observed
comparing T-catch™ and Ficoll-Paque.
Conclusions: The T-CatchTM method presents to be an ideal tool for
selection of cells for further cell analysis on mass cytometer, due to
its ability to yield very pure target cell suspension with minimum
platelet to cell aggregation and amenable to automation. Unlike for
Ficoll-Paque, the T-CatchTM columns need to be matched to sample
size (1ml blood needs 0.1ml agarose beads matrix). T-CatchTM cell
isolation is thus suitable for pre-selection of target cells to shorten
the sample acquisition time and extending the lifetime of a detector
in a mass cytometer.
http://www.uhnres.utoronto.ca/facilities/wcif/PDF/Autofluorescence
.pdf
172/B35
A Novel Method for Single Cell Deposition Setup:
Target Practice
2. Denk W, Strickler JH, Webb WW. Two-photon laser scanning
microscopy. Science 248:73-76 (1990)
Cytometry, Walter and Eliza Hall Institute, Parkville,
Australia
Index
1. Autofluorescence: Causes and cures: Wright Cell Imaging Facility
Toronto Research Institute.
169
Speaker/Author
Conclusion: We describe a novel sorting method for size separation
of gametes allowing for high throughput isolation of live gametes,
providing a way of selecting cell size that can be used to address a
wide range of evolutionary and ecological questions.
Poster Sess on
Abstracts
A Spectra-Physics Tsunami laser system was configured to
femtosecond pulse mode and used to evaluate TPE and sorting of
Clonetech AcGFP 6 peak Flow Cytometry Calibration Beads on a
modified Beckman Coulter Moflo legacy sorter.
Oral Session
Abstracts
In cytometers, the background autofluorescence decreases the
resolution of the instrument, that is, the ability of the cytometer to
rank changes in small numbers of fluorescent molecules. A number
of papers describe TPE sources on custom built platforms fitted with
flow chambers or micro-fluidics chips with low flow rates (4,5).
This poster evaluates a TPE laser system built on a high speed
sense-in-air sorter, and the system's potential to sort on the TPE
emission of GFP beads.
Results: The size of gametes was observed to span from ~5μm to
<10μm, based on size standard particles. The gametes also
autofluoresced in spectrum >650nm, when excited with any laser.
In combination with pre-gating on FSC-Par A for small and large
cells, the biggest separation in MFI was in a detector with 750LP
filter, when cells were excited with a 561nm laser. Three sorting
strategies resulted in a significant separation of size, all with
p<0.001. However the combination of FSC-Par-A with
autofluorescence resulted in the largest mean size separation
(1.7μm vs 0.6μm and 0.7μm). Surprisingly, we observed, when
gametes were sorted on FSC-Par H signals, there was a significant
separation in flagella length (6.4μm vs 7.6μm).
Commercial
Tutorials &
Exhibits
TPE has been utilized in microscopy over the last few decades (2,3).
It has been successful in improving the image resolution by
restricting the excitation volume and reducing background
contributions such as off axis fluorescence.
Scholars &
Emerging
Leaders
Two photon excitation (TPE) light sources have the potential to
improve the signal resolution by reducing the auto-fluorescence
background contribution. This background autofluorescence comes
from flavins and porphyrins in animal cells or Chlorophyll in plants
(1).
Introduction: Clamydomonas reinhardtii is motile single cell bluegreen algae, which may be used as a model for the study of
different aspects of evolutionary biology and ecology, such as cell
cycle, cell physiology and biofuel modelling. Evolutionary, in
higher-evolved organisms, female gamete “eggs” evolve to be larger
than the male gamete “sperm”. It is postulated that this size
difference may have an impact on the survival and physiological
and reproductive fitness of an offspring, in algae as well as in other
species. To study this hypothesis, we developed a flow cytometry
based method for sorting on small cell size variation, using the BD
Influx, without the need for extrinsic fluorophores.
Method: Gamete size distribution, based on FSC and SSC
parameters, was determined using polystyrene size standard
particles in combination with samples. The gametes were also
assessed for their autofluorescent properties, due to the presence of
chlorophyll, by using excitation from 5 lasers (355nm – 642nm),
and emission across 18 fluorescent detectors spaning in spectrum
from 420nm to >750nm. Three sort gating strategies were assigned.
Low (small cells) and high (large cells) 15% signal distribution in
FSC-Par-Area or High parameters, or low and high MFI of >750nm
emission from 561nm laser excitation, following prior FSC-Par-A
gating. Sorts was performed using an 86μm nozzle, at 30psi sheath
pressure, with 30.2psi sample pressure, and 1 drop purity mask.
50,000 gametes from each gate were sorted directly onto slides.
Cells were fixed in a 10% final concentration Lugols Iodine solution
and imaged under 40x objective on an Olympus IX81 microscope
to determine size.
Poster
Session
Wellcome Trust-MRC Cambridge Stem Cell Institute,
University of Cambridge, Cambridge, United Kingdom
University of Western Australia, Nedland, Australia,
Centre for Evolutionary Biology, University of Western
Australia, Perth, Australia
2
Wednesday
15 June
Andy Riddell
1
Tuesday
14 June
173/B36
Two Photon Flow Cytometry and Sorting on a
Beckman Coulter MoFlo High Speed Sorter: A
Provisional Study
Irma Larma-Cornwall1, Catherine E Seed2, Joseph E
Tomkins2
Monday
13 June
This is a simple technique used to reassure the investigator and the
sorter operator that the sorted droplets are being delivered to their
target.
174/B37
A Size Restricted Cell Sorting Strategy for
Applications in Modeling Assisted Evolution in the
Algae, Chlamydomonas reinhardtii
Sunday
12 June
Using 3,3ƍ,5,5ƍ-Tetramethylbenzidine (TMB) the substrate used for
ELISA detection and particles either microspheres or cells
suspended in horse radish peroxidase (HRP) solution a visible
colorimetric change can be produced in each well of a microtiter
plate from a single sorted droplet. The particles play no part in the
reaction are used merely to trigger the instrument. The technique is
robust and quantitative. Although the colour change is easily visible
to the human eye plates can also be read on an ELISA plate reader,
the absorbance being dependent on the concentration of HRP, the
sample differential, the nozzle size and the volume of TMB. The
method works for any cell sorter and all nozzle sizes. The signal is
less for smaller nozzle sizes but the concentration of HRP can be
increased if necessary.
5. Zhong CF, Ye JY, Myc A, Norris TB. Quantitative Two-photon
Flow Cytometry 2005 Quantum Electronics and Laser Science
Conference (QELS).
Saturday
11 June
Studying single cells reveals biology that cannot be explored using
bulk techniques. Cell sorters provide the opportunity of separating
single cells either for cell culture or for downstream molecular
studies such as qPCR to study specific gene expression or single cell
mRNA sequencing. Some of these molecular studies can be very
expensive so the investigator will often want reassurance that the
cell sorter can indeed deposit a single cell into each well of a
microtiter plate which may contain a small volume. To miss some
of the wells could leave the data set incomplete requiring costly
repetition. To verify this by microscopy is at best very time
consuming and at worst impossible.
4. Tkaczyk ER, AH Tkaczyk, Katnik S, Yong Ye J, Luker KE, Luker
GD, Myc A, Baker JR. T Norris TB. Extended cavity laser enhanced
two-photon flow cytometry J. BioMed. Opt. 13(4):041319 (2008)
Special
Lectures
Simon Monard
3. So PTC, Dong CY, Masters BR. Two photon excitation
Fluorescence Microscopy. Ann-Rev Biomed. Eng. 02:339-429
(2000)
Congress
Overview
Supported by Ministry of Health Czech Republic grant number 1526588A and by Charles University Grant Agency number 376214
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Index
Abstracts
175/B38
Innovations in Fluorescence Activated Cell Sorting
for Genomic Studies of Single Cells
177/B40
Cell Differentiation Approaches Using 5-Way Cell
Sorting: Facts and Artifacts
Matthew Crow1, Timothy Petersen1, Mina Rohani1,
Daniel Horner1, Amy Tam2, J. Clark Mason2
Alvaro Luiz Bertho1,2, Raquel Ferraz3,4
1
Becton, Dickinson & Co., Seattle, WA, United States,
2
Becton, Dickinson & Co., San Jose, CA, United States
The new BD FACSseq™ Cell Sorter enables the precise isolation of
cells prior to sequencing the cell's genome, allowing targeted
sequencing and gene expression analysis of single cells. We report
on the sorter's performance in detecting single cells using a patentpending method of collecting reflected fluorescent light. The carrier
fluid of the cell sorter acts as an optical fiber, trapping fluorescent
and scattered light from the excitation of the cells using total
internal reflection (TIR). This unique arrangement of the excitation
laser and the optical detectors allows for simplified device setup
and alignment, and also makes possible an exchangeable fluidics
path for different applications. We also describe the automated
monitoring of droplet formation - critical for high purity sorting and the instrument feedback that automatically adjusts to maintain
stability when sorting in an unattended mode. These technical
innovations enable rapid, advanced isolation and characterization
of single cells, for successful genomic studies. This new sorter is
configured with a single laser and supports up to four parameters
for measurement and deposition of cells into tubes or plates.
176/B39
Clonogenic Potential of a Rare Marrow-Derived
Mesenchymal Stromal Cell after Purification with
Droplet versus Microchip Sorting
Lisa O'Flynn1, Emma Horan1, Evelyn Rodriguez-Mesa2,
Jack Dunne3, Steve Elliman1
1
Orbsen Therapeutics, Galway, Ireland, 2Miltenyi Biotec,
Santa Barbara, CA, United States, 3Miltenyi Biotec,
Livermore, CA, United States
We have described a rare Mesenchymal Stromal Cell (MSC) in
human bone marrow, defined as CD271+CD362+ (ORBCEL-M™),
including explorations of the therapeutic potential of these defined
MSCs in multiple pre-clinical models of inflammatory disease
(REDDSTAR.eu). The clonogenic potential (colony-forming unitfibroblasts, CFU-F) of these MSCs is commonly employed to assess
the sort performance and the health of the cells after processing. As
well, CFU-F is an important indication of the potential therapeutic
benefit and cost of production of these cells for therapeutic
applications after expansion. The current methods of enriching
stromal cells by plastic adherence results in a CFU-F ratio of 1 MSC
in 30,000 cells. Typically BD FACSAria four-colour sorting of
CD271+CD362+ stromal cells enables the isolation of stromal cells
to a frequency of 1 MSC in every 3-4 cells sorted. However, we
have compared CFU-F frequencies of these cells after purification
on a BD FACSAria and on the Miltenyi MACSQuant Tyto, using 2
colours for both systems, and found substantially higher frequencies
with the Tyto system. Practical aspects of sort performance were
similar, though various aspects important for GMP manufacturing
(closed, single use sterile fluidics, ease-of-use, and reliability) were
also substantially improved with the Tyto. We are now proceeding
with Tyto-processed cells in early human clinical trials for studies of
safety and indications of efficacy in the repair of non-healing
ulceration secondary to chronic diabetes.
[OL1]Like for like? For a fair comparison!!
1
Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil,
Flow Cytometry Core Facility, Oswaldo Cruz Institute,
FIOCRUZ, Rio de Janeiro, Brazil, 3Lab. of
Immunoparasitology, Oswaldo Cruz Institute, FIOCRUZ,
Rio de Janeiro, Brazil, 4Flow Cytometry Core Facility,
Oswaldo Cruz Institute, Rio de Janeiro, Brazil
2
Due to the important role of effector and memory CD8+ T
lymphocytes in the immunopathogenesis in cutaneous
leishmaniasis (CL), we standardized a flow cytometry protocol to
determine the profile of CD8+ T-lymphocyte subsets and their in
vitro differentiation under specific-antigenic stimulation. For this,
we obtained peripheral blood mononuclear cells from CL patients
evaluated before and during treatment and after cure, in order to
investigate the process of in vitro differentiation of CD8 T-cell
subsets to effector and memory cells and correlate their functions in
the CL healing process. We performed a flow cytometric staining
protocol using monoclonal antibodies anti-CD3, anti-CD8, antiCD56, anti-CD45RA and anti-CD27, in order to identify effector
and memory CD8+ T-lymphocyte subsets. MoFlo Astrios Cell Sorter
(Beckman Coulter) allowed us to sort 5-different cell populations: a
positive sorting of effector CD8+ T lymphocytes; effector memory
CD8+ T lymphocytes, central memory CD8+ T lymphocytes and
naï ve CD8+ T lymphocytes, as well as a negative sorting of CD8 T
lymphocytes. After sorting, CD8- cells were cultivated with different
CD8+ T-cell subsets, with or without Leishmania braziliensis
antigens. After 5 days of culture, we evaluated by flow cytometry, if
there was changes in the initial frequency of each CD8+ T-cell
subset and in which subset the cells would be differentiated. This
process generates a variety of issues, such as the necessity to verify
the frequency of subsets pos-sorting with the same monoclonal
antibodies used in pre-sorting staining; how efficient is the shedding
process?; may we use another monoclonal-antibody combinations
to evaluate these subsets? Besides, the use of detaching reagent; the
time required for shedding; and comparing the frequencies of
subsets submitted to different staining combination, such as antiCD28 and anti-CD45RO will be discussed.
178/B41
Nodexus' Label-Free Cell Screening and Isolation
Platform Allows for Unprecedented Clinical Utility
Karthik Balakrishnan
Nodexus Inc., Berkeley, CA, United States
Nodexus Inc. is a Berkeley-born Angel-, SBIR-, and Venture-backed
startup that is developing an integrated platform that consists of a
hardware device and single-use, disposable microfluidic cartridges
that reliably and rapidly detect and isolate rare cell types. Our
technology allows for purification of rare cells of interest from
complex, heterogeneous samples without altering or adversely
affecting their viability – a highly valuable feature that we are
currently utilizing to enumerate and isolate circulating tumor cells
(CTCs) as well as other rare cell types from an 8 mL patient blood
draw. Nodexus’ technology suite possesses a number of advantages
including reduced hands-on time, single-cell resolution, unbiased
isolation (independent of epithelial marker expression like many
existing CTC technologies), and prioritization of recovery and
viability – all critical performance parameters which have been
validated by clinical leaders and collaborators.
Cell identification, isolation, and characterization has been
essential across the spectrum of biomedicine: from basic research
to clinical practice to even clinical trials. Screening and isolating
cells of interest has allowed for improvements in disease diagnosis,
staging, and monitoring, even leading to advances in drug
discovery and the development of novel therapies.
More recently, numerous systems to detect and characterize CTCs
using methods such as immunofluorescent imaging and
170
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Monica DeLay1, Huirong Xie2, Yinhuai Chen2, Sherry
Thornton3, Yueh-Chiang Hu2
1
Research Flow Cytometry Core, Cincinnati Children's
Hospital, Cincinnati, OH, United States, 2Transgenic Animal
and Genome Editing Core, Cincinnati Children's Hospital,
Cincinnati, OH, United States, 3Cincinnati Children's
Hospital Medical Center, Cincinnati
Index
171
Speaker/Author
Genome engineering tools such as CRISPR/Cas9 introduce a sitespecific double strand break, enabling precise gene insertion and
gene replacement in cells via homology-directed repair (HDR).
However, efficiency of HDR is limited as the more dominant
nonhomologous end joining (NHEJ) repair pathway competes with
Poster Session
Abstracts
Scholars &
Emerging
Leaders
Sorting is achieved with a 1064 nm optical trap controlled by a
piezo-tilt mirror. Several site-directed and error-prone PCR libraries
based on mCherry/FusionRed FPs were subjected to microfluidic
screening and sorting to select clones with improved properties. So
far, we have found variants with enhanced brightness, lifetime and
DSC kinetics, which can be useful for lifetime-based or superresolution-based imaging modalities. For example, a clone with 2fold higher lifetime than the parent mCherry (1.87 ns vs. 3.8 ns) has
been identified after several rounds of mutagenesis and subsequent
microfluidic enrichments. Further improvement and
characterizations of the mutants are underway. In conclusion, our
181/B44
Enrichment of CRISPR-Mediated HomologousDirected Repair in Mammalian Cells by Cell CycleBased Flow Sorting
Poster
Session
In this instrument, 532 or 561 nm laser output is split into multiple
beams (8-10) and focused into the microfluidic device in an
inverted microscope. The first beam is amplitude-modulated at 29.5
MHz for the in-flow measurement of excited-state lifetime of the
FPs by phase fluorimetry. The ratio of the fluorescence intensities
from the last and first beam is used as a metric for irreversible
photobleaching as the inter-beam delay is controlled to eliminate
the contribution of reversible photobleaching. We also developed a
frequency-domain method to quantify the DSC time-scales of the
FPs by means of measuring the “phase-advance” from an excitation
beam modulated at ~ 20 kHz. This microfluidic platform has the
capability to screen 105 variants at a typical rate of ~20 cells per
second. This rate is constrained by the photophysical timescales of
the FPs. Screening results on various site-directed and randommutagenesis libraries reveal correlations among excited-state
lifetime, irreversible photobleaching and DSC processes. These
findings illuminate the inter-dependence of various photophysical
properties and their dependence on the environment of the FP
chromophore, which give further clues regarding library design.
Wednesday
15 June
Though their genetic encodability makes them powerful tools for
live-cell imaging, fluorescent proteins (FP) with improved
photophysical properties such as higher photostability and
brightness are in high demand. Often times, new FPs with
improvement in one parameter show reduced performance in
others. Hence, we designed a multi-parametric microfluidic flow
system for simultaneous measurement and sorting of cell-based
libraries on various photophysical properties of the expressed FPs
e.g. brightness, photostability, excited-state lifetime (proxy for
quantum yield), dark state conversion (DSC) etc.
The Flow Cytometry Research Group through the Association for
Biomolecular Resource Facilities (ABRF) has continued with the
goal of establishing best practice guidelines for cell sorting
conditions that minimize cell stress, perturbation, or injury to the
sorted cell populations. In prior FCRG studies, gene expression
changes in Jurkat T lymphoblast cells were measured following cell
sorting with different system pressures and nozzle sizes where
minimal effects observed resolved over time in culture. A more
recent study examined the effect sorting has on primary cells
(C57Bl/6 mouse splenic B lymphocytes). B lymphocytes were
isolated using multiple flow sorters and different pressure/nozzle
configurations. Genome-wide gene expression analysis was
performed on selected samples using affymetrix microarrays and a
small number of candidate genes were identified as responding
differentially in high or low pressure conditions. Here we describe
a study in which B cell samples from the same batch of sorting runs
along with sorted and unsorted mouse ES cells and mouse dendritic
cells were assayed by eBioscience QuantiGene Plex (QGP). We
sought to validate the significance of the candidate genes identified
in microarray data and test additional genes known to respond to
cell stress and damage were also evaluated for changes as a result
of cell sorting. In the cell types tested here, we found minimal gene
changes as a result of cell sorting across all instrument and settings.
Genes previously identified by microarray to be differentially
expressed in B cells after sorting were validated using the QGP
assay.
Tuesday
14 June
Chemistry & Biochemistry, University of Colorado at
Boulder, Boulder, CO, United States, 2Chemistry &
Biochemistry, JILA, NIST and University of Colorado,
Boulder, CO, United States, 3Chemistry & Biochemistry,
BioFrontiers Institute, University of Colorado, Boulder,
Boulder, CO, United States
Monday
13 June
1
1
Research Flow Cytometry Core, Cincinnati Children's
Hospital, Cincinnati, OH, United States, 2Immune
Monitoring and Flow Cytometry Resource, Dartmouth
College, Lebanon, NH, United States, 3Cytometry Shared
Resource Laboratory, Stowers Institute for Medical
Research, Kansas City, MO, United States, 4West Virginia
Univ HSC, Morgantown, 5Univ of Rochester, Rochester,
6
Flow Cytometry and Cell Sorting Facility, University of
Vermont, Burlington, VT, United States, 7MGH
Massachusetts General Hospital, Brookline, 8New York
Univ, New York, NY, United States, 9Univ of Pittsburgh,
Pittsburgh, PA, United States, 10The Scripps Research
Institute, La Jolla, CA, United States
Sunday
12 June
Premashis Manna1,2, Felix Vietmeyer2, Amy Palmer1,3,
Ralph Jimenez1,2
Monica DeLay1, Alan Bergeron2, Andrew Box3, Kathleen
Brundage4, Matthew Cochran5, Roxana del Rio-Guerra6,
Maris Anne Handley7, Peter Lopez8, Ernest Michael M.
Meyer9, Alan Saluk10
Saturday
11 June
179/B42
Multi-parametric Microfluidic Flow Cytometer for
Directed Evolution and Characterization of
Fluorescent Protein Libraries
180/B43
Evaluating the Effects of Cell Sorting on Gene
Expression
Special
Lectures
In the past years, the growing need for improved cell-screening
platforms has also led to the emergence of microfluidic
technologies for cell screening. Although these systems have
demonstrated success in certain targeted applications, they suffer
from broad limitations, making their commercialization difficult.
Ultimately, the Nodexus platform will deliver purified, viable, and
unlabeled cells, for characterization and further provide mutagenic
profile information as desired by clinical, industrial, and academic
researchers. These highly purified samples are amenable to
downstream analysis, enabling personalized therapies and
therapeutics, which has been impossible due to the limitations of
industry standard technologies.
platform is capable of simultaneous improvement of photophysical properties of large FP libraries, which are not possible in
conventional plate-based technique or commercial flow
cytometers.
Congress
Overview
immunomagnetic-based isolation have emerged, but many of these
suffer from key limitations, including the need for exogenous
labeling, elaborate experimental design, non-intuitive data
interpretation, and the difficulty of viably sorting extremely rare cell
sub-populations from complex samples (e.g. blood) for researchers’
desired downstream analysis and/or culture.
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
HDR. One of the main reasons this competition happens is due to
NHEJ occurring throughout the entire cell cycle, while HDR takes
place only during S and G2 phase. Methods have been reported to
enhance HDR by synchronizing cell cycle or blocking the NHEJ
pathway using chemicals or additional DNA vectors, which
potentially results in undesirable side effects. Here we present a
simple, cell cycle-based FACS sorting method to enrich cells
undergoing HDR. We used a pX458-Dazl vector that contains a
Cas9-2A-GFP expression cassette and a U6 promoter-driven sgRNA
targeting the 3’ end of Dazl coding sequence. Mouse embryonic
stem cells (mESCs) were electroporated with pX458-Dazl, along
with a circular donor plasmid that contains 2A-tdTomato flanked by
homologous arms of 2.9 kb and 2 kb, respectively. Five hours
following electroporation, when Cas9-2A-GFP became visible
under a fluorescence microscope, cells were labeled with Hoechst
33342 and sorted according to GFP expression and DNA content.
A week later, mESCs were analyzed for Dazl-2A-tdTomato
expression, as a readout for HDR. Results show that 20.9% of S
phase-sorted mESCs were positive for Dazl-2A-tdTomato as
compared to 6.9% of G1 or 14.3% of G2/M phase-sorted cells. This
result indicates that at the start of sgRNA/Cas9 expression, cell
cycle stage affects the frequency of HDR in cells. Our cell cyclebased FACS sorting can significantly enrich cells undergoing HDR
following CRISPR-Cas9 targeting, resulting in a more rapid selection
of precisely targeted cell populations.
182/B45
Dilute Sample and High Flow Rate Maximize Sort
Recovery
Index
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Scholars &
Emerging
Leaders
Poster
Session
Wednesday
15 June
Tuesday
14 June
Giri Buruzula, Angela Wood, Thomas Serwold
Immunobiology, Joslin Diabetes Center, Boston, MA,
United States
Purity, recovery and viability are all factors that describe cell sorter
performance. Purity and recovery are dependent on sample
concentration, threshold event processing rate, coincidence events
and electronic aborts; viability is primarily influenced by initial
sample conditions (prep time, method, experience, etc.).
Evaluating recovery is not trivial: absolute counts of sorted particles
are often unfeasible when dealing with rare samples, and inefficient
counting methods can lead to large errors or conflicting results. By
directly counting particles that were lost to waste under carefully
controlled conditions, we were able to accurately quantify the
effects of sample concentration, event rates, and flow rate on sorted
particle recovery. We propose that sample concentration, flow rate
and conflict/ abort rate can be used to estimate the loss of particles
of interest to waste. The effects of these parameters suggest
guidelines for determining optimal sample concentration and flow
rate to maximize sort recovery.
Sort performance was tested on a BD FACSAria II and Beckman
Coulter MoFlo Legacy. First, the effect of increased flow and
threshold rates was assessed on the frequency of cells lost to waste,
when sorted from a constant sample concentration. Then the effect
of flow rate was independently assessed by sorting from
progressively more dilute samples, such that, even as flow rate
increased, the threshold rate remained constant.
Our data shows a direct correlation between sample concentration
and particles of interest lost to waste on both instruments.
However, there is no correlation between flow rate and events lost
to waste when threshold rate is maintained by sample dilution.
This indicates that sample concentration can be optimized to
minimize loss to waste even at high flow rates. Thus, very high
flow rates and diluted cell samples maximize cell recovery during
cell sorting. This sorting setup will be especially useful to users
who sort rare populations from large samples.
172
183/B46
Development of a Sensitive Flow Cytometry-Based
Platform for Isolation and Molecular
Characterization of Circulating Tumor Single Cells
and Clusters
Charles Pletcher1, Neha Bhagwat2, Ling Wang3,
Stephanie Yee4, Liping Yu5, Jonni Moore1, Ben Stanger2,
Eric Dixon3, Erica Carpenter4
1
Pathology & Laboratory Medicine, University of
Pennsylvania, Philadelphia, PA, United States, 2Division of
Gastroenterology, Department of Medicine, University of
Pennsylvania, Philadelphia, PA, United States, 3Cell and
Tissue Technologies, BD Technologies, Durham, NC,
United States, 4Division of Hematology and Oncology,
Department of Medicine, University of Pennsylvania,
Philadelphia, PA, United States, 5Research and
Development, BD Biosciences, San Jose, CA, United States
Circulating tumor cells are cells shed from solid tumors and found
at extremely low frequencies in the bloodstream of patients in most
cancer types. A subset of these cells can seed distant organs in the
body and give rise to metastases, which is the primary cause of
cancer-related mortality. Isolation and characterization of these
cells from blood can be a sensitive and non-invasive method for
early detection and disease monitoring. Data from preclinical
models as well as patients have shown that circulating tumor cells
can be found in the blood even at early disease stages. There is
increasing evidence that clusters of tumor cells in the blood are
associated with higher metastatic potential. Efficient isolation and
interrogation of these rare clusters from large blood volumes
remains a challenge in the field. In this study, we utilize an in-line
rare cell enrichment platform developed by BD, coupled with the
BD FACSTM Influx cell sorter to rapidly isolate both circulating
single cells as well as clusters from blood. The enrichment platform
utilizes magnetic particle-based depletion of unwanted leukocytes
and combines acoustic focusing to remove red blood cell debris
while enriching for rare cells. The in-line enrichment process
streamlines cell isolation and minimizes loss of rare cells.
Importantly, the use of a large 200um nozzle on the Influx
minimizes shear forces and maintains cell viability and integrity of
cell clusters. Finally, this workflow can be seamlessly integrated
with downstream molecular analysis such as next-generation
sequencing (NGS) at the single-cell level.
As proof of principle, we utilized this platform to isolate and
characterize circulating tumor cells from a genetically engineered
pancreatic cancer mouse model in which all the tumor cells are
labeled with YFP (KPCY). In initial studies using YFP+ pancreatic
cell lines spiked into normal blood, a >23 fold enrichment of tumor
cells was observed with the in-line enrichment process as
compared to without. By using a 200um nozzle and low flow rates,
we could distinguish between and sort single cells as well as cell
clusters ranging from 2-10 cells/cluster. We also successfully
prepared high quality cDNA libraries for RNA sequencing from
single cells.
We then used the optimized workflow to sort YFP+ circulating
tumor cells and CD45+ white blood cells from the blood of tumorbearing KPCY mice. The molecular identity of the sorted cells was
confirmed by expression of YFP and epithelial markers such as
Krt19 and Krt18 by RT-PCR. NGS cDNA libraries were successfully
generated from low input or single cells for whole exome RNA-Seq
analysis. The resulting RNA-Seq analysis will be used to identify
pancreatic cancer-specific cell surface markers and these markers
will be evaluated for their ability to detect CTCs in blood and for
development of a clinically relevant pancreatic cancer diagnostic
assay.
2
3
Liping Yu , Rachelle Johnson , Marty Bigos , Chip
Lomas1, Alice (Xiaoyang) Wang1
1
1
Flow Cytometry Core Facility, DRFZ, Berlin, Germany,
APE Angewandte Physik & Elektronik GmbH, 3DRFZ, 4Cell
Biology, Deutsches Rheuma-Forschungszentrum Berlin
(DRFZ), Berlin, Germany, 5Deutsches RheumaForschungszentrum Berlin (DRFZ), Berlin, Germany
2
Index
173
Speaker/Author
We are able to efficiently filter out cell doublets and non-specific
pulses (due to electronic artefacts) to increase data quality.
Furthermore, we are able to identify erythrocytes which we confirm
with a specific erythrocyte staining. This method shows great
Poster Session
Abstracts
Based on this standard practice, we suppose that a more complete
analysis of the shape could yield novel information about cell type
without having to stain extra markers. Using custom hardware, we
capture these pulses and analyse their shapes using a discrete
wavelet transform. We confirm the stability of this method with the
quantiFlashTM device as well as with microspheres.
Oral Session
Abstracts
In standard flow cytometry, cells are characterised by using an
estimate of scatter and fluorescence intensities. These estimates are
derived from an electronic pulse corresponding to the physical
response of a detector (PMT or Photo diode), which in turn
corresponds to the characteristics of emitted and scattered light
from a cell. The estimates are based on pulse area, height and
width. A very simple method of using pulse shape is to compare the
relationships of area, height and width to each other, e.g. the pulse
height and width are typically used to distinguish between single
cells and doublets.
Commercial
Tutorials &
Exhibits
Kristen Feher1, Konrad von Volkmann2, Jenny Kirsch3,
Richard Addo4, Andreas Radbruch5, Jan Popien2, Toralf
Kaiser3
Scholars &
Emerging
Leaders
Background: Vaccines constitute one of the biggest achievements
in medicine. While the importance of antibody producing plasma
cells and T cells for protective immunity have been established, it
Poster
Session
1
Flow Cytometry Shared Resource Laboratory, Baylor
Institute for Immunology Research, Dallas, TX, United
States, 2Dynavax, Dynavax Technologies Corporation,
Berkeley, CA, United States, 3Institute for Immunity,
Transplantation, and Infection Operations, Stanford
Wednesday
15 June
Jean-Philippe Blanck1, Elizabeth Gatewood1, Jessica
Cardozo1, Nandhini Raman1, Emily Ruchaud1, Robert
Coffman2, Gerlinde Obermoser3, Kay Kayembe1
186/B49
A Novel Method for Characterising Cell Properties
Based on Pulse Shapes
Tuesday
14 June
185/B48
BD INFLUX 6-Way 13 Population Sorting. Vaccine
Signature Deconvolution on Leukocyte Subsets.
Tracking Immune Response to Heplisav®
Results and Conclusions: Since the target populations were sorted
directly into RNA stabilizing lysis buffer, purities post sorts were
assessed on separate populations collected into PBS. Target sort
numbers were 100K for PBMCs, 50K for major and 10K for small
populations. Duplicates were sorted whenever possible. Purity was
on average 97.6%. 2055 sorted samples were generated in just a
month. Complex and massive sort series are labor intensive, time
consuming, require optimized SOPs, scaled-up workflow
procedures and dedicated personnel for high-quality results. Our
investigator was able to complete the downstream genomic
profiling on sorted individual leukocytes of interest.
Monday
13 June
Single cell gene expression analysis is a powerful method to resolve
tumor cell heterogeneity and reveal biology that is hidden in bulk
sample measurement. BD fluorescence activated cell sorting
(FACSTM) has been shown to be able to accurately identify and
isolate single cells for next generation sequencing (NGS). Combined
with molecular barcoding technology of BDTM Precise Assays, a
high-throughput, high precision single cell gene expression analysis
workflow is established. Furthermore, we have incorporated an inline cell enrichment technology in FACSTM to enrich for target cells
and remove cellular debris to simplify the preparation process of
complex samples, such as bone marrow homogenates, and to
speed the sorting of rare target tumor cells. Here we conducted a
pilot study in which MCF7 control and MCF7shLIFR cells were
spiked into primary mouse bone marrow samples and were
individually sorted into 96-well PreciseTM encoding plates using a
BD FACSTM Influx sorter. Each cell was lysed and the mRNA
content was barcoded with a unique sample and molecular index
during the reverse transcription step. 100 breast cancer relevant
genes were interrogated to identify the cellular composition of the
sorted cells. cDNA products in two 96-well plates were pooled
together to run on an Illumina MiSeq instrument. Automated data
analysis was performed on the Seven BridgeTM Genomics Platform.
The work-flow developed in the pilot study will be ultimately
validated by harvesting MCF7 control and MCF7shLIFR tumor cells
disseminated to the bone marrow in mice and examining these
populations for changes in single-cell mRNA levels. These results
demonstrate a stream-lined workflow for single cell isolation and
genomic analysis, and its application in cancer biology research.
Methods: 13 Leukocyte subsets from patient PBMCs with 9 visits
each were sorted directly into RNA lysis buffer with predetermined
target number for microarray profiling. Prior to sorting, we
optimized a sequential thawing with a total of 9 time points a
patient. We tested and validated our 13-color antibody cocktail
which included a viability marker and a dump channel. While
sorting on average from 11.30am to 11pm, the sorter was
optimized throughout the day to account for temperature
fluctuations. Our workflow was also optimized to flash freeze the
sorted cells on dry ice and then stored in -80°C.
Sunday
12 June
Breast cancer cells frequently metastasize to distant organs,
including the lung and bone marrow. Previous studies have
demonstrated a crucial role for the leukemia inhibitory factor
receptor (LIFR) in maintaining a dormancy phenotype in breast
cancer cells disseminated to the bone marrow and loss of the LIFR
enables dormant breast cancer cells to aggressively colonize the
bone marrow; however, the gene expression changes that drive
these cells to induce osteolytic bone destruction and proliferate at
sites of bone metastasis remain unclear. We therefore aim to
determine differential gene expression in MCF7 control and MCF7
LIFR knockdown tumor cells (MCF7shLIFR) disseminated to the
bone marrow using single-cell mRNA sequencing.
Saturday
11 June
R&D, BD Biosciences, SAN JOSE, CA, United States,
Radiation Oncology, Stanford University, Stanford, CA,
United States, 3Stanford Shared FACS Facility, Stanford
2
In a cohort of 25 adults who received the hepatitis B vaccine
Heplisav®, which contains HBs antigen and CpG-ODN adjuvant;
our researcher noticed that the gene expression arrays on blood
samples taken immediately before and on days 1 – 56 post
immunization revealed a distinct signature to vaccination. We were
asked to investigate the vaccination response by phenotyping and
sorting individual leukocyte subsets from cryopreserved PBMCs
before and after immunization and address the question of how
these profiles contribute to overall leukocyte transcriptional
signature. To get the most out of the limited samples we developed
a multi-parameter panel on the BD Influx cell sorter to delineate
and sort these populations.
Special
Lectures
1
remains unclear how the innate immune system shapes the
adaptive responses after vaccination. As a flow cytometry Shared
Resource Laboratory (SRL); part of our goal is to partner with
investigators and assist them with resources and expertise necessary
to answer these fundamental questions.
Congress
Overview
184/B47
New Sorting and Genomics Technologies to
Facilitate High-Throughput, High-Precision, Single
Cell Gene Expression Analysis of Breast Cancer Cells
in Bone Marrow Samples
Congress
Overview
Special
Lectures
187/B50
Translation of the Human Immunophenotyping
Panels from a FACS Aria Fusion to an Influx Platform:
Evolving Polychromatic Cell Sorting Services in SRL
Esther Perez, Laura Bergamachi, Chris Bowman, Natalia
Savinykh, Lorinda Turner, Anna Petrunkina
Department of Medicine, University of Cambridge,
Cambrdige, United Kingdom
The capacity of multicolour Immunophenotyping panels has been
rapidly expanding owing to the growing number of newly
developed fluorescent dyes. New generations of dyes produce
higher resolution due to their low signal-to-noise ratio and
enhanced brightness, thus enabling more populations to be
resolved, and thereby, reliably identified and successfully sorted.
Any phenotyping experiment design requires careful planning in
order to optimise the combination of fluorochromes and cell
markers compatible with the SRL equipment to enable sensitive
detection and high resolution of the target cell subsets.
The aim of this project was to transfer an optimized 15 colour panel
previously validated and on a BD FACS Aria Fusion to a BD Influx
platform so that the jet-in-air technology can be exploited in full
extent to reduce the time of the sort and to conduct 6 ways sorting
to maximise the utilization of the patient sample that require
simultaneous collection of multiple subsets.
The Influx sorter platform in the Cell Phenotyping Hub based on a
collection optics set up in octagon- and trigon-shaped pathways
allowed a closely matching alignment of configurations for Fusion
and Influx. After optimising the voltages for each channel in order
to get the best resolution and reduce the spectral overlap between
the fluorochromes, a comparable resolution of the populations and
a nearly identical compensation matrix (with all coefficients <50%)
were obtained. Split aliquots of the same sample (PBMCs stained
with 15 different markers) were run at the same time, and an
unified gating strategy was used on both sorters. The resolution and
the percentages of the populations obtained were comparable
between both platforms (Tregs, cDCs, pDCs, NKs). The quality of
the sort was verified in for both systems by a purity check of the
sorted populations. In all the cases the populations sorted were well
above the 95% threshold.
An obvious shortcoming of using a Jet-in-air sorter is a daily need
for the stream adjustment and lasers alignment. That introduces a
risk of affecting consistency between the experiments. To ensure
the best performance, a QC template for 8 peaks beads were
applied. CVs of the brightest peak was markedly below the minimal
threshold 5% for each laser and the different peaks remained in the
same position as compared to a reference sample.
These results illustrate how the polychromatic human
immunophenotyping panels could be successfully transferred to a
flexible Influx sorter platform to enable routine phenotyping
operations in a translational research environment. Not only the
overall quality of the sorting services, capacity, and flexible
fulfilment in SRL were enhanced but as well as a markedly impact
on research quality was achieved due to the noticeable increase in
the final yields of sorted cells, the significant reduction in the
sorting length and, a decrease in cell death associated with the
lengthy sorting.
Speaker/Author Poster Sess oni
Index
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Scholars &
Emerging
Leaders
Poster
Session
Wednesday
15 June
Tuesday
14 June
Monday
13 June
Sunday
12 June
Saturday
11 June
promise in identifying a greater range of cell types, as well the
potential to be implemented in a sorter yielding high purity sorts.
Furthermore, this approach could be an interesting tool for the
measurement of small particles, e.g. bacteria.
188/B51
Evaluation of Methods to Verify Aerosol
Containment in Cell Sorters: Collection of Aerosols
with a Cyclone Air Sampler
Kevin Holmes1, Jan Baijer2, David Ambrozak3, Richard
Nguyen3, Stephen Perfetto3
1
Flow Cytometry Section, RTB, National Institutes of Health,
Bethesda, MD, United States, 2CEA-DSV, 3NIH / NIAID
It is well established that stream-in-air cell sorters may produce
high concentrations of aerosols during fail mode (partial nozzle
obstruction) (1). Due to the potential exposure of the operator to
these aerosols while sorting potentially infectious samples, cell
sorting is considered a high risk laboratory hazard. Instrument
manufacturers have designed aerosol containment systems to
mitigate this risk. However, as recommended in the latest ISAC Cell
Sorter Biosafety Standards (2), aerosol containment testing of these
systems must be performed at intervals determined by a risk
assessment. Current published method for containment testing
utilizes Glo-Germ beads and the Aerotech impactor (3). However,
this method has several drawbacks and alternate methods have
been actively pursued with the goal of establishing a sensitive assay
for aerosol containment testing.
Previously (CYTO 2015) we presented results of tests with Cyclex -d
non-viable impactors and fluorescent beads and compared these
with a novel assay. This assay measured phosphate, contained
within aerosols,that were captured on the Cyclex-d impactors.
Sensitivity of this phosphate-based assay was 1.0 particle/cm3 (3.7
ȝM [phosphate]).
Although the spectrophotometric phosphate assay can measure as
low as 1.6 pmoles, inherent background levels of phosphate in the
Cyclex-d impactor limited the sensitivity of this assay and
necessitated a search for an alternative aerosol capture device.
Recently (CYTO 2015, abstract by Jan Baijer), an automated
cyclonic liquid impinger that captures aerosols in liquid was
described that had sensitivity and efficiency characteristics
sufficient to be considered as a replacement technology for the
current methods (Coriolis ȝ Air Sampler system, Bertin
Technologies). The Coriolis ȝ is a high volume (air flow: 100-300
L/min) collection device with a D50 of 0.5 ȝm and a collection
efficiency of 70- 80% for aerosols in the size range produced by
cell sorters. Results of tests to determine the sensitivity of this
device to detect cell sorter generated aerosols will be presented.
Aerosols generated from a cell sorter in fail mode and housed in a
Class II Biological Safety Cabinet, were collected using the Coriolis
ȝ and subsequently quantitated with a spectrophotometric assay for
phosphate (a constituent of the Phosphate Buffered Saline in the
sheath fluid). Failure of containment was simulated by a reduction
of the airflow rate of the Aerosol Management System, which also
permitted a more consistent aerosol concentration for measurement
in the Coriolus/Phosphate Assay (CPA). Sensitivity of the CPA was
determined by correlating absorbance readings with aerosol
concentration as measured with a UV-APS aerodynamic particle
sizer (TSI, Inc). Tests were performed to determine the starting
volume of the collection liquid (5, 10 or 15ml) that provided the
best sensitivity in the phosphate assay. Preliminary results show
that at 5ml starting volume, aerosol concentrations of
approximately 0.05 particles/cm3 could be detected, corresponding
to 0.6 ȝM [phosphate]. Sensitivity of the CPA for cell sorters not
housed in a BSC, as well as comparisons of the Cyclexd/fluorescent bead assay with the CPA will also be presented.
References:
1. Holmes KL. Cytometry A 2011;79:1000-1008.
2. Holmes KL, Fontes B, Hogarth P, Konz R, Monard S, Pletcher
CH, Jr., Wadley RB, Schmid I, Perfetto SP.Cytometry A 2014.
3. Perfetto SP, Ambrozak DR, Koup RA, Roederer M. Cytometry
A 2003;52:122-130.
174
Fort Collins Development Center, Beckman Coulter, Inc.,
Fort Collins, CO, United States
Oral Session
Abstracts
Poster Session
Abstracts
Index
175
Speaker/Author
Commercial
Tutorials &
Exhibits
StemCell Technologies Inc, StemCell Technologies Inc,
Scholars &
Emerging
Leaders
Catherine Ewen, Andy I. Kokaji, Samuel Clarke, Drew
Kellerman, Martina Chambers, Mandy Chan, Steven
Woodside
We herein present a new microfluidic platform for high thoughtput
single cell analysis. The platform is designed to capture individual
cells in sub-nanoliter traps. Subsequently, the captured cells can be
stained on chip for molecular analyses or treated with
chemotherapeutic agents for drug screening. Then, wide-field
fluorescent imaging and image processing are carried for analysis.
With molecular assays performed on chip, the system considerably
minimizes cell-loss associated with sample washing and transfer
steps in conventional methods. As a proof of concept, we applied
the system to detect central nervous system (CNS) lymphoma in
cerebrospinal fluids, a challenging clinical task due to low cell
density and limited sample volumes. Using the developed system,
we captured individual lymphoid cells, and profiled them
according to a three-step labeling strategy: 1) the use of CD19
and/or CD20 to determine B cells; 2) the use of kappa or lambda
light chains to identify different clonal populations of lymphomas;
and 3) additional phenotypic markers for subtyping and prognostic
tasks. To facilitate such analyses, we also established an automated
image processing algorithm for clonality assessment. The developed
platform achieved the capture efficiency of >90%. Through on-chip
staining, we could image both intracellular and extracellular
protein markers from individual lymphoma cells; the image
processing algorithm further allowed for quantitation of target
markers at the single cell level. The entire assay was complete in
less than one hour, demonstrating the potential for on-site disease
diagnosis and characterization. In the future, this platform will be
integrated with a single-cell micromanipulation to enable target cell
retrieval for further analysis
Poster
Session
190/B53
Fast and Easy Immunomagnetic Positive Selection of
PE- or Biotin-Conjugated Antibody Labelled Cells
with Releaseable RapidSpheres™ Results in
Functional, Pure, and Particle-Free Cells
Center for Systems Biology, Massachusetts General
Hospital / Harvard Medical School, Boston, MA, United
States, 2Department of Pathology, assachusetts General
Hospital, Boston, MA, United States, 3Cancer Center,
assachusetts General Hospital, Boston, MA, United States
Wednesday
15 June
Beckman Coulter, the stylized logo, and Astrios, are trademarks of
Beckman Coulter, Inc. Beckman Coulter and the stylized logo are
registered in the USPTO.
1
Tuesday
14 June
Conclusion: Demonstrating the AstriosEQ capabilities for sorting
with small and large tips at high and low pressures presents
multiple advantages of using tip diameters outside of the standard
range. For the 50 micron tip the ability to sort at high speeds with
high efficiency is necessary for sorting high concentrations of low
volume submicron particles or rare event sorting. The 200 micron
tip allows sorting of sort large fragile cells and organisms. The use
of 50 and 200 micron tips expands the other attributes of the
AstriosEQ such as unique FSC detection and up to 42 SSC
parameter.
Kyungheon Lee1, Anna Truetsky1, Jun Song1, Randy J
Giedt1, Eunha Kim1, Alexandra E Kovach2, Ephraim P
Hochberg3, Cesar M Castro1, Ralph Weissleder1, Hakho
Lee1
Monday
13 June
Results: The AstriosEQ equipped with the 50 um tip sorted
polystyrene and silica beads with a higher yield compared to using
a 70 um tip. EVs also sorted faster with a lower sort output volume
than larger nozzle tips. Large beads and biological samples were
also successfully sorted.
191/B54
Microfluidic Platform for On-Chip Single Cell
Analysis
Sunday
12 June
Methods: Submicron - The AstriosEQ was set up to sort submicron
samples by first installing a 50 micron tip and increasing the
pressure to 80 psi. To detect these small particles it is critical that
the nozzle tip and glass surfaces are clean to reduce instrument
noise. Signal to noise separation was checked using submicron
beads before analysis of EVs. AstriosEQ IntelliSort feature allows for
beadless sort setup (typically 120,000 Hz, 15 volts and drop delay
of 32) which generate over 150,000 drops per second. Samples
were sorted at over 70,000 eps above 50% efficiency and post-sort
purity determined. Large Particles - The first step of setting up the
AstriosEQ for sorting large particles is to reduce system pressure to
5 psi and install a 200 micron tip. To allow proper laser delay
determination only three laser can be present in the software. Sort
setting are set manually using the Summit drop delay wizard
(typically 7,000 Hz, 50 volts, and drop delay of 15). Beads and
different biological samples were sorted and post sort purity and
viability determined.
The system is highly adaptable, targeting cells through the use of
any biotin or PE-conjugated primary antibody or ligand. We have
thoroughly tested the EasySep™ Release kits with biotin and PEconjugated to antibodies to human CD19 and human CD3,
obtaining 93% to 98% purity and 35% to 60% recovery.
Additionally, the technology has been used to isolate numerous
other human immune cell subsets such as CD4, CD45RO, CD20,
and HLA-DR expressing cells from leukopak samples and
cryopreserved peripheral blood mononuclear cells. The EasySep™
Release Biotin and PE positive selection kits have also undergone
preliminary testing on rat splenocytes, and various mouse tissues
such as spleen, thymus, lung, and liver. EasySep™ Release
technology is amenable to sequential isolations, which can enable
the isolation of immune cell subsets with complex cell surface
marker phenotypes. Ultimately, this will offer a gentle and cost
effective, magnetic particle-free isolation method for the isolation of
virtually any cell population.
Saturday
11 June
Background: Sorting submicron samples such as extracellular
vesicles (EVs) requires instruments with high analysis and sort
speeds for processing high sample concentrations. Using nozzle
tips with smaller diameters (50 nm) and higher instrument pressure
increases the number of drops allowing increased sort speeds and
efficiencies. Additionally, the smaller drop volume helps maintain
high post sort sample concentrations. Conversely, large fragile cells
and organisms (>30 microns), require low system pressure and
larger diameter tips (200 micron). The MoFlo Astrios EQ (Beckman
Coulter, Inc.) introduced bead-free sort set-up and monitoring with
IntelliSort for 70 and 100 micron tips but is capable of sorting with
tips smaller and larger than this range by following a few unique
steps.
Special
Lectures
Alan Dean, Carley Ross, Robin Morris, Aliaksandr
Kachynski
Immunomagnetic cell isolation has long been recognized as a costeffective and gentle method of obtaining purified cell populations.
However, isolating novel or rare cell populations often relies on
time consuming cell sorting techniques due to the lack of off-theshelf immunomagnetic cell isolation kits. To bridge this gap, we
developed the EasySep Release isolation kits for PE- or biotinconjugated antibodies. EasySep™ Release incorporates our latest
magnetic particles that have low non-specific binding
characteristics, and are rapidly removed from isolated cells after
positive selection using a simple reagent.
Congress
Overview
189/B52
Beyond the Norm with the Beckman Coulter
AstriosEQ: Sorting with 50 and 200 micron Tips
Congress
Overview
Special
Lectures
John Tigges, Virginia Camacho, Vasilis Toxavidis
Index
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Scholars &
Emerging
Leaders
Poster
Session
Wednesday
15 June
Tuesday
14 June
Monday
13 June
Sunday
12 June
Saturday
11 June
Cell-Derived Microvesicles (B55 – B64)
192/B55
A Methodological Approach for Characterization of
Extracellular Vesicles: Small-Particle Flow Cytometry
Beth Israel Deaconess Medical Center, Boston, MA, United
States
Flow cytometry is an advantageous tool for the analysis and
characterization of extracellular vesicles (EVs) because of its robust
statistical power and its multiparametric capabilities. The goal of
nanoscale flow cytometry analysis and nanoscale sorting, termed
Small Particle Flow Cytometry (SPFC), is to accurately represent the
size distribution and scatter profiles of these vesicles. As with
conventional flow cytometry, the suspended vesicles pass through a
chamber and are hydrodynamically focused. As vesicles pass
through, the laser light is refracted and scattered in all directions.
Their scatter properties are measured by detectors; concurrently
fluorescent dyes can be used to tag various properties of interest.
Characterization of extracellular vesicles (EVs) is greatly impeded
by several factors: their size (below 100nm), their overlapping size
distribution, particle polydispersity, and an overall low refractive
index (Orzoco, Van Der Pol). Because conventional flow
cytometers are not equipped with necessary optical capacities to
reach this lower threshold, instruments that are used for SPFC
typically have specific adaptations. In light of these enhancements,
there are a number of modifications to the conventional form of
analysis that should be considered.
Here we describe a comprehensive methodology for the set up and
standardization of EV analysis using SPFC. Controls of different size
ranges, fluorescent intensities, and materials can be used to set up
distribution curves that are then used for instrument optimization
and as a reference guide. Using these controls, FACS instruments
can be primed for the detection, analysis and sorting of specific EV
populations. This allows for cross platform comparison and the
ability to monitor both Quality Control (QC) and Quality Assurance
(QA). The method here will describe the use of nanoparticles to
optimize a flow cytometer for small particle detection. It will also
outline the procedures necessary to recover EVs for downstream
applications.
193/B56
Limitations of Microvesicle Analysis with LightScattering Flow Cytometry
Anastasiya Konokhova1, Maxim Yurkin1,2, Denis
Korneev3, Valeri Maltsev1,2
1
Voevodsky Institute of Chemical Kinetics and Combustion
SB RAS, Novosibirsk, Russia, 2Novosibirsk State University,
Novosibirsk, Russia, 3SRC VB Vector, Koltsovo, Novosibirsk
region, Russia
Background: Light-scattering is the most widely used method for
microvesicle’s (MVs) analysis, in particular, in flow cytometry (FC).
Still, complete understanding and interpretation of these
measurements is lacking. The key open questions are: (1) how
sensitive are standard measurements of scattering in forward (FS)
and side directions (SS) for MV detection? (2) Which MV’s
characteristics can potentially be estimated from these signals? (3)
What is the reliability of these estimates (especially, of FS-based
sizing) given uncertainties in MV’s refractive index (RI) and shape
(possibility of their aggregation) and commonly incomplete
knowledge of the instrument optical configuration: (4) What are the
options, when FS and SS fail, i.e. do not provide sufficient
information?
scattering profiles (LSPs) and standard FS and SS signals. Lightscattering simulations for particles with spherical and non-spherical
(aggregates of spheres) shapes were performed using the Mie theory
and the discrete-dipole approximation, respectively. Transmission
electron microscopy was used for control sizing of polystyrene
microspheres.
Results: We developed a general method to estimate collection
angular ranges for FS and SS signals, based on measurement of
preliminary characterized microspheres and consequent non-linear
fitting. Applied to SFC, this method allowed us to determine
detection limits in terms of size and RI. In particular, FS is capable
to detect single MVs in a more broad range than SS. Additionally,
when both FS and SS signals are above noise level, we
demonstrated that size and RI can be deducted. However, the the
solution is unique only for some combinations of particle
characteristics, even if particle sphericity is assumed, and this
region complexly depends on a specific instrument. By contrast,
LSP-based characterization is always unique and accounts for
particle shape. Moreover, when combined with FS and SS
measurements it results in excellent precision – typically tens
nanometers for size and several thousandths for RI.
Conclusions: Accurate estimation of the angular range of collected
light-scatter data is a necessary practice for MV analysis with any
flow cytometer. However, even at its best, a combination of FS and
SS signals leads to reliable determination of size and RI of single
spheres only in a limited range of those characteristics. Enhanced
MV analysis, including their accurate identification and
characterization by shape, size and RI, is only possible with
additional light-scattering signals, such as LSPs measured by a SFC.
194/B57
Identifying Exosome Binding and Internalization in
Blood Cell Subsets by Imaging Flow Cytometry
Haley Pugsley, Sherree Friend, Christine Probst, Phil
Morrissey
Amnis part of MilliporeSigma, Seattle, WA, United States
Only recently has the importance of extracellular vesicles as key
mediators of intercellular communication been appreciated.
Extracellular vesicles are membrane derived structures that include
exosomes, microvesicles and apoptotic bodies. Exosomes have
been shown to transfer molecules between cells and have the
potential to transfer signals between cells. Exosomes are released
under normal physiological conditions; however, they are also
believed to serve as mediators in the pathogenesis of neurological,
vascular, hematological and autoimmune diseases as well as
cancer. Quantifying and characterizing exosomes in a
reproducible and reliable manner has been difficult due to their
small size (50 – 100 nm in diameter). Exosomes analysis can be
done using high-magnification microscopy however this technique
has a very low throughput. Attempts to analyze exosomes using
traditional flow cytometers has been hampered by the limit of
detection of such small particles and low refractive index. To
overcome these limitations we have employed multispectral
imaging flow cytometry that has the advantage of combining high
throughput flow cytometry with higher sensitivity to small particles
and the added benefit of imaging that can provide visual
confirmation of particle integrity and characterization. In this study
we use multispectral imaging flow cytometry to investigate the
interaction of exosomes with white blood cells. Exosomes derived
from different cell types will be investigated for their preferential
interactions with blood cell subsets by combining
immunophenotyping with morphological parameters to measure
their binding and internalization.
Methods: Light-scattering measurements from individual particles
(polystyrene microspheres and MVs in platelet-rich plasma) were
performed using a Scanning Flow Cytometer (SFC) fabricated by
CytoNova Ltd (Novosibirsk, Russia, http://cyto.kinetics.nsc.ru),
which provides simultaneous measurement of angle-resolved light-
176
1
2
Academic Medical Center, Amsterdam, Netherlands,
University of Twente, Enschede, Netherlands
For many years, studies have shown that tumor-derived exosomes
(TEX) have a role in cancer development. TEX are released and
influence several tumor development paths such as angiogenesis
and metastasis. TEX are also responsible for suppressing the
immune response in the tumor microenvironment. TEX cultured
with primary human lymphocytes demonstrate reduced NK
proliferation, apoptosis of CD8+ T cells (Tcyto) and transition of
CD4+ T cells (Tconv) cells to a T-regulatory CD4+CD39+(Treg)
phenotype. Down-regulation of the immune system by both
cellular and molecular mechanisms is evaluated in this study.
Index
Speaker/Author
177
Poster Session
Abstracts
T cells subsets were cultured with TEX or exosomes released from
cultured dendritic cells (DEX) and gene profiling was performed.
The response was evaluated by measuring immune response genes
using qRT-PCR. Both TEX and DEX up-regulated immune response
expression in multiple genes. Analysis demonstrated that the source
of exosomes was not determinant of the gene expression response,
whereas cell type and activation status were. Tregs were more
sensitive to TEX than other T-cell subsets as indicated by the
increase in CD39 expression and adenosine production. In Tconv,
CD69 expression decreased, inhibitory genes increased, and
function declined. Visualization with the ImageStream revealed that
exosomes were not internalized by Treg cells, unlike B cells and
Oral Session
Abstracts
1
FACS Core Facility Research Services, Loyola University
Chicago, Maywood, IL, United States, 2Department of
Otolaryngology and Head & Neck Surgery, University
Hospital, Basel, Switzerland, 3Departments of Pathology,
Immunology and Otolaryngology, Univeristy of Pittsburgh
School of Medicine, Pittsburgh, PA, United States,
4
Department of Microbiology and Immunology, Loyola
University Chicago, Maywood, IL, United States
Commercial
Tutorials &
Exhibits
Introduction: Extracellular vesicles, which include exosomes,
microvesicles (MVs), apoptotic vesicles (AVs), range in size from 30
to ~3000nm. Differentiation between EV populations remains a
problem due to overlapping characteristics between vesicles,
particularly in techniques such as flow cytometry, where sizing is
not straight forward. Furthermore, flow cytometry standardisation
techniques for sizing, though shown with beads has not made the
step to linking modelling with biological phenotypes. Here we use
CD41a, caspase 3/7, mitochondrial stain, intracellular calcium
concentration and membrane staining, combined with Mie theory
as a standardisation tool to differentiate between EV subsets.
Methods: 6mL of blood was drawn from 3 healthy individuals into
citrated tubes. Tubes were centrifuged twice at 1500g. Supernatants
were drawn off and stored at -80oC. Supernatants were thawed at
Patricia Simms1, Laurent Muller2, Theresa Whitesides3,
Phong Le4
Scholars &
Emerging
Leaders
1
Faculty of Medicine, University of Southampton,
Southampton, United Kingdom, 2Physical Sciences and
Engineering, University of Southampton, Southampton,
United Kingdom
197/B60
Exosomes Alter mRNA Gene Profile in T
Lymphocytes
Poster
Session
Joshua Welsh1, Peter Horak2, David Smith1, James
Wilkinson2, Judith Holloway1, Nicola Englyst1
Wednesday
15 June
196/B59
Novel Extracellular Vesicle Assay Combined with
Mie Scatter Allows Differentiation between
Apoptotic Vesicles, Microvesicles and Residual
Platelets
Tuesday
14 June
Conclusion: SPRi detects lower antigen densities on EV than FCM.
Monday
13 June
Results: On intact cells the MFI and SPRi signals obtained with the
eleven antibodies correlate for HS578T (R2=0.66), MCF7 (R2=0.82)
and SK-BR-3 (R2=0.96). Due to the limited number of antigens with
MFI above background for EV, correlation between FCM and SPRi
could only be determined for HS578T derived EV (R2=0.65). These
EV had five antigens with MFI above background, while MCF7 and
SK-BR-3 derived EV only had one and two, respectively.
Interestingly, SPRi signals were above background for not only
these eight antigens, but for in total respectively seven (HS578T),
seven (MCF7) and six (SKBR3) antigens. The remaining four, four
and five antigens respectively had signals below background for
both techniques. These undetectable antigens also had signals
below background for intact cells, except for CD227 on SK-BR-3
cells.
Conclusion: It can be seen that markers of AVs, caspase 3/7 and
mitochondrial staining predominantly appear in the theorized size
range of >1000nm. These markers can also be seen to co-localize
with CD41a in this region, inferring that residual platelets are
present within standard MV preparations, as well as larger bodies.
This combined staining validates the use of Mie theory with
biological phenotyping as a standardization tool to verify flow
cytometer resolution and as method for determining analysis gating
strategies. We have also shown differentiation between MVs, AVs
and residual platelets within the same sample for the first time.
Sunday
12 June
Methods: To compare cell surface antigen exposure measured by
FCM and SPRi, human breast cancer cell lines HS578T, MCF7, and
SK-BR-3 were used. The MFI was compared with the SPRi signal for
eleven antibodies on intact cells and EV derived from each cell
line. The antibodies for CD9, CD44, CD49e, CD63, CD71, CD221,
CD227, EGFR, EpCAM, Her2 and Her3 recognize cell surface
antigens that are expressed on these cell lines.
Results: Modelling revealed, that in terms of collected light scatter,
~200nm polystyrene beads (200nm P) were equivalent to ~1000nm
biological vesicles, with the Fortessa having a lower detection limit
of 100nm for polystyrene beads (100nm P). Sample analysis
showed a mean±SD of 93.8±0.00% of BAPTA-1-AM+ve events,
99.1±0.012% of saspase 3/7 events, and 100±0.00% of
mitochondrial stain events were above the 200nm polystyrene
beads. CD41a can be seen to co-localize with BAPTA-1, Caspase
3/7 and mitchondrial staining above 200nm P. The MV gate
(100nm P to 200nm P) contained 36.8±0.14% of CD41a+ve
events. If a lower limiting gate was not used, this increased to
56.5±1.39%.
Saturday
11 June
Introduction: Flow cytometry (FCM) mean fluorescence intensity
(MFI) is proportional to the mean number of fluorochrome-antigen
conjugates. However, the MFI of such conjugates on extracellular
vesicles (EV) is often undistinguishable from background due to low
antigen quantities. Surface plasmon resonance imaging (SPRi) is an
alternative for analyzing antigen exposure, which captures EV on a
surface with an antibody array. The captured EV cause an increase
in refractive index (RI) at the surface. This RI is directly proportional
to the SPRi signal, which was shown to correlate with antigen
exposure on cells. Here we compare antigen exposure on EV
measured with FCM and SPRi.
Special
Lectures
Frank Coumans1, Elmar Gool1, Ivan Stojanovic2, Anita
Böing1, Richard Schasfoort2, Edwin van der Pol1, Guus
Sturk1, Ton van Leeuwen1, Rienk Nieuwland1
37°C for 10 minutes. 50μL of supernatant was added to 50μL
0.1μm double-filtered HEPES Buffered Saline (HBS) with either: 2μL
Cell Light Mitochondria-GFP (3 hours), 2μL 500μM BAPTA-1 (30
mins) or CellEvent Caspase 3/7 (30 mins) and incubated at 37°C
before pelleting at 18,000g for 30 mins. Supernatants were
discarded and 50μL 0.1μm double-filtered HBS was used to
resuspend the MV pellet. 20μL of 25μg/mL CD41a PerCP-Cy5.5
was added and incubated on ice for 15 mins. 1μL of 200μM VRD
was added and incubated for 5 min before analysis. Samples were
serially diluted until event rates were below 500E/sec. A Fortessa X20 was used for analysis, with Megamix Plus FSC, 100, 200, 400,
700, 1000nm polystyrene NIST and 400, 700, 1000nm silica NIST
beads for calibration and Mie scatter modelling.
Congress
Overview
195/B58
Surface Plasmon Resonance Imaging Detects Lower
Antigen Densities Than Flow Cytometry
Congress
Overview
Results: In cell culture supernatant of cancer cell lines, we detected
differing expression of melanoma and carcinoma markers for EVs
captured on the multiplex beads. MCSP is seen on all melanoma
samples but the differing levels can be well discriminated. Likewise,
melanoma exosomes hardly show CD326 (EpCAM) as compared to
colon cancer exosomes.
Special
Lectures
198/B61
Tumor-Derived Exosomes Regulate Expression of
Immune Function-Related Genes in Human T Cell
Subsets
We investigated surface proteins of EVs directly from ascites of
ovarian cancer patients. Besides the common exosome markers
CD9, CD63 and CD81, we detected markers indicating blood cell
derived vesicles like CD4 and CD8 for T cells and CD14 for
monocytes. Interestingly, discrimination of positive signals was seen
between patients for key tumor makers such as for EpCAM, CD133
(PROM1), CD44 and CD49e showing tumor derived exosomes in
the ascites. The multiplex assay was successfully used in other
sample types such as urine to analyze multiple exosomes within the
samples (data not shown). For the analysis of in vitro activated B
cell exosomes, we used specific staining antibodies to investigate
potential exosome subpopulations. The signals for the activation
markers CD80 and CD86 demonstrate that the activation status of
the cells is reflected by the secreted vesicles. The platelet marker
CD42a indicates vesicles from contaminating platelets that have
been present in the primary B cell culture. B cell marker stainings
indicate that besides CD42a, also CD9 is likely not present on B
cell derived vesicles but of platelet origin.
Index
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Scholars &
Emerging
Leaders
Poster
Session
Wednesday
15 June
Tuesday
14 June
Monday
13 June
Sunday
12 June
Saturday
11 June
monocytes, even after an extended co-culture time. As TEX were
not internalized, the immune response may be mediated by
expressed surface receptors and ligands. Initial work to identify
exosomes bound to cell surface molecules demonstrated that the
Amnis ImageStream is able to visualize these surface bound
exosomes.
Patricia Simms1, Muller Laurent2, Theresa Whiteside3
1
FACS Core Facility, Loyola University Chicago, Maywood,
IL, United States, 2Otolaryngology and Head & Neck
Surgery, University Hospital in Basel, Basel, Switzerland,
3
Department of Pathology, Immunology and
Otolaryngology, University of Pittsburgh, Pittsburgh, PA,
United States
For many years, studies have shown that tumor-derived exosomes
(TEX) have a role in cancer development. TEX are released and
influence several tumor development paths such as angiogenesis
and metastasis. TEX are also responsible for suppressing the
immune response in the tumor microenvironment. TEX cultured
with primary human lymphocytes demonstrate reduced NK
proliferation, apoptosis of CD8+ T cells (Tcyto) and transition of
CD4+ T cells (Tconv) cells to a T-regulatory CD4+CD39+(Treg)
phenotype. Down-regulation of the immune system by both
cellular and molecular mechanisms is evaluated in this study.
T cells subsets were cultured with TEX or exosomes released from
cultured dendritic cells (DEX) and gene profiling was performed.
The response was evaluated by measuring immune response genes
using qRT-PCR. Both TEX and DEX up-regulated immune response
expression in multiple genes. Analysis demonstrated that the source
of exosomes was not determinant of the gene expression response,
whereas cell type and activation status were. Tregs were more
sensitive to TEX than other T-cell subsets as indicated by the
increase in CD39 expression and adenosine production. In Tconv,
CD69 expression decreased, inhibitory genes increased, and
function declined. Visualization with the ImageStream revealed that
exosomes were not internalized by Treg cells, unlike B cells and
monocytes, even after an extended co-culture time. As TEX were
not internalized, the immune response may be mediated by
expressed surface receptors and ligands. Initial work to identify
exosomes bound to cell surface molecules demonstrated that the
Amnis ImageStream is able to visualize these surface bound
exosomes.
199/B62
A Multiplex Bead Platform for Protein Profiling of
Exosomes by Flow Cytometry
Stefan Wild, Christian Dose, Nina Koliha, Yvonne
Wiencek, Ute Heider, Andreas Bosio
R&D, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany
Background: Exosomes or extracellular vesicles (EVs) are loaded
with specific sets of proteins, lipids and nucleic acids. The EV
composition depends on the originating cell and different EVs can
be distinguished by surface marker profiling. We established a
multiplex bead-based assay consisting of capture and detection
antibodies to analyze the composition of exosomal surface proteins
by flow cytometry.
Methods: Exosome samples are incubated with a mix of 39 distinct
fluorescently labeled capture antibody beads. Capture antibodies
were chosen with focus on blood cell and potential tumor markers.
The bead populations can be discriminated by flow cytometry and
specifically bound exosomes are detected by fluorescently labeled
antibodies e.g. a set recognizing the exosome markers CD9, CD63
and CD81. By splitting a sample, alternative staining antibodies can
be used in addition for further exosome characterization.
178
To enable the analysis of potentially infectious material, we
established a protocol to fix bound and stained vesicles on the
multiplex beads for later flow cytometric analysis.
Conclusions: We developed the multiplex bead platform to simplify
the analysis of the surface protein composition of exosomes.
Vesicles can be captured directly form body fluids like ascites or
urine. The exosomes, which are bound to specific beads and
stained using common exosome markers, can be detected using
standard flow cytometry. Additional staining antibodies can reveal
information on potential exosome subpopulations.
200/B63
Submicron Particle Analysis and Counting Is Highly
Favored by the Use of Side Scatter from the Violet
Laser on the CytoFLEX Flow Cytometer
Philippe Poncelet1, Tarik Bouriche1, Stéphane Robert2,
Corentin Franco1, Romaric Lacroix2,3
1
R&T, Biocytex, Marseille, France, 2VRCM, UMR-S1076,
Aix-Marseille Université, INSERM, Faculté de Pharmacie,
Marseille, France, 3Hematology and Vascular Biology
Department, CHU La Conception, APHM, Marseille, France
Flow cytometry (FCM)-based analysis of submicron particles (SMPs)
is a rapidly evolving field with major technological challenges.
Whereas FCMrs best analyze cells in the 1-50 μm size range, new
doors are now opening for reliable SMP detection and counting
down-to 0.1 μm possibly covering the size range of i) cell-derived
microparticles (MPs, 0.1 to 1 μm in size), ii) bacteria and giant
viruses (1), and also iii) synthetic objects such as latex or metallic
beads and liposomes.
Aim: to challenge the capabilities of a new commercial FCMr,
CytoFLEX from Beckman-Coulter (CFX, BC), which provides access
to side scatter information from the violet laser (VSSC configuration)
in addition to conventional blue SSC. A 3-laser CFX with microplate option was tested for a few days aside a BC Gallios in FS W²
mode. Ready-to-use blends of fluorescent submicron polystyrene
beads designed to optimize scatter settings using FSC (MegamixPlus FSC, BioCytex) or SSC (Megamix-Plus SSC) were mixed V/V to
create i) Gigamix (2,3), a SMP-oriented QC system featuring 7
reference bead sizes of 100, 160, 200, 240, 300, 500 and 900 nm
and ii) with 75 nm beads to challenge scatter sensitivity. Both
purified and plasma-contained MPs were used as testing models,
after dual staining with Annexin V-FITC and either i) CD142-PE for
TF+ bxPC3 MPs, ii) CD59-PE for EMPs or iii) CD41-PE for plasmatic
PMPs. 0.22 μm-filtered water was used as sheath fluid to reduce
non-specific background (BkGd) from particulate matter and as
sample to measure intrinsic BkGd in each scatter setting. 3 μm
counting beads (MP-Count beads, BioCytex) served to compare
Results: Data from 42 patients from two CAR therapy trials have
been entered into the SCRI LabKey server for analysis. Parameters
from over ten polychromatic flow cytometry panels (6-11 colors),
deep sequencing data, and clinical data have been merged for
comprehensive analysis. With this approach, we have discovered
novel relationships between the intrinsic properties of CAR T cells
at the start of therapy and therapy success.
Oral Session
Abstracts
Poster Session
Abstracts
203/B66
Cell-Based Biomarkers in Clinical Research:
Developing, Validating and Implementing Sample
Preparation, Biobanking and Image Cytometry Assays
to Support Drug Development
Commercial
Tutorials &
Exhibits
Conclusions: Utilizing data integration software such as LabKey
provides a high-throughput method for flow cytometry analysis as
well as a highly scalable and comprehensive analysis model for
translational research. By centralizing these multidisciplinary data,
we have the unique capacity to perform rapid and dynamic analysis
of over fifty datasets. The comprehensive analysis of CAR T cells
will provide a better understanding of the function of CAR T cells in
vivo, and help improve future CAR T cell products for maximized
therapeutic success.
Scholars &
Emerging
Leaders
Jan Detmers1, Anja Mirenska2, Christian Hennig3
1
179
Index
Central Lab Services, Zellkraftwerk GmbH, Leipzig,
Germany, 2Hannover Medical School, Hannover, Germany,
3
Zellkraftwerk GmbH, Hannover, Germany
Speaker/Author
Methods: Standard multiparameter flow gating strategies are
developed before entry into the database. Quality control and
further analysis is then performed on the LabKey server. Because
LabKey utilizes a relational database model, FACS, luminex and
clinical data can be merged and comprehensively studied using
SQL queries. In addition, we are able to leverage the R server on
the LabKey platform for dynamic and thorough statistical analysis.
Poster
Session
Extracellular Vesicles (EVs) are released into biofluids by most cells
and express biomarkers on their surface membranes that enable
classification based on the cell of origin. For example, an EV
expressing CD3 antigens on its surface membrane most likely came
from a T lymphocyte, whereas an EV expressing CD41 on its
surface most likely originated in a platelet. Since phenotypic
markers reveal the likely origin of an EV, flow cytometers are an
obvious instrument choice for characterizing EVs in blood and
other biofluids, but the scientific community has raised significant
controversy over the reliability of flow-based EV detection and
characterization, due in part to their small size (most are less than 1
micron in diameter) and the potential for coincident events in the
flow cytometer. At <0.3-micron diameter, the smallest EVs are
below the light scatter detection limit of most flow cytometers and
can only be detected if they occur as a coincident event with other
particles or are detected by a fluorescent marker. With so much
controversy, the subtle differences in light scatter characteristics of
EVs have widely been ignored. We conducted multiple studies of
EVs from human blood samples on a custom B-D FACSCanto Plus
specifically modified to enhance light scatter and fluorescence
sensitivity. We have found that 488nm side light scatter (SSC) for
EVs of specific phenotypes have distinct, reproducible patterns. For
example, most endothelial cell-derived EVs appear much
larger/denser by SSC than platelet-derived EVs and T cell-derived
EVs have a much more varied SSC distribution than either
endothelial cell-derived or platelet-derived EVs. These findings
suggest that EV size and density can vary based on their cell of
origin, which has serious implications for the comparison of EVs
prepared by different centrifugal separation methods. In addition,
the finding that smaller, less dense EVs are different phenotypically
from larger, denser EVs means that the phenotypic characterization
of EVs can be dramatically affected by the flow cytometer’s SSC
sensitivity.
To do this, we have developed in-depth molecular and cellular
studies (with heavy emphasis on FACS) on patient specimens
obtained during clinical trials to determine prerequisites,
biomarkers or correlates of successful treatment. We have utilized
the LabKey Software platform to integrate these diverse datasets in
real-time with both manufacturing and clinical datasets in order to
analyze, visualize and share trial data.
Wednesday
15 June
Laboratory Operations, CytoVas, LLC, Philadelphia, PA,
United States
Immunotherapy using chimeric antigen receptor (CAR) modified T
cells is a promising new cancer therapy. Given that the factors
affecting the success or failure of a treatment in a given patient are
largely unknown, the Correlative Studies Laboratory (CSL) focuses
on defining intrinsic properties of T cells that determine their ability
to function and persist in vivo.
Tuesday
14 June
W. Roy Overton, Michael McGrane, Maggie WesberrySchmierer, Tatiana Arango, Todd Johnson
Background: While innovative methods such as clustering
algorithms for analyzing flow cytometry data are increasingly
prevalent, traditional sequential gating remains the primary
approach for FACS analysis. This is particularly important in
longitudinal clinical trials where changes in fluorescence due to
natural equipment fluctuation or protocol changes can occur
throughout the duration of the study.
Monday
13 June
201/B64
Differences in Light Scatter Characteristics of
Phenotypic Subsets of Extracellular Vesicles
1
Seattle Children's Research Institute, Seattle, WA, United
States, 2Seattle Children's Hospital, Seattle, WA, United
States, 3Department of Pediatrics, University of Washington,
Seattle, WA, United States
Sunday
12 June
2,3) Poncelet et al, Cytometry A 2015 and TRASCI 2015.
Hannah Smithers1, Olivia Finney1, Stephanie RawlingsRhea1, Roxana Hicks1, Ben Futrell1, Danielle Doolittle1,
Linda Ton1, Julie Park2,3, Rebecca Gardner2,3, Michael
Jensen1,3
Saturday
11 June
1) Khalil et al, Frontiers Microbiol 2016
202/B65
Utilizing Data Integration Software to Create an
Analysis Platform for Immunotherapy Clinical Trials
Special
Lectures
References:
Clinical Trials (B65 – B69)
Congress
Overview
absolute MP counts between CFX and Gallios. Gallios FSC cutoff
was set at 0.3 μm bead equivalent (μm-eq) with Mgx+ FSC and CFX
SSC cutoff was set at a MP-homologous value of ~0.17 μm-eq with
Mgx+ SSC (2,3), providing acceptable BkGd levels of respectively
~3,000 and ~10,000 evts/s (abort rate <10%). Not only VSSC
displayed clearer resolution than SSC for beads below 200 nm (100
vs 160 nm and 75 vs 100 nm) but pure water could be analyzed
with acceptable BkGd using a VSSC threshold below 75 nm beads.
A VSSC threshold including all 100 nm beads was selected for
sample analysis on CFX with BkGd < 100 evts/s in water. This
easily attainable low cutoff provided higher absolute PMP counts (>
3-5x) than the standardized settings set at either i) 0.17 μm-eq on
blue SSC in CFX or ii) 0.3 μm-eq on W² FSC in Gallios, which
were comparable. Same, more TF+ or CD59+ MPs were seen in
purified MP suspensions. Noteworthy, comparable PMP counts
were obtained on the same sample in 5 min vs 1 min runs and in
single tube vs microplate configurations but MP-count beads were
needed for reliable absolute counts. Using SSC from a violet laser,
as permitted on the BC CytoFLEX, brings much promise to reduce
the size limits of FCM for SMP analysis. It significantly increases
resolution below 200 nm-eq, allowing clear-cut discrimination of
75 vs 100 nm beads and routine analysis of plasma-derived PMPs
with a VSSC threshold below 100 nm-eq. Note that detection of
100 nm plastic beads does not infer detection of 100 nm-sized cellderived MPs, especially when using SSC (2,3), but this at least
denotes a real breakthrough towards the targeted sensitivity limit.
Whether this option can be generalized on other FCMrs remains to
be demonstrated.
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Index
Abstracts
In the pharmaceutical industry, cytometry is increasingly
recognized as a powerful analytical tool for the analysis of multiple
biological parameters of individual cells within heterogeneous cell
populations. The implementation of standardized cytometry-based
biomarker assays in clinical trials remains a challenge due to the
limited stability of clinical specimens and inter-instrument
variations. To participate in industry-sponsored clinical studies
(Phase I-III) it is crucial to develop robust sample biobanking and
validated clinical assays.
Chipcytometry is an image cytometry platform that has previously
been used in biomedical research to perform immunophenotyping,
cell counting and numerous cell function assessments, such as
intracellular cytokine production, protein phosphorylation, cell
proliferation, apoptosis and tissue cytometry.
In this talk, we summarize current platform-independent practice in
developing, validating and implementing cytometry assays to
support clinical trials. Furthermore we present two case studies
(autoimmune disease, infectious disease) including the
implementation of sample preparation and sample biobanking to
evaluate cell-based biomarkers. Both image cytometry assays
underwent a clinical assay validation according to ICH guidelines
and typical CVs at the LLOQ were 10-15% (inter-assay) and 12 %
(inter-operator).
204/B67
Isolation of Antigen Specific CD8+ T cells Using the
MACSQuant Tyto: A Closed, Sterile, Single-Use
Microchip-Based Cell Sorter
Megan Ragland1, Steffen Walter2, Ivy Lai3, Cassian Yee3
1
Research and Development, Owl Biomedical, Goleta, CA,
United States, 2Chief Scientific Officer, Immatics US, Inc.,
Houston, TX, United States, 3Melanoma Medical Oncology,
The University of Texas MD Anderson Cancer Center,
With Adoptive T Cell Therapies (ACTs) for cancer becoming more
prevalent, the safe and simple isolation of candidate cells in a
clinical setting represents an important technical and regulatory
challenge. To address this and related cell sorting needs, we
developed the MACSQuant Tyto, a benchtop cytometer with a
closed, single-use cartridge built around a microchip sorting valve.
We used the MACSQuant Tyto to sort cancer antigen specific
cytotoxic T cells from two different sample preparations. In the first,
we performed exploratory work with antigen specific T cell lines.
We spiked one T cell line into another at frequency of .5% into a
background of 2 million cells. In the second, we sorted the desired
cells out of patient-derived peripheral blood mononuclear cells
which were stimulated before with the respective antigen. In this
case, sorted cells were transferred directly from the cartridge into
culture for in vitro expansion and autologous infusion in an
ongoing Phase II clinical trial with melanoma patients. Target
populations ranged from .2% - 1.5% targets out of a background of
800,000 – 2 million PBMCs. In both samples we labeled the
desired T cells with PE conjugated HLA tetramers. Under these
conditions, we repeatedly achieved purities 89% and yields
70%. The design features and sorting performance characteristics
of the MACSQuant Tyto are well-aligned with requirements of this
important therapeutic strategy and will be implemented in related
treatment protocols with a variety of other cancer related peptide
antigens.
205/B68
Stability Assessment of Various Flow Cytometry
Markers in Stabilized Whole Blood
Background: Multiparametric flow cytometry has become a tool of
choice given its ability to analyze cellular phenotypes and functions
in complex mixtures of cells such as in peripheral blood and
tissues. During drug development, particularly in the later stages,
clinical trials are designed to include multiple investigative sites
distributed globally requiring the need for specimens to be shipped
for centralized analysis. The primary challenge is the delay between
specimen collection and sample processing and analysis. Therefore,
the assessment of sample stability becomes a requirement during
assay development to assure that the data collected is comparable
to freshly isolated and analyzed samples. The data presented herein
reports on the stability assessment of various phenotypic markers
following blood collection in Cytochex® collection tubes.
Methods: Blood was collected from 3 volunteers and tested at
various time points following venipuncture. Our assessment
included various parameters such as time post-venipuncture,
storage temperature, clones and fluorophore/antibody pairs from
different providers. Main lineage cell subsets such as CD3, CD4,
CD8, Monocytes, NKT, and NK cells were analyzed along with
other molecules of immune activation and maturation.
Results: Table I shows the stability data obtained for a subset of the
markers when blood was stored at 2-8°C. Stability was calculated
in three different donors in triplicate by determining the average %
difference between the total frequencies detected over time
compared to day 0 (freshly analyzed blood samples). We show that
6/8 markers varied within 30% at Day 7 while some markers like
PD-1, ICOS CD69 and CD45RA were stable for up to 14 days postvenipuncture. CD62L, a homing marker for secondary lymphoid
tissue, was rapidly lost when blood was collected in Cytochex®
vacutainers.
Conclusions: Taken together, both the frequencies and fluorescence
intensities (data not shown) of the markers detected by flow
cytometry showed varying stability profiles suggesting that
Cytochex® blood collection during clinical trial testing is a viable
option for extending the stability of samples post-venipuncture.
However, robust testing is required to validate the selection of
clones and fluorochromes multiplexed in the panel.
Table I: Average percent differences (n = 3 donors) obtained in the
detection of the listed markers compared to freshly isolated
samples. Samples were store at 2-8°C and tested in triplicate.
D0
D3
D7
D14
D28
CD27
0.0
24.1
24.3
33.7
53.7
CD45RA
0.0
11.6
3.0
-0.6
-6.6
CD69
0.0
-15.9
-6.4
11.0
26.6
HLA-DR
0.0
-18.8
-6.4
14.2
41.5
ICOS
0.0
-1.0
11.7
19.8
31.7
PD-1
0.0
-1.9
7.0
18.1
30.8
TIM-3
0.0
-41.8
-26.7
-7.4
12.6
CD62L
0.0
38.9
42.2
N/Ap
N/Ap
206/B69
Oral Supplementation with Baker’s Yeast b Glucan is
Associated with Alterations in Granulocyte Function
following Strenuous Exercise in a General Population
Cohort
Erin Bowman1, Andrea Henning1, Adam Venable2, Brian
McFarlin1
Dominic Gagnon, Philippe Pouliot, Bader Yassine-Diab,
Rose-Marie Brito, Valerie Hebert, Audrey Gagnon, JeanFrancois Poulin, Yoav Peretz
Applied Physiology Laboratory, University of North Texas,
Denton, TX, United States, 2Denton
ImmuneCarta, Caprion Biosciences Inc., Montreal, QC,
Canada
Background: Strenuous exercise is associated with an increased the
risk of developing upper respiratory tract infections (URTI). Baker’s
yeast b 1,3/1,6 glucans (BG; Wellmune®), a known
immunomodulator, has been shown to reduce the risk of URTI after
180
1
Index
Speaker/Author
181
Poster Session
Abstracts
To comply with the clinical trial settings, ZellGate was validated by
virtual spiking of target cells (Th17, regulatory T and plasma cells)
into target cell-free PBMCs. Sensitivity and specificity are sufficient
to reliably gate less than 0.1% plasma cells. Benchmarking against
manual gating confirms that automated gating decreases the interassay and intra-assay variation.
Oral Session
Abstracts
ZellGate was developed specifically for image cytometry in clinical
study settings but can be easily adapted to other cytometry methods
(source code in R available upon request). The tool is integrated
into the ZellExplorer software and is executed automatically. First, a
set of tests is performed to detect outlier images and correct for
illumination differences between images. For binary markers,
staining quality for each biomarker is assessed based on Fisher’s
discrimination ratio. If the staining doesn’t fit one of the QC criteria,
the user gets immediate detailed feedback on the detected
problems, otherwise ZellGate automatically proceeds with the
gating according to a hierarchical gating template that was defined
prior to the begin of the study. The algorithm assumes that each cell
population consists of negative and positive cells, and that their
fluorescence value distributions can be approximated by Gaussian
distributions. Thus, a Gaussian mixture model is fitted based on a
set of characteristics of the kernel density estimate of the
fluorescence values. Depending on the anticipated biomarker
expression of the target population, the algorithm uses the standard
deviations either of the positive or of the negative cell populations
to set the gates.
Commercial
Tutorials &
Exhibits
More and more clustering and visualization algorithms are
proposed to detect the cell types that are present in flow cytometry
samples. However, the interpretation of the resulting clusters can be
challenging. For example, in the SPADE algorithm, you need to
look at each node and compare plots for all the different markers to
determine the marker presence on the cells represented by the node
and convert this to a cell type. In FlowSOM, we combine
information about the different markers in a “star”-glyph for an
easier overview, but you still need to interpret the marker
combinations for each node. However, when conducting an
experiment, the researcher often has already some cell types in
mind, defined by marker properties such as “CD4 T cells are CD3
While a number of computational gating algorithms are available,
they are rarely used routinely in clinical studies. Possible reasons
are the lack of an easy-to-use graphical user interface, the need for
supervision and difficulties to gate rare cell populations.
Additionally, to our knowledge none of the existing algorithms has
been validated for its sensitivity and specificity in real-world
clinical study settings.
Scholars &
Emerging
Leaders
1
Department of Information Technology, Ghent University,
iMinds, Ghent, Belgium, 2Data Mining and Modelling for
Biomedicine group, VIB Inflammation Research Center,
Ghent, Belgium, 3Unit of Immunoregulation and Mucosal
Immunology, VIB Inflammation Research Center, Ghent,
Belgium, 4Department of Internal Medicine, Ghent
University, Ghent, Belgium, 5Department of Biomedical
Molecular Biology, Ghent University, Ghent, Belgium
In clinical trials (phase I-III), often deep phenotyping is necessary to
identify a number of different target populations, be it to assess drug
efficacy and safety or to stratify patients. To generate such data, an
increasing number of biomarkers has to be combined per sample to
identify and measure the population size of the desired target
populations. High-dimensional cytometry methods allow to record
the expression of 50 markers and more on a single-cell level, and
are thus very well suited for this purpose. However among others,
the level of precision that is required by regulatory bodies is
difficult to achieve due the bias that is introduced by manual
gating. Furthermore, manual quality control and manual gating of
high-dimensional cytometry data are extremely time-consuming.
Poster
Session
Sofie Van Gassen1,2, Mary J Van Helden3,4, Martin
Guilliams3,5, Tom Dhaene1, Yvan Saeys2,4
Zellkraftwerk GmbH, Hannover, Germany
Wednesday
15 June
207/B70
Automated Cell Type Annotation
Anja Mirenska, Christian Hennig
Tuesday
14 June
Computation and Informatics (B70 – B83)
208/B71
ZellGate: Automated Quality Control and Gating of
High-Dimensional Cytometry Data in Clinical
Validation Settings
Monday
13 June
Funding Disclosure: This project was funded in part by a grant from
Kerry to the University of North Texas. The authors were not
directly compensated for their completion of this project.
We tested this method on a flow cytometry dataset with several
stainings. For example, in a lymphocyte staining, B cells, CD4 T
cells, CD8 T cells, NK cells and gamma delta T cells were
successfully automatically identified. Additionally, we also used
this method on a separate experiment to detect conventional
dendritic cells type 1 and 2 across tissues.
Sunday
12 June
Discussion: This study demonstrates that image-based flow
cytometry may be used to detect improvements in granulocyte
function. Additionally, this is the first report that BG may modulate
granulocyte function following a stressful bout of exercise. Such a
change would be consistent with a reduced “open window”
response and overall reduction in infection risk following strenuous
exercise.
Saturday
11 June
Results: BG supplementation was associated with a modulation of
granulocyte activity during recovery from strenuous exercise
compared to placebo. Shifts were observed in both “moderate” and
“high” active granulocyte subsets.
We developed a scoring function which can automatically test for
each node how well it corresponds with the proposed definitions,
by comparing the node’s MFI’s with the highest or lowest values
present for the markers. By selecting only the nodes which get the
highest scores, an overview figure can be presented indicating all
probable cell types.
Special
Lectures
Methods: A subset of 36 healthy, non-obese, sedentary individuals
from a larger exercise-induced-stress study of the effects of BG
supplementation on the immune system (double blind, placebo
controlled, crossover design) were assessed for changes in
granulocyte function. Briefly, venous blood samples were collected
in heparin tubes from volunteers prior to supplementation (BASE),
after 10 days of supplementation before exercise (PRE), immediately
post exercise (POST), and 2 and 4-h post exercise (2H, 4H). Whole
blood was incubated with pHrodo-Red conjugated S.aureus
bioparticles at 37°C before DHE was added. Image-based flow
cytometry (Amnis FlowSight) was used to assess two measures of
granulocyte function, phagocytosis and oxidative burst, on
CD66b+/CD45- cells. Subsets of activated granulocytes were
identified by comparing bright detail intensity of phagocytosis
(pHrodo-Red+) vs. bright detail intensity of oxidative burst (DHE+).
high, CD4 high and CD8 low”. Instead of using this knowledge to
identify the cell type for each node, we propose to let the
researcher define these cell type definitions in advance. By
automatically matching nodes to these definitions, it becomes
much quicker to interpret the FlowSOM tree, while you can still
easily detect unexpected marker combinations and cell types.
Congress
Overview
marathon running. We have previously reported modulation of
immune system proteins and circulating leukocyte concentrations
after strenuous exercise in athletes supplemented with BG. Preclinical research on BG mechanism of action suggests that
granulocyte function may also be modulated by supplementation.
Historically, assays to measure granulocyte function have been
developed to identify major deficits in granulocyte function but not
to detect improvement. Our laboratory has previously developed a
novel image-based flow cytometry assay that allows for detection of
improvements in granulocyte function in healthy human subjects.
The purpose of the study was to use this novel assay to determine if
granulocyte function is modulated in response to BG
supplementation in healthy sedentary human subjects after
strenuous exercise.
Congress
Overview
209/B72
Characterizing Cell Subsets in Heterogeneous Tissues
Using Marker Enrichment Modeling
Index
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Scholars &
Emerging
Leaders
Poster
Session
Wednesday
15 June
Tuesday
14 June
Monday
13 June
Sunday
12 June
Saturday
11 June
Special
Lectures
Kirsten Diggins, Jonathan Michael Irish
Vanderbilt University, Nashville, TN, United States
Background: Advances in single cell biology have enabled
measurement of 35 or more features on millions of single cells.
Analysis of single cell data typically has focused on grouping cells
into populations, either by manual gating or computational
clustering, followed by evaluation of measured features using
median expression values. This strategy works well when feature
variance within clustered populations is low. However, in analysis
of phenotypically diverse cells from primary tissues, medians tend
to over-represent the contribution of highly expressed features and
overlook features that are highly enriched but lower in expression.
This issue is an acute problem in research and clinical fields
involving heterogeneous samples, including cell type discovery and
characterization, disease diagnosis, risk stratification, expression
profiling and biomarker identification, cell sorting, and drug
response testing.
Methods: Marker Enrichment Modeling (MEM) is a metric that
quantifies context-specific feature enrichment on cell subpopulations. MEM provides an enrichment score for each feature
and combines these into a descriptive, unbiased label for each
subset. MEM accounts for both feature magnitude and feature
variance between and within populations and assigns high values
to markers that are specifically enriched on a given subset
compared to others. An additional advantage of MEM is that scores
can be positive or negative, where positive enrichment indicates
population specific expression and negative enrichment indicates a
specific lack of expression on that subset.
Results: Four datasets were analyzed to highlight specific use cases
of MEM. Three of the four datasets, including a bone marrow
CyTOF dataset from Bendall et al., a murine myeloid CyTOF
dataset from Becher et al., and a fluorescent phospho-flow AML
dataset from Irish et al, were published and had been studied
previously using expert and computational analysis. MEM analysis
provided an automatic label that effectively quantified all
populations from each of these studies, including novel biologically
and clinically relevant cells. MEM assigned high scores to
canonical markers on normal human blood and bone marrow cell
subsets and assigned low scores to markers that were not enriched
on any specific population. MEM analysis of the murine myeloid
dataset identified markers that were enriched on formerly
overlooked immune cells from mice. Finally, MEM highlighted
intracellular signaling patterns that were enriched on response
groups of AML patients.
Conclusions: MEM accurately quantified the features that best
distinguished biologically relevant cell subsets and AML patient
response groups. MEM is an alternative metric to median that can
be applied immediately in cell biology to quantify context-specific
feature enrichment in areas including machine learning of cell
identity in healthy and diseased tissues, biomarker discovery, and
clinical monitoring.
210/B73
High Dimensional Cytometry Data Visualization
Using Parallel Coordinates
Jeffrey Hokanson1, Chris Wakefield2, John Weinstein2,
Michael Andreeff1, Jared Burks3
1
Department of Leukemia, MD Anderson Cancer Center,
Houston, TX, United States, 2Department of Bioinformatics
and Computational Biology, MD Anderson Cancer Center,
Houston, TX, United States, 3Flow Cytometry & Cell
Imaging Core Facility, MD Anderson Cancer Center,
populations of interest. With the increasing number of channels
available with modern fluorescent and mass cytometers, this
manual gating process has become time consuming. Identifying a
population now requires viewing dozens of biaxial plots and
correctly placing gates in each. Recent work has focused on
reducing this burden using two different techniques: automated
clustering and new visualizations. Clustering algorithms
automatically assign each event in a flow cytometry experiment to
one of a small number of classes which are then labeled and
analyzed. This approach eliminates the potential hazards inherent
in manual gating, improving repeatability and reducing biases.
However, the loss of information inherent in this approach makes it
difficult for the user to identify mistaken clustering of distinct cell
types. The alternative data analysis approach, adopted here, is to
present a better visualization for human analysis. We use an
interactive parallel coordinates plot that concisely displays all of the
features of the data with minimal loss of information. A parallel
coordinate plot displays the coordinates of data as parallel axes;
then, each event is shown as a series of line segments connecting
each coordinate axis at the value taken by that event. Gates can
then be interactively drawn on each axis and only those events
remaining in all the gates will be shown. This permits the user to
identify high-dimensional structure in the data without having to
examine dozens of biaxial plots. Moreover unlike SPADE and
viSNE, the original, high-dimensional data is displayed, aiding in
interpretation. This new visualization has the potential to speed the
manual analysis of flow cytometry data while returning control to
the user and preserving the nuances of the original data set.
211/B74
A New Statistical Agglomerative Clustering
Algorithm for High Dimensional Single-Cell Data
Andrei Terekidi, Christina Loh, Olga Ornatsky, Vladimir
Baranov
Fluidigm Canada Inc., Markham, ON, Canada
A typical mass cytometry experiment includes multiparametric
antibody panel, metal containing stains, internal bead-based
standards, and barcoding reagents. The complexity of the dataset
represents a significant challenge for supervised analysis, as well as
for automatic hierarchical clustering that is looking for similarities
in the set. Mass cytometry transforms the common goal of grouping
cells into cell populations into a complicated task of separation of
barcoded samples, application of the internal standards and
clustering of each sample into groups, which can be compared and
quantified. Mass cytometry data sets elevate requirements for
computational methods such as SPADE, viSNE, ACCENSE, and
others by putting emphasis on reproducibility, robustness, and
transparency for biologists. We are developing a complete solution
from raw instrument data to biological insight to diminish tension
between the desire to use enabling cutting-edge technology and the
need to understand the resulting data.
The approach, which will be presented in this work uses a new
induction principle. From the most well-known algorithms, our
method diverges by employing a combination of agglomerative and
divisive hierarchical clustering. Formation of clusters is performed
by merging of preliminary subclusters. In opposite to agglomerative
method, where each object initially represents a cluster of its own,
our method assumes a certain statistical analysis of the entire
system to establish the most optimal size (radius in
multidimensional space with Euclidian metrics) of the initial
subclusters. Thus, it does not require the number of clusters and
their size to be pre-set by the user, as in typical partitioning
methods. The merging is provided by the iterative procedure, which
lasts until the cluster structure becomes unaltered. On each iterative
step, the merging of the subclusters is performed according to some
similarity and connectivity measures (parameters). These
parameters are chosen on the basis of statistical analysis and
represent a numerical measure of the notion of "clustering". In other
words, it brings some objective criteria to consider subclusters as a
group forming a cluster. Note that our method is particularly
applicable for any data sets containing chain-like and concentric
clusters when the manual gating is impossible.
Traditionally, flow cytometry has analyzed data by creating a
hierarchy of manual gates on biaxial dot plots to identify
182
John Quinn, Jay Almarode, Mike Stadnisky
Flowjo, LLC, Ashland, OR, United States
Poster Session
Abstracts
Index
Speaker/Author
183
Oral Session
Abstracts
The second, ‘per-cluster’ registration method seeks to register all
clusters with the corresponding target sample clusters, without
linking nearby clusters. This provides more complete registration
for all clusters, and is useful for removing individual sample
variation (e.g. genetic variation) in cluster positions, to improve the
accuracy of measuring the number of cells per cluster in each
sample. However, cluster-specific registration may remove real
biological differences between samples in the fluorescence
intensity of particular populations. Thus the optimal amount of
registration (i.e. reduction of variability in fluorescence intensity)
Commercial
Tutorials &
Exhibits
Methodology: The verification tool allows the user to specify the
number of events, number and density of clusters and parameter
counts and titrated events and parameters between 10K-50K and
10-210, respectively. We extended this analysis to high-parameter
flow and mass cytometry data as well as next-gen sequencing data
with known populations. Each dataset was run through different
configuration settings of the algorithm for batch verification which
allowed us to determine optimal settings for tSNE on both synthetic
and biological data sets, increasing the efficiency of initiation.
Both methods start with a cluster template generated by the SWIFT
clustering algorithm, and use information from all clusters. No
operator intervention is required, thus maintaining objectivity. The
'per-channel' registration method compares cluster locations in the
target and the sample to be normalized, and seeks to reconcile
'global' differences between samples while maintaining local
fluorescence intensity differences. The global normalization is
particularly useful for correcting global artifacts introduced e.g. by
variations in staining or cytometer settings.
Scholars &
Emerging
Leaders
While this algorithm has been increasingly adopted in the
community due to its retention of event-level data and representing
n-dimensions in two dimensions, challenges remain. Less effort has
been made towards addressing (1) verification of tSNE algorithms
by comparing its results with synthetic data to ensure the cell
populations known to be distinct are correctly separated and (2)
validation of the tSNE approach applied on biological data to show
that the algorithm identifies known cell populations. For
verification, we designed a tool to create synthetic data sets that
contained pre-defined clusters, and then used the F-Measure
statistic to verify that each continent in tSNE space mapped to a
known cluster. To perform validation of the tSNE calculations, we
used biological datasets and compared tSNE clusters with known
populations.
Technical, machine, and true biological variation in flow cytometry
data can confuse classification of similar populations across
samples for both automated and manual modes of analysis. Flow
cytometry studies of sufficient size or complexity will contain levels
of uncontrolled variability that manifest as changes in fluorescence
intensity. This variability normally requires expert analysts to
inspect all samples on a case-by-case basis, and using either purely
manual or semi-automated tools, reduce variability to improve
subsequent statistical analysis. Previous approaches have
attempted to treat the problem on a per-channel ‘global’, or perpopulation ‘local’ basis, but both rely on targeting specific features
(histogram peaks or valleys) of manually gated data. Because it is
critically important to maintain objectivity as long as possible
during the analysis of flow cytometry data, methods that could
register cluster positions without manual intervention would be
very useful. Therefore, we propose two complementary registration
methods that rely on an objective, data-derived Gaussian model
description of the full unimodal semantics of the multidimensional
data space.
Poster
Session
High-parameter single cell data from single cell sequencing,
transcriptomics, flow and mass cytometry has challenged
conventional visualization and analysis methods. To analyze this
data, dimensionality reduction techniques (PCA, SPADE, and tSNE)
are used; the trade-offs for increased insight include determinism,
computational efficiency, retaining event-level data, and to date - a
high-quality, verified, and validated approach to tSNE (tDistribution stochastic neighbor embedding (tSNE).
1
Center for Vaccine Biology and Immunology, University of
Rochester, Rochester, NY, United States, 2Electrical and
Computer Engineering, University of Rochester, Rochester,
NY, United States, 3Center for Vaccine Biology and
Immunology, Univ of Rochester, Rochester, NY, United
States
Wednesday
15 June
FlowJo, LLC, Ashland, OR, United States
Jonathan Rebhahn1, Sally Quataert1, Gaurav Sharma2,
Tim Mosmann3
Tuesday
14 June
Shahid Siddiq, Jay Almarode, Michael Golden, Mike
Stadnisky
214/B77
Fully Automated Fluorescence Intensity
Normalization (Cluster Registration) for Flow
Cytometry Data to Minimize Batch Effects and
Optionally Remove Variation in Individual Cluster
Locations
Monday
13 June
213/B76
Verification and Validation of tSNE Results
We also validate that the cell populations produced by the tSNE
algorithm are the same as identified by a biologist and show that
using our validation technique we can map human-defined
populations to continents.
Sunday
12 June
Herein, we describe the development and linking to FlowJo of a
NoSQL database for rapid derivative data exploration in
conjunction with an analysis pipeline centered around the server
version of FlowJo, the most cited third-party cytometric analysis
software package. The pipeline consists of data collection on a
cytometer, direct uploading of the data to a server via 4th Wall
software, automated analysis through FlowJo Enterprise, and direct
database updating using a Java plugin. FlowJo version 10 supports a
plugin architecture that can be used to create custom functionality,
including database interaction. The plugin we have created extracts
a specified set of statistics from the FlowJo workspace XML and
populates the database with values and categorical information.
Data were then able to be queried via a front end database
visualization tool, run through a browser.
Results: Our results show that the tSNE algorithm can be verified to
identify correctly known clusters in a statistically measureable way.
Using a rigorous and reproducible approach, we were able to
quantify the precision and recall of the tSNE algorithm across a
wide range of cluster types and algorithm settings.
Saturday
11 June
Cytometric analyses continue to grow in both complexity and
nuance, and increasingly rely on a database backend to explore
and curate data. Databases provide the ability to store processed
information in a manner that easily organized, sorted, and updated.
This is a critical resource for larger data analysis operations. Tying
this capability to frontend flow cytometry analysis software such as
FlowJo is important for streamlining and automating the analysis
pipeline to lead to fewer manual errors and a faster turnaround for
turning data into discovery.
To validate with biological data, we analyzed datasets using
traditional gating techniques and we ran tSNE several times with
different settings of the algorithm. Finally, we compared the results
of the traditional gating with the results of tSNE, using comparison
statistics and visualizations.
Special
Lectures
212/B75
Data to Discovery: A ‘Nuts-and-Bolts’ level
Discussion on Linking Flow Analysis to a Database
Finally, we calculated F-Measure statistics to quantify how well the
tSNE algorithm identified known clusters.
Congress
Overview
We will represent results of analysis of PBMC samples from 20
different donors barcoded and pooled in one vial for
immunostaining. This representative set will be analyzed and
compared with manual gating using transparent statistical criterion.
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
may depend on the experimental design, and so we provide the
option of blending both methods such that at every step each
method is given a user-specified weight. This method can
substantially remove variations in cluster channel medians between
batches of samples, and can optionally remove even individual
differences between clusters, if cluster membership numbers are the
main desired experimental outcome.
215/B78
CyteGeist: An Open-Source Project for Integration,
Visualization, and Collaborative Analysis of Diverse
Biomedical Data
Adam Treister1, Anja Mirenska2, Christian Hennig3
1
CyteGeist, Ashland, OR, United States, 2ZellKraftWerk,
GmbH, Hannover, Germany, 3ZellKraftWerk, GmbH
Several different technologies are often applied to investigate
components of a biological study. Because the analysis software
generally focuses on a single technology and data format, the
integration of the pieces is outside the scope of any analysis
software and left to the scientist to combine. Moreover, the lab is
left responsible for the management and administration of
increasingly large data sets of raw and digested experimental
information.
Business applications have learned to appreciate the Data
Warehouse as a tool to pre-process data sets to normalize and
rationalize data before analysis. They can provide a blueprint of an
analogous toolbox for scientists. A flexible MongoDB repository
supports varying data sets easily, and a XML-based Experiment
Description Language (EDL) provides the structured description of
methods and objects. The analysis architecture includes a
Cytometry-domain Specific Language (CSL) to simplify the
specification of common operations (e.g., gating, graphing, and
statistics) and embed ontological and analytical elements within
HTML5 documents.
This open-source and extensible platform contains the following
components:
Index
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Scholars &
Emerging
Leaders
Poster
Session
A tool that helps to perform literature search, save and share the
results in a structured, searchable way;
216/B79
Automated Comparison of 200 Surface Marker
Stainings to Identify Markers Specific for Memory B
Lymphocytes by Flow Cytometric Screening Assay
Hyun-Dong Chang, Rene Riedel, Stefan Kröger, Ulrik
Stervbo, Andreas Radbruch
DRFZ Berlin, Berlin, Germany
In mouse, the identification of antigen-experienced memory B cells
is done by staining the class-switched B cell receptor or by staining
of the B cells with the fluorescently labelled cognate antigen.
However, these markers cannot discriminate recently activated B
cells and resting memory B cells and also do not a possible
heterogeneity among memory B cells. In order to identify novel
discriminating surface markers on memory B cells, we screened for
the expression of more than 200 surface markers by staining with
phycoerythrin (PE)-conjugated antibodies. We compared the
expression of these markers on splenic naï ve and memory B cells
identified according to IgG2b surface expression. To allow for the
automated identification of differentially expressed markers
between naï ve and memory B cells, we devised a composite index
for the evaluation of the degree of similarity between two sets of
frequency distributions of PE fluorescence signals. This index allows
the unbiased ranking of the distribution of the different markers
between our two populations of cells based on the degree of
similarity integrating mean fluorescence values and the range of a
frequency distribution. The composite similarity index was
developed by evaluation of a test set of markers, which based on
previous measurements were known to be or not be differentially
expressed between naive and memory B cells. The similarity index
ranges from 0 to 1, where 1 denotes a high degree of similarity and
a score converging 0 indicates high dissimilarity. The automated
comparison between memory and naive B cells identified the
differential expression of several surface molecules, e.g. CD80 or
CD73, and represents a novel method for the identification of
differential marker expression in comparative screening studies.
217/B80
The Characterization of Receptor Density on Human
Blood Cells
A lightweight, extensible ontology-style data structure that describes
biological and computational methods, objects and relations in a
machine-operable syntax;
Robert Balderas
A data uploader and converter;
The lack of data describing the characterization of cell surface
antigen density, as well as, the expression profiles of cell surface
molecules on the surface of terminally differentiated blood cells has
posed a number of challenges for the development of multi-color
flow panels. With the expansion of new colors and the
development of flow cytometers with greater than 25 fluorescence
parameters, additional information on co-expression of receptors
and receptor density will provide a much needed parameter in flow
cytometry. Today we will focus on a study to enable a detailed
characterization of receptor expression on multiple subsets of T
cells, B cells and non T/B cell populations. A detailed analysis or T
cells, T regulatory cells and B cell populations will be presented.
This presentation will also describe a technique for antigen receptor
characterization and a study outline for the characterization of
more than 400 surface receptors. Using information from this
detailed study, including the parameters of receptor co-expression
and receptor density, we will construct a high dimensional flow
cytometry panel by matching a corresponding fluorochrome with
each specific antibody.
Data analysis tools for single sample and comparative sample
analysis;
An image server that stores, processes and serves image data;
A structured text editor to facilitate individual and collaborative
paper writing and embedding domain objects into documents;
A web publisher to make data available to collaborators and to the
public.
Additional services and modules are envisioned, based on feedback
from the community.
This study is based on a use case that integrates genomic data and
cytometry data from a hematological research project about
neutrophil differentiation demonstrating that integration and
visualization of different life science data sources from the same
sample improves our understanding of complex disease processes
that are barely explained by just the genomic view or the cytomic
view.
184
BD Biosciences, San Jose, CA, United States
Software Development, Verity Software House Inc,
Topsham, ME, United States
Elisa Nemes1, Wendy Burgers1, Catherine Riou1, Erica
Andersen-Nissen2, Guido Ferrari3, Clive Gray1, Thomas
Scriba1
1
Pathology, University of Cape Town, Cape Town, South
Africa, 2Cape Town HVTN Immunology Laboratory,
Hutchinson Centre Research Institute of South Africa, Cape
Town, South Africa, 3Surgery, Duke University, Durham,
NC, United States
Index
185
Speaker/Author
The main goal of the 5-day workshop is to provide training in flow
cytometry through lectures and hands-on sessions on the following
topics: basic principles; instrument set-up, optimization and quality
Poster Session
Abstracts
Africa is particularly affected by infectious diseases. Improvement
of regional capacity to design, conduct and interpret clinical studies
is urgently needed. To enhance technical and theoretical
knowledge of African scientists, and in recognition of lack of local
training opportunities, the first ‘Intracellular Cytokine Staining’
workshop was held in 2005 and then bi-annually in South Africa.
The aim of the workshop is to build scientific and technical literacy
about measurement of immune responses to infectious diseases, to
ultimately empower a new generation of African researchers to
apply cutting edge technology and locally train their peers.
Oral Session
Abstracts
221/B84
Teaching Advanced Flow Cytometry in Africa:
Lessons Learned over the Last 10 Years
Commercial
Tutorials &
Exhibits
The primary problem with log transforms is that they are not
defined for signals that are less than or equal to zero due to the 1/x
slope not being defined at x=0. Unfortunately, most signal unmixing and base-line restore algorithms can create zero or less than
zero numbers. Truncating these negative signals to the first channel
of display systems has created numerous issues for cytometry - the
worst being a strong tendency to over compensate data. In 2002
Parks and Moore described a generalized hyperbolic sine function
that was log-like at higher signal intensities and linear-like through
the origin. Since then, there have been a number of other log-like
transforms that have been published or described in earlier
literature. Many of these transforms are quite complex and present
challenges to software implementers.
Cytometry in Resource Poor Settings (B84)
Scholars &
Emerging
Leaders
Typically, the magnitude of a measurement’s uncertainty is
proportion to the magnitude of the signal. The proportionality
constant that relates uncertainties to signal magnitudes is coefficient
of variation or CV and is given by the simple equation, SD = CV*x
where x is the magnitude of the measurement and SD is its
uncertainty or standard deviation. Because biologic signals have
ranges that span four to five decades, the uncertainties of
population measurements also span many decades. If you wanted
to design a seemingly perfect transformation for cytometry where
the linearly increasing SDs would be converted to uniform SDs, you
would find a function whose slope at each x value would be 1/x.
This function is the well-known log function, z=log(x), and has
been routinely employed for many years to enable the visualization
of cytometrically-derived cellular populations.
We demonstrate the properties of a number of different tests for
unimodality on synthetic data as well as real flow cytometry data,
gated by eight different analysts, from the FlowCAP I challenge.
Poster
Session
C. Bruce Bagwell
This study attacks the problem of determining when a group of
measurements should be split into two or more cell populations.
Due to the various possible shapes of flow cytometry cell
populations it is in general not possible to assume that a population
has a given distribution, e.g. Gaussian. But we can use the single
assumption that the density of a cell population is unimodal and
apply non-parametric tests from the rich statistics literature on
unimodality to find out how confident we should be that a density
is multimodal and thus must comprise multiple cell populations.
Wednesday
15 June
219/B82
Sometimes Simpler Is Better: An Easy-to-Implement
Log-Like Transform for Cytometry
Arguably, the biggest problem with manual gating of flow
cytometry data is its inherent subjectivity. Automated gated
algorithms can remove this subjectivity — but when multiple sets of
gates are plausible, decisions are made in a more or less arbitrary
manner as determined by the algorithm. There is a striking need for
statistically grounded measures which can describe the confidence
in a given partition of cells into cell populations. Such measures
could be used in conjunction with manual gating to enable
informed gating decisions and consistent gating strategies. In
conjunction with automated gating such measures could be a
powerful tool for quality control.
Tuesday
14 June
The advantages of modeling systems like PSM are 1) scalability, 2)
accuracy, 3) reproducibility, and 4) automation. A new forcedirected graphics system summarizes much of the information in
the model and thereby provides an informative and reproducible
method for evaluating the results.
1
Centre for Mathematical Sciences, Lund University, Lund,
Sweden, 2Centre de Bioinformatique, Biostatistique et
Biologie Intégrative, Institut Pasteur, Paris, France
Monday
13 June
This study uses the technique of probability state modeling (PSM) to
automatically model and extract information from PBMC samples.
The major cell types defined in the model are 1) CD8 T cells, 2)
CD4 T cells, 3) Gamma-Delta T cells, 4) B cells, 5) NK cells, 6)
Monocytes, 7) Dendritic cells, 8) Basophils, and 9) Granulocytes.
Within many of these cell types, populations are sub-classified into
stages or categories and within each of these categories additional
populations are defined with the TriCOM combinatory analysis
system such as TH1, TH2, TH17, NKT, TReg, and others. These
population classifications are also compared with other published
approaches such as SPADE and viSNE.
Kerstin Johnsson1, Magnus Fontes1,2
Sunday
12 June
Analyzing data that comprises 48 correlated measurements on cells
presents major issues for classical gating analysis systems. The
number of two-dimensional measurement plots necessary to
investigate all correlations is as high as 2,256 – a number that may
require investigators to consider different analysis paradigms to
extract useful information from the sample.
220/B83
Statistical Tests of Unimodality Supporting Difficult
Gating Decisions
Saturday
11 June
1
Software Development, Verity Software House Inc,
Topsham, ME, United States, 2ITI_HIMC, Stanford
University School of Medicine, Stanford, CA, United States,
3
ITI-HIMC, Stanford University, Stanford, CA, United States,
4
VP Cellular Proteomics, Applied Markers, Fluidigm, South
San Francisco, CA, United States
Special
Lectures
C. Bruce Bagwell1, Holden Maecker2, Michael Leipold3,
Gregory Stelzer4
This study examines a simple log-like transform that is very easy to
implement and that has the desirable attributes of being log-like at
higher intensities and linear-like through the origin. The arguments
to the transform are 1) number of desired decades, 2) a scaleindependent coefficient that determines the slope through zero, 3)
desired maximum transform range, and 4) the maximum linear
range. Implementers are free to use and modify this transform if
they don’t have access to other more complex transformation
systems.
Congress
Overview
218/B81
High-Dimensional Modeling of Peripheral Blood
Mononuclear Cells from a Helios Instrument
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
control; experiment design and optimization; data visualization,
analysis and interpretation.
Applicants based in Africa are selected through an international
peer-review process, based on the following criteria: scientific
merit, training needs (at both individual and institutional levels) and
impact on career advancement. All selected participants receive an
award to cover all expenses. Learning is assessed by initial and final
exams and daily homework assignments, corrected during highly
interactive sessions. Participants provide immediate feedback about
the workshop, to ensure continuous improvement. Workshop’s
impact on participants’ work is evaluated by surveys over time.
Scientists based within and outside Africa form the organizing
committee and faculty team. Funding has been secured through a
range of sources (educational grants, academic funding, private
foundations and vendors).
During the last 10 years, over 70 scientists based in more than 15
different African countries have been trained. Over time, some of
the more experienced participants joined the faculty team, whose
members were mostly Africa-based in 2015. Participants leave the
workshop with a better knowledge of current techniques (two fold
increase in pre- to post-assessment scores in 2015), teaching
materials to train their peers and, importantly, a network of regional
and international contacts.
Due to funding limitations and the practical nature of the
interactive workshop, participant selection is highly competitive:
only 25% of applications were selected in 2015. This suggests that
there is demand for advanced flow cytometry training in Africa.
Most applicants cannot afford or do not have access to alternative
training opportunities. This unmet need for flow cytometry training
in Africa could be fulfilled by additional, free-of-charge, frontal and
E-learning-based courses.
In light of the increasing needs and efforts to conduct clinical
research in Africa, we believe that these workshops serve not only
to enhance local African scientific capacity, but also contribute to
the global health agenda.
Diagnostics (B85 – B90)
222/B85
Macrophages Gold Nanoparticle Uptake Analysis by
Image and Flow Cytometry
Attila Tarnok1, Susanne Melzer1, Rinat Ankri2, Dror
Fixler2
Index
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Scholars &
Emerging
Leaders
1
Heart Center Leipzig, Leipzig, Germany, 2Bar Ilan
University, Ramat-Gan, Israel
Background: In atherosclerosis stable and vulnerable
atherosclerotic plaque types are distinguished that behave
differently concerning rupture, thrombosis and clinical events. The
stable are rich in M2 macrophages. The unstable are rich in
inflammatory M1 macrophages and are highly susceptible to
rupture, setting patients at risk for thrombotic events when they
undergo invasive diagnosis such as coronary angiography.
Therefore, novel approaches for non-invasive detection and
classification of vulnerable plaques in vivo are needed. Whereas
classical approaches fail to differentiate between both plaque types,
a new biophotonic method (combination of the diffusion reflection
(DR) method with flow cytometry (FCM) or image cytometry (IC)) to
analyze gold nanoparticle (GNP) loading of plaques could
overcome this limitation.
Methods: Two types of GNP were used three variants of gold
nanorods (GNRI with 40x18nm, II 65x25nm and III 52x13nm in
size) and gold nanospheres (GNS with an average diameter of 18.5
nm). The GNS had an absorption peak at 520 nm and the GNR at
630 nm. Monocytes were isolated from human buffy blood
samples, differentiated into macrophages and their subtypes and
labelled with GNR and GNS for 3 and 24h. GNS and GNR loading
were determined by FCM and/or IC. Macrophages within tissue-like
phantoms were analyzed by the DR system.
Results: After GNR labelling of macrophages the FCM light scatter
values increased up to 3.7 fold and the DR slope changed from an
186
average slope of 0.196 (macrophages only) to an average slope of
0.827 (macrophages labelled with GNR). But, GNRIII did not
present much higher DR slopes than the control phantoms,
indicating that macrophages take up GNRIII in a lower amount than
GNRI or II. IC and microscopy showed that all particle variants
were taken up by the cells in a heterogeneous fashion.
Conclusion: The combination of FCM and DR measurements
provides a potential novel, highly sensitive and non-invasive
method for the identification of atherosclerotic vulnerable plaques,
aimed to develop a potential tool for in vivo tracking. Further
experiments will show, if different macrophage subtypes (M1 or
M2) take up the particles differently and may thereby serve to
distinguish stable from vulnerable plaques.
223/B86
Epithelial and Mesenchymal Circulating Tumor Cells
Isolated from Breast Cancer Patients and Injected at
High Cell Dose Are Not Tumorigenic in NOD-scid
Il2rȖnull Mice
Albert Donnenberg1,2, Per Basse3, Vera Donnenberg4
1
Medicine, University of Pittsburgh School of Medicine,
Pittsburgh, PA, United States, 2McGowan Institute for
Regenerative Medicine, Pittsburgh, PA, United States,
3
Immunology, University of Pittsburgh Cancer Institute,
Pittsburgh, PA, United States, 4Cardiothoracic Surgery,
United States
The quantitative evaluation of circulating EpCAM+ tumor cells
(CTCs) in the peripheral blood of breast cancer patients provides an
independent predictor of risk of progression in patients with
metastatic disease. Here we investigated the tumorigenic potential
of CTCs from cryopreserved mobilized leukapheresis products
obtained from three metastatic breast cancer patients in remission.
Cells were immunomagnetically separated if they expressed either
the epithelial cell surface marker EpCAM, or CD90, a mesenchymal
stromal cell marker associated with tumorigenic stem-like cancer
cells. The numbers of cells injected are shown (Figure). Cells were
injected into the mammary fat pads of NOD-scid Il2rȖnull mice. The
injection of very large numbers of CTCs (0.3 - 1.5 x 106 CTCs per
site, 20 sites per sample) in an optimized xenograft model did not
result in the establishment of human-derived tumor xenografts. Four
orders of magnitude fewer cells of the same CD90+ phenotype, but
obtained from metastatic breast cancer pleural effusions, were
highly tumorigenic in the same model system. These results favor
the interpretation that circulating tumor cell load does not directly
bear on metastatic potential, and that tumorigenic circulating breast
cancer cells in patients with metastatic breast cancer are
exceedingly rare. Further, the CD44+/CD90+ phenotypic signature
indicative of tumorigenicity in cells separated from metastatic or
primary breast tumors does not have the same significance in
circulating tumor cells. We speculate that the tumor cell niche
itself, and particularly tumor associated macrophages, play an
important role in determining which cells are tumorigenic in the
natural host, and which survive to form tumors in xenograft models.
224/B87
Utilization of Imaging Flow Cytometry for the
Identification of Rare Spermatozoa in Patients with
Non-obstructive Azoospermia
Melissa Filice1, Andree Gauthier-Fisher1, Kirah Hahn1,
Sonja Swanson1, Kirk Lo2, Sergey Moskovtsev1,3, Clifford
Librach1,3
1
Create Fertility Centre, Toronto, ON, Canada, 2Mount Sinai
Hospital, Toronto, ON, Canada, 3Department of Obstetrics
and Gynecology, University of Toronto, Toronto, ON,
Canada
R&D, MilliporeSigma, Hayward, CA, United States
Index
187
Speaker/Author
Clinical diagnostics requires access to simplified instrumentation
and technologies that can provide test results on complex cellular
samples with minimal user intervention or expertise but still provide
high reproducibility and accuracy. The Muse® Auto CD4/CD4%
System is a low cost, small footprint cell analyzer, based on
microcapillary cytometry that uses guided touchscreens to provide
CD4 T cell count and CD4% of lymphocytes results from 10 μL of
Poster Session
Abstracts
Julie Clor, Patrick De Borja, Dave Perry, Catherine
Karaman, Kamala Tyagarajan
Oral Session
Abstracts
227/B90
Simplified and Affordable CD4 Monitoring Using the
Touch-Screen Based Muse® AutoCD4/CD4% System
Commercial
Tutorials &
Exhibits
Conclusions: PSM is a validated analysis method to enumerate
CD34+ hematopoietic stem cells, and to ascertain T cell and NK
cell population percentages in biological specimens. PSM is wellcorrelated with the ISHAGE single platform methodology, and it
also exhibits excellent reproducibility from operator to operator.
Further, the automated PSM method has met our goals of reducing
the time required to complete an analysis, and in eliminating repeat
analyses due to mistakes in region placement.
Scholars &
Emerging
Leaders
Here we present the different steps undertaken to integrate flow
cytometry into our Quality Control release program. The initial
validation of the technology by means of comparison to classical
biological techniques, such as plating (colony forming units) and
image cytometry, will be presented. Data will be presented on the
analysis of several yeast commercial formats: cream, stabilized
liquid yeast, cake, and dry. Also, an overview of the importance of
implementing proper Standard Operating Procedures between the
research laboratory and the production facility will be highlighted
by providing data obtained while developing the protocols
currently used for yeast viability assessment.
Results: We observed excellent correlation between the manual
ISHAGE analysis method and the automatic PSM method. For the
measured absolute number of CD34+ SCs per microliter of sample,
r² = 0.990; and for the percentage of CD34+ SCs from all
acquired viable leukocytes, r² = 0.999. The automated PSM
method was 100% reproducible from operator to operator (CV = 0;
SD = 0), while significant variability was measured with the
ISHAGE manual analysis method (CV = 0.8 - 7.6; SD = 79.6).
Separately, the two methods also exhibited very good correlation in
an independent assay component that quantified the percentages of
residual T cells and NK cells in the apheresis products. For total
CD3+ events, r² = 0.930; for CD4+ lymphocytes, r² = 0.976; for
CD8+ lymphocytes, r² = 0.953; and for NK cells, r² = 0.912.
Poster
Session
In recent years, the use of flow cytometry as a research and
development tool as well as a yeast quantification system, has been
introduced in the Distilled Spirits industry. As a modern yeast
manufacturer, Lallemand Biofuels & Distilled Spirits developed its
own methodologies, research and development projects, and
databases on yeast strains utilizing flow cytometry. Due to the
power of the technology, its accuracy, and yeast characterization
possibilities, multiple flow cytometry features are now exploited
within our manufacturing processes.
Methods: We used 612 data files that were generated from the
apheresis samples of 204 patients. The Flow Cytometry Standard
(FCS) file sets for each patient were separately analyzed by trained
analysts using both the ISHAGE manual analysis method; and also a
recently-described, automatic probability state modeling (PSM)
analysis method. Numeric values for CD34+ counts that were
obtained from either method were compared in order to determine
the level of congruence between the two enumeration
methodologies. We also tested the reproducibility of both methods,
by having multiple trained analysts workup the same set of 15 files.
Separately, 269 additional files were analyzed for T cell and NK
cell percentages using both methods.
Wednesday
15 June
Lallemand Biofuels & Distilled Spirits, Lallemand, Montréal,
QC, Canada
Background: Flow cytometry (FCM) is the preferred technology for
enumerating CD34+ stem cells (SC) in therapeutic products that are
used in hematopoietic stem cell transplants (HSCT). For the flow
cytometric enumeration of CD34+ SCs, the ISHAGE protocol is
considered to be the standard method, but like most FCM protocols
that rely on manual analysis strategies, it suffers from subjective
errors that arise from variability in the placement of regions by
different data analysts.
Tuesday
14 June
Annick Mercier, Fédéric D'Aoust
Flow and Image Cytometry, Roswell Park Cancer Institute,
Buffalo, NY, United States, 2Software Development, Verity
Software House Inc, Topsham, ME, United States
Monday
13 June
225/B88
Flow Cytometry Knowledge Transfer: From the
Research Laboratory to the Yeast Production Facility
1
Sunday
12 June
Conclusion: Imaging flow cytometry provides an efficient and more
advanced option in confirming the presence of sperm for patients
with NOA. Further analysis of sperm from NOA patients is required
to assess their viability and maturation, as well as to investigate the
use of biomarkers to capture these sperm for potential use in
assisted reproduction.
Joseph Tario, Jr. 1, Jennifer Greenway1, Paul Wallace1,
Beth Hill2, C. Bruce Bagwell2, Donald Herbert2
Saturday
11 June
Results: In Patient 1, 12 ejaculated spermatozoa were identified.
For Patient 2 (provided ejaculate and testicular sample), 1 sperm
was identified in the ejaculate and 3 sperm in the testicular sample.
The percentage of sperm within the total population of cells imaged
by the cytometer represent less than 0.005%. Sperm visualized
from these samples suggest immature morphology indicated by the
presence of large heads and short tails.
226/B89
CD34+ Stem Cell Enumeration with Supplemental T
Cell and NK Cell Evaluation: A Comparison between
Manual and Automated Analysis Methods
Special
Lectures
Methods: With institutional Research Ethics Board approval and
written informed consent, ejaculate (n=2) and testicular samples
(n=1), were obtained from NOA patients. Samples were processed
by centrifugation and extensive searching using a micromanipulator
performed by embryologists. When no spermatozoa were found,
working media and cell pellets were incubated with 70% ethanol
for permeabilization and fixation followed by staining with PI.
Imaging flow cytometry was performed using ImageStream and
later analysed using IDEAS software. Sperm were visually identified
using area vs. aspect ratio for gating, and confirmed using PI
staining.
Overall, we will provide examples of the utilization of this
technology and how Lallemand is integrating this cutting edge
technology into its industry.
Congress
Overview
Background: Azoospermia, the absence of sperm in semen, affects
approximately 15% of infertile men. Non-obstructive azoospermia
(NOA) occurs in approximately 60% of azoospermic cases and can
be caused by failure or arrest of spermatogenesis in the testis.
Retrieval of spermatozoa by microdissection testicular sperm
extraction (mTESE) is the only option for men with NOA. The
recovery of spermatozoa from testicular tissue has low success rates
and the method is time-consuming, highly variable and labour
intensive. Therefore, there is a need to develop efficient automated
methods for retrieval of spermatozoa from surgically retrieved tissue
samples as well as rare ejaculated spermatozoa in cases of severe
cryptozoospermia. The currently available method of cell
suspension flow cytometry with propidium iodide staining (PI) is
limited to ploidy assessment of spermatozoa. However, it does not
offer insight into morphology or the maturational state of sperm as
provided by imaging flow cytometry. The purpose of this project
was to investigate the use of imaging flow cytometry for the
identification of rare ejaculated and testicular spermatozoa as well
as their morphological and maturation assessment in NOA patients.
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
adult or pediatric whole blood samples. The instrument features
small sample size requirement, low biohazardous waste generation,
and user guided software with automated settings and gating that
greatly transforms and simplifies the user experience. The
combination of simplified assay steps and automated software,
which can minimize training needs and testing errors, presents the
potential for the instrument to be an easy to use, rapid, and
affordable system for CD4 enumeration in resource constrained
settings. In this study, we present data from the Muse® Auto
CD4/CD4% System on 38 adult whole blood samples. Method
comparison studies demonstrate equivalent results to clinically
approved comparative test methods with R2 for CD4 count
measurements of 0.99 and 0.98 for CD4% measurements. Average
% Difference was 4.13% for CD4 count measurements and average
absolute difference for CD4 % measurements was 0.99. In
precision studies, low average coefficient of variation (%CV) data
was obtained on triplicates of 38 samples shows CVs from 3.09%
for CD4 count, 2.03% for CD4% and 2.75% for total lymphocyte
count. Studies with low and high blood controls demonstrates an
inter-instrument and inter-assay %CV <7%. Linearity studies
demonstrate broad linear counting range for CD4 count and total
lymphocyte count measurements. The powerful technical and
performance features of the low-cost, small foot-print Muse® Auto
CD4/CD4% System combined with the highly simplified user
interface can enable accurate and precise CD4 monitoring in a
variety of user settings.
0-100 %) in 55 sorted monocyte populations (CD34+CD33CD14+CD19-) from 17 swALL patients.
The Muse AutoCD4/CD4% System is not for sale in the United
States.
229/B92
CD4+ Naive T Cells from HIV+ Patients with
Suppressed Viremia Contain Fewer Copies of Proviral
DNA and Greater Numbers of sjTRECs Compared to
Central or Effector Memory Lymphocytes
228/B91
Transcriptomic and Polychromatic Analysis of New
Subtype of B Cell Acute Lymphoblastic Leukemia
with a Switch to Monocytic Lineage
Poster
Session
Acute lymphoblastic leukemia (ALL) is the most common cancer
type in children. We identified a subtype of childhood B cell
precursor (BCP) ALL with a switch towards monocytic lineage
within the first month of treatment (swALL). During the lineage
switch blasts continually lose B cell precursor phenotype
characterised by CD19 and CD34 and acquire expression of
monocytic markers CD14 and CD33 (Slámová et al., Leukemia
2014).
Monitoring of leukemia patient treatment process is based on
measurement of minimal residual disease (MRD) which could be
detected by qPCR (assessment of Ig-TCR rearrangements) or by flow
cytometry (FC).
Index
Abstracts
Oral Session
Abstracts
CLIP-Dpt. of Paediatric Haematology and Oncology, 2nd
Faculty of Medicine, Charles University in Prague and
University Hospital Motol, Prague, Czech Republic
Scholars &
Emerging
Leaders
Alena Dobiasova, Michaela Novakova, Tomas Kalina,
Martina Vaskova, Eva Frookova, Lucie Slamova, Marketa
Kubreanova Zaliova, Ondoej Hrusak, Karel Fiser, Ester
Mejstoikova
Commercial
Tutorials &
Exhibits
Wednesday
15 June
Tuesday
14 June
Disease Progression Monitoring (B91 – B94)
We investigated qPCR and FC MRD levels in swALL bone marrow
(BM) and peripheral blood (PB) samples at day 8, day 15, and day
33 after diagnosis and we found unexpected discrepancies between
the two methods compared to typical BCP-ALLs. Spearman rank
correlation coefficients in respective time points were: day 8 BM
R=0.34 (p=ns), day 8 PB R=0.63 (p=0.02), day 15 BM R=0.44
(p=0.03), day 15 PB R=0.44 (p=0.03), day 33 BM R=0.18 (p=0.37).
Our results show that FC MRD frequently underestimates total MRD
level in comparison to qPCR. We hypothesised that it is caused by
the loss of CD19 which is crucial for B cell oriented FC MRD
during the first phase of treatment in swALL patients.
To assess the level of leukemic involvement in monocytoid cells we
performed FACS sorting experiment using 8-colour combination
(CD45, CD14, CD10, CD34, CD19, CD33, CD3, CD20). We
confirmed presence of Ig-TCR rearragements (median 16 %, range
188
With the aim to identify potential markers usable for improvement
of FC diagnostics and MRD distinction of switched monocytoids
from healthy monocytes in swALLs we analyzed transcriptomes of
swALL and BCP-ALL diagnosis samples (n=8, 8) and swALL sorted
monocytoid populations (n=2). We found significantly increased
expression of CLEC12B and CLEC12A (CLL1) that both encode a Ctype lectin receptors normally expressed on mature monocytes and
CEACAM1 (CD66) already at diagnosis before the lineage switch.
The loss of B cell specific markers (CD19, CD20) observed on
protein level by FC was also found on transcriptome level in
monocytoid blasts.
We conclude that swALLs are characterised by discordance
between B cell oriented FC and qPCR MRD levels. The
involvement of clonal Ig-TCR rearrangement in monocytoid cells
prove the presence of blasts that basically lost the B cell phenotype.
Using transcriptome data we identified potential swALL markers
and we designed a 16-18 colour panel for Sony SP 6800 spectral
cytometer to prospectively identify immunophenotypic signature
distinguishing monocytoid blasts and healthy monocytes.
Supported by Ministry of Health of the Czech Republic, grant nr.
15-28525A, GAUK 802214, UNCE 204012, OPPK
CZ.2.16/3.1.00/24505
Andrea Cossarizza1, Sara De Biasi1, Elena Bianchini2,
Margherita Digaetano1, Simone Pecorini1, Marcello
Pinti2, Lara Gibellini1, Vanni Borghi3, Cristina Mussini1,3,
Milena Nasi1
1
Dept. of Surgery, Medicine, Dentistry, Univ. of Modena
and Reggio Emilia, Modena, Italy, 2Department of Life
Sciences, University of Modena and Reggio Emilia,
Modena, Italy, 3Infectious Diseases Clinics, University
Hospital, Modena, Italy
Background: The progression of HIV infection can be halted by
potent drugs that block the production of the virus and inhibit its
integration into the host DNA. Response to therapy is typically
monitored by counting CD4+ T cells and measuring plasma viral
load, which becomes undetectable in most patients. Since at
present HIV cannot be eradicated and persists in the host, robust
data are needed on the importance of monitoring residual viral
activity and intracellular HIV reservoirs in patients with
undetectable viremia. Taking into account that the virus establishes
latent infection at different degrees within central (CM) and effector
memory (EM) or naï ve (TN) CD4+ T cells, a possible and novel
approach would be to measure HIV DNA in different lymphocyte
populations. Similarly, the residual capacity to reconstitute the
immune system needs to be accurately studied and monitored,
possibly by measuring cellular concentrations of the signal-joint T
cell receptor rearrangement excision circle (sjTREC), a marker of
thymic functionality. Thus, using flow cytometry and cell sorting
along with a molecular biology approach based on droplet digital
PCR (ddPCR), in different subsets of CD4+ T cells such as TN, CM
and EM we have quantified HIV proviral DNA and the presence of
sjTREC.
Methods: We enrolled 20 HIV+ patients (mean age 48.7±7.9 years,
6 females) successfully treated for at least two years, with a CD4+ T
cell count >500 cells/uL and with plasma viremia undetectable
from at least 15 months. TN, CM and EM CD4+ T cells were sorted
with a S3e sorter (Bio-Rad, CA, USA) with a purity always >95% in
a specifically designed biosafety containment hood (Biobubble,
UK). HIV proviral DNA and sjTREC DNA were quantified in each
subset with QX200 droplet digital PCR (Bio-Rad).
To correlate immune dysregulation in astronauts with terrestrial
disease, a clinical study was initiated translating the same panel of
assays which define immune dysregulation in astronauts to
Terrestrial Shingles patients. To date, 48 Shingles patients have
enrolled in this study. Saliva was collected to determine viral load
by quantitative PCR, and blood was collected for a variety of flowcytometry based assays including comprehensive peripheral
leukocyte distribution, T cell function (early blastogenesis) and
cytokine profiles (plasma and mitogen stimulated).
Poster Session
Abstracts
Index
189
Speaker/Author
After two months of abatacept administration, we detected a shift to
naï ve CCR7+CD45RA+ forms within T-cell compartment. Absolute
counts of naï ve T-cells increased 2.1 times in CD4+ and 1.6 times
in CD8+ population. Surprisingly, CD4+CD127-CD25+ Treg cells
decreased 1.6 times. In contrast, sirolimus induced Treg cells (they
increased 2.7 times), but the shift from the effector to naï ve T-cells
was similar as in the abatacept treated patient. Moreover, both
abatacept and sirolimus influenced the loss of T-cell activation and
plasmablast counts. Finally, mTOR kinase activity was successfully
inhibited with sirolimus. Phosphorylation of Akt remained
Oral Session
Abstracts
These information indicate that Shingles patients exhibit a pattern
of immune system dysregulation, aspects of which are similar to
astronauts during spaceflight. Planned deep-space exploration
missions beyond the Van Allen belt may exacerbate persistent
immune dysregulation and, coupled with other factors such as
increased radiation exposure, result in increased clinical risks to
astronauts. The development of immune countermeasures may be
warranted to mitigate this phenomenon for exploration missions.
Commercial
Tutorials &
Exhibits
The Shingles patients demonstrated alterations in central memory T
cell distribution, as well as increases in constitutively activated T
cells, reductions in general T cell function, and increases in a
variety of plasma cytokine concentrations. Mitogen stimulated
cytokine concentrations were also dysregulated in Shingles patients.
VZV DNA in the saliva of zoster patients ranged between 1,046 to
129,569,080 copies per ml. Live virus (VZV) ranging from 5.8% to
92.4% was detected by pre-polymerase chain reaction (PCR)
propidium monoazide (PMA) treatment in 9 of the VZV positive
Shingles patients.
Scholars &
Emerging
Leaders
We used single-cell phospho-flow and hyperspectral flow
cytometry to monitor the treatment response and optimized the
tests to be used in a 4mL sample of peripheral blood from pediatric
patients. The hyperspectral 13-colors antibody panel
(CD127/CD3/CD8/CD19/CD4/CD45RA/CD25/Ki-67/CD45/HLADR/CD27/CCR7/CD38) was introduced to detect the changes in the
lymphocytes composition, proliferation, and activation. Single-cell
phospho-flow revealing the activation of PI3K signaling pathways
(expressed as Akt (S473), mTOR (S2448), and S6 (S235/236) w/wo
anti-CD3/CD28/CD49d stimulation) was optimized to monitor the
mTOR inhibition efficacy.
Dysregulation of the immune system, characterized as altered
peripheral immunophenotype, reductions in T cell function, and
altered cytokine profiles occurs in astronauts and is known to result
in subclinical reactivation of latent herpesviruses.
Poster
Session
In the present study, c.178T>A, p. Y60N mutation in cytotoxic Tlymphocyte-associated protein 4 (CTLA-4) associated with CTLA-4
protein down-regulation was found in 5 members of the Czech
family. This mutation causes the primary immunodeficiency in all
affected members manifested as multiple autoimmunities. Based on
the laboratory and clinical findings administration of the specific
drugs (abatacept - recombinant CTLA-4/Ig fusion protein, and
sirolimus - inhibitor of Mammalian target of rapamycin (mTOR))
was approved by the institutional review board (in accordance with
the Declaration of Helsinki) in two family members aged 9 and 5
years, respectively.
Biomedical Research and Environmental Sciences, NASA,
Houston, TX, United States, 2Microbiology, JES Tech,
Houston, TX, United States, 3Health Sciences, University of
Texas, Houston, TX, United States, 4Biomedical Research,
Wyle Laboratories, Houston, TX, United States, 5Biomedical
Research, JES Tech, Houston, TX, United States
Wednesday
15 June
In the era of personalized medicine, the challenge of clinical
cytometry is to apply new emerging technologies and establish the
tests to monitor the response of the patients’ cells to modern
treatment modalities (e.g. recombinant receptors, cytokines, or
specific kinase inhibitors).
1
Tuesday
14 June
CLIP-Dpt. of Paediatric Haematology and Oncology, 2nd
Faculty of Medicine, Charles University in Prague and
University Hospital Motol, Prague, Czech Republic, 2Dpt. of
Immunology, 2nd Faculty of Medicine, Charles University
in Prague and University Hospital Motol, Prague, Czech
Republic
Brian Crucian1, Satish Mehta2, Stephen Tyring3, Hawley
Kunz4, Darilyn Chew5, Heather Quiriarte5, Ashlie
Renner4, Duane Pierson1
Monday
13 June
1
231/B94
Flow Cytometric and PCR Based Characterization of
Immune Dysregulation in Shingles Patients
Sunday
12 June
Veronika Kanderova1, Michaela Novakova1, Eva
Fronkova1, Michael Svaton1, Jana Kayserova2, Petr
Smisek1, Ondrej Hrusak1, Tomas Kalina1
Supported by P302/12/G101, 15-28541A, UNCE 204012,
CZ.2.16/3.1.00/24505, MH CZ – DRO, University Hospital Motol,
Prague, Czech Republic 00064203
Saturday
11 June
230/B93
Revealing the Treatment Response in Patients with
CTLA-4 Immunodeficiency with Single-Cell Phosphoflow and Hyperspectral Flow Cytometry
In summary, we developed simple tests where the innovative
cytometric technology was used to bring the clinically important
information about composition and function of cells in peripheral
blood of children with CTLA-4 haploinsufficiency treated with
abatacept and sirolimus, respectively.
Special
Lectures
Conclusions: In HIV patients, by linking flow cytometry and cell
sorting with a molecular biology approach based on ddPCR, we
can now combine precious information on the amount of virus
present in different CD4+ T cell populations (by quantifying HIV
DNA) and on the regenerative capability of the immune system (by
quantifying TREC), to provide crucial guidance on optimal
monitoring of HIV+ patients. This combined approach not only will
lead to a better comprehension of the features of HIV reservoir and
its monitoring, but could also be useful for the development of
eradication strategies.
unchanged in unstimulated cells, but the phosphorylation of mTOR
increased 4.5 times and phosphorylation of its target, S6 ribosomal
protein, decreased 2.8 times. The similar effect was detected in
anti-CD3/CD28/CD49d stimulated T-cells, phosphorylation of
mTOR target was diminished twice compared to healthy control.
Congress
Overview
Results: HIV proviral DNA, measured as LTR copies/1,000 cells,
was significantly lower in TN cells (mean±SEM: 0.59±0.24)
compared to CM (3.74±0.83) or to EM (2.17±0.47); p<0.0001 or
p=0.0010, respectively. Conversely, TN cells contained a higher
number of sjTREC copies/1,000 cells (12.08±2.26) compared to CM
(1.30±0.38) or to EM (1.91±0.70); p<0.0001 in both comparisons.
No correlations were present among clinical (actual CD4+ T cell
count, CD4+ T cell nadir or time to reach undetectable plasma
viremia) and the molecular parameters we have investigated.
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Index
Abstracts
DNA Damage and Repair (B95)
232/B95
Automation of Cytokinesis-Block Micronucleus Assay
Using Imaging Flow Cytometry
1
1
Christine Probst , Matthew Rodrigues , Lindsay BeatonGreen2, Barbara Kutzner2, Ruth Wilkins2
1
Amnis Biology R&D, Millipore Sigma, Seattle, WA, United
States, 2Consumer and Clinical Radiation Protection Bureau,
Health Canada, Ottawa, ON, Canada
The cytokinesis-block micronucleus (CBMN) assay is used to
measure chromosome damage by enumerating the number of
micronuclei in binucleated cells, and has been successfully applied
for in vitro studies of molecular toxicity as well as biodosimetry of
individuals exposed to radiation. A major bottleneck for the
conventional CBMN assay is that slides must be manually scored
under a microscope by trained personnel, limiting its applicability
for large-scale pharmaceutical screens or radiation incidents. While
automated and semi-automated microscopy has been successfully
used for the CBMN assay, slide preparation and scanning steps
remain time consuming. Here we describe the development of a
high-throughput, automated CBMN assay using the ImageStreamX
imaging flow cytometer (CBMN-ISX). The ImageStreamX is a highspeed microscope that collects brightfield, side-scatter, and
fluorescent imagery for objects in suspension at rates up to 1,000
objects per second. Using a suspension-based CBMN protocol,
human blood was irradiated with known doses from 0-4 Gy to
generate a dose response calibration curve, which was then used to
generate dose estimations in 9 blinded samples. Brightfield and
DRAQ5-labelled DNA imagery were collected for a minimum of
50,000 events for each sample using ISX, and an analysis template
was developed in IDEAS® image analysis software to quantify the
number of micronuclei per binucleated lymphocyte. The results
demonstrate that the CBMN-ISX assay is able to generate doseresponse calibration curves comparable to those previously
reported for automated and semi-automated methods, and
accurately predicts dose in all blinded samples to within 0.5 Gy. In
summary, the novel CBMN-ISX method addresses throughput
limitations of the traditional CBMN assay while retaining high
performance, and may enable large-scale studies of cell cytotoxicity
and patient biodosimetry.
Facility Management (B96 - B103)
233/B96
Merging Two Large Flow Cores; Expectations and
Challenges
Derek Davies, Graham Preece
Flow Cytometry, The Francis Crick Institute, London, United
Kingdom
The Francis Crick Institute in the UK was recently formed by the
amalgamation of two long-established Institutes, the London
Research Institute and the National Institute for Medical Research.
Both Institutes are leaving their buildings and moving to a purpose
built Institute in mid-2016. New STPs (Science Technology
Platforms) have been formed to serve the needs of researchers so
two well-established flow cores are merging. This has presented
problems and challenges but also opportunities.
The initial task was to assess working practices at both sites and
begin to align SOPs. There was also the identification of redundant
technology, areas where economies of scale could benefit the new
Lab and pinpointing areas where new technology may be able to
be implemented - this was in particular the development of a
Containment Level 3 analyser and sorter. There is also a need to
manage expectations of users and staff of the facility. It is important
to determine and utilise the skill set of the members of the Lab (and
at initial operation there will be 11.6 FTEs in the facility), ensure no
skills are lost and to build in a recognisable career path with
individual areas of responsibility, including a clear path of
management training.
190
There needs to be an increased emphasis on education (of staff, of
users and of partner organisations) and dissemination of information
both by traditional methods (websites, mailing lists) and the
increasing use of social media.
The remit of the new Lab will be to provide a cell sorter operation
service, train users to self-operate analytical flow and imaging
platforms, develop new techniques (often in collaboration with
commercial partners), develop and integrate data analysis and data
management into the workflow and fulfil the ethos of the Institute
as being seen as a world leader in cytometry.
This poster will describe the path taken so far to bring about the
changes needed and with less than 6 months till the new Institute is
fully functional, show the progress that has been made.
234/B97
The Need to Know: Cytometry Education for
Biotechs
Rachael Walker1, Derek Davies2
1
Babraham Institute, Cambridge, United Kingdom, 2Lincolns
Inn Fields Laboratory, The Francis Crick Institute, London,
United Kingdom
Flow cytometry is a widespread resource in many areas of
biomedical research and SRLs (Shared Resource Laboratories) exist
in many research settings. However this is less common in the
commercial sector and many start up biotechs, small CROs and
even small Pharmaceutical companies do not always have in-house
expertise. Flow cytometry has a central place in drug development,
antibody screening, target validation, and biomarker discovery but
an understanding of the technology is crucial. The advent of
simpler, automated cytometers has led to many biotech companies
without an SRL or flow cytometry expert, purchasing the systems.
Within a Research Campus, which houses both academia and
industry, a central core facility is used to meet flow cytometry
needs. However, some biotech companies have purchased their
own instruments. We found that many companies were in need of
education – basic and more advanced – in order to use their own
equipment efficiently and effectively. So in 2013 we applied for
and were awarded a Modular Training Partnership grant from the
Biotechnology and Biological Sciences Research Council (BBSRC)
to develop Flow Cytometry courses specifically for small Industry.
To show the market need for this grant, we surveyed over 20
companies as to their requirements, relevance of courses and
whether they would send delegates on the course. A Scientific
Advisory Board was also formed to help the development and
direction of the courses. We also surveyed current best educational
practices and developed a financial three-year plan to enable
delivery of courses.
To date we have delivered 10 courses which are a mixture of
classroom based lecture, activities and demonstrations; the courses
are portable and intimate allowing time for re-cap and are aimed at
the level of the delegates to allow learning at different paces.
Here we highlight the processes that enabled the development of
the course, the metrics used for success and the assessment of
deliverables to the funding body.
235/B98
Purdue Cytometry Email Discussion-27 Years and
Growing
J. Paul Robinson1, Bartek Rajwa2
1
Basic Medical Sciences, Purdue University, West Lafayette,
IN, United States, 2Bindley Bioscience Center, Purdue
University, West Lafayette, IN, United States
The Purdue Cytometry EMAIL list has reached 4200 members as it
continues to support the cytometry community. Over the past 27
years of operation, the list has become one of the most vibrant, and
high quality science discussion forum in the internet. The reason for
this is the selfless attitude of many of our cytometry colleagues who
continuously offer assistance to younger, less experienced
Membership to the list is free, but members must subscribe to the
email distribution list in order to participate in the list. The reason
for this is that the list is a monitored list that ensures the high quality
content, relevance to cytometry, and prevention of email
advertisers and spammers from gaining access. The list does not
allow attachements and the alternative is for submitters to use a
URL link that will achieve the same goal.
Kristen Leone
Anna M. Petrunkina
Cambridge, United Kingdom
Index
Traditionally, user education and training at flow cytometry core
facilities has taken the form of a lecture detailing fundamental
concepts and an instruction session at the instrument. Using this
191
Speaker/Author
1
Memorial Sloan-Kettering Cancer Center, New York, NY,
United States, 2Research and Development, STEMCELL
Technologies, Inc, Vancouver, BC, Canada
Poster Session
Abstracts
Tomas Baumgarther1, Jennifer Wilshire2
Oral Session
Abstracts
239/B102
Education Overhaul: Active Learning Approaches to
User Education in a Core Facility Setting
Commercial
Tutorials &
Exhibits
With cores in multiple locations, maintaining quality and standards
for both core operation and technician training is crucial. By further
streamlining our training procedures, we can increase
comprehension and reproducibility. Sharing our experiences with
standardizing aspects of our core operations can help other cores to
develop and improve their own training and QC procedures.
Scholars &
Emerging
Leaders
Customized sort reports will also be standardized to track other
critical factors that are not included in OEM sort reports such as
issues with users, cell types or instrumentation. Tracking by this
method will improve communication with researchers when there
are questions or concerns about the sort output.
Poster
Session
The formalism laid down here could initiate and trigger a
discussion how the optimisation of SRL performance could be
addressed in a systematic way.
New implementations in the core can increase administrative task
efficiency and improve our overall core functioning. Currently,
researchers reserve time on the instruments by using iLabs online
calendar and the core uploads the BD FACSDiva instrument usage
logs into iLabs. These uploads allow us to track and invoice users
who have not created reservations in the scheduler. Implementing
iLabs Kiosk will require users to create reservations in the scheduler
prior to accessing the instruments, thereby lessening the staff hours
required to upload and confirm usage logs.
Wednesday
15 June
A generalized mathematical formalism describing the customizable
metrics of service (i.e. for efficiency E and practical capacity C) is
defined in the current model as E=OD/N and C=(t-d)·E where OD is
the number of operational days (working days less authorized
absences for any operational or individual reason), N is the product
of nominal working days and the number of staff, and multidimensional vectors t and d reflect the cumulative staff time
assigned to each service and the non-bookable non-operative time
for each service, respectively). This formalism would be applicable
to any service, including cytometry, provided a matrix P
(distribution of duties according to the role profile/job description)
is carefully gathered for each staff member of SRL and regularly
reviewed to reflect changes in operational landscape. The general
strategy how to increase performance within framework of this
formalism has been identified through breaking down these metrics
into components and maximizing them according to the specific
requirements. These strategies could be potentially adopted for
different operational or local procedures, in line with institutional
or national policies. Maximizing of these metrics will be illustrated
using two specific examples: 1) under-performance of a SRL due to
poor operational practices, lack of communications and poor staff
attendance and 2) under-performance due to the shortage of
capacity caused by the outdated sub-optimal operational strategy
and the shortage of equipment.
Implementation of a standardized rigorous daily QC program has
improved monitoring instrument performance and maintaining
consistency critical in longitudinal studies. Technicians preform
bench in instrument assays utilizing cell lines within the core. On a
weekly basis cellular standards are stained for 14 fluorochromes,
fixed and are available to be used by researchers who do not have
proper controls. These are instrumental for trouble shooting and
setting up pilot experiments as well.
Tuesday
14 June
An existing general belief that no generalized strategy is possible
per se has been so far a major concern because the specialist
requirements (e.g. for a dedicated operator or particular aspects of a
service) are too specific for the core services of different nature to
establish and apply unified metrics across the broad range of
services. A further concern is related to the notion that any
particular approach adopted in one country may collide with
institutional or country-specific policies elsewhere and thereby
prevent translational applications across the institutions and
borders.
Standardized training protocols are also implemented for self-run
users on both sorters and analyzers. Competency and
troubleshooting skills are required for all users that use the facility
during non-staffed hours. All analyzers users are trained on a one
on one basis with a user specific experiment, which has proven to
be more effective than group training.
Monday
13 June
Many shared laboratory resources are faced with the increased
demand in usage which may potentially represent a major
operational challenge, especially in the infrastructure-lacking
environment and in context of competing for limited funding.
Operational optimization and performance benchmarking
including but not limited to tracking utilization have been identified
as key challenges faced by the core facilities and institutions in
which they operate. Strategies to optimize operations and
management of such facilities, especially against a backdrop of
creating a generalist approach which could be applied to a plethora
of services using a mathematical formalism compatible with the
local specifics and policies, would be instrumental in increasing
performance of SRL.
New core technicians follow a 12-week training guide culminating
in a performance review. This serves as a targeted list of skills
necessary to become a competent technician. Supervisors meet
with the technician to review the guide and set weekly goals to
clarify and expand upon concepts and assess any concerns. While
working alongside senior technicians, the new technician receives
constant feedback ensuring they reach the milestones of
understanding necessary to be successful.
Sunday
12 June
236/B99
Mathematical Model for Optimising the Operational
Performance of Shared Laboratory Resources
Flow cytometry core facilities that are comprised of many
instruments in multiple locations require rigorous standardization
and quality control (QC) procedures to insure consistency. The
Flow Cytometry Cores of Dana Farber Cancer Institute consist of a
staff of 7 scientists, nine analyzers and five sorters in two separate
buildings that are utilized by approximately 1,500 researchers. The
cores are constantly evolving and implementing new methods to
increase QC and administrative task efficiency needed in a cross
core platform.
Saturday
11 June
The address for subscription to the list is
[email protected] the website for review of the
archive is at http://www.cyto.purdue.edu/hmarchiv/index.htm.
There is a search function available for the general list as well.
Flow Cytometry, Dana-Farber Cancer Institute, Boston, MA,
United States
Special
Lectures
238/B101
Standardization in a Multi-core Platform
Congress
Overview
colleagues. The list has a fully searchable database for each section
on the list. This allows date, author or subject searches on all or any
subunit of the list.
Sunday
12 June
240/B103
Evaluating Cell Sorter Cleaning Procedures across
ABRF-Flow Cytometry Research Group Institutions
by Testing for Common Contaminants
Roxana del Rio1, Kathleen Brundage2, Alan Bergeron3,
Andrew Box4, Matthew Cochran5, Monica DeLay6, Maris
Anne Handley7, Ernest Michael M. Meyer8, Alan Saluk9,
Peter Lopez10
1
Surgery, University of Vermont, Burlington, VT, United
States, 2Microbiology, Immunology and Cell Biology, West
Virginia University, West Virginia, VA, United States,
3
Dartmouth College, 4Stowers Institute for Medical
Research, 5Univ of Rochester, 6Cincinnati Children’s
Hospital Medical Center, 7MGH Massachusetts General
Hospital, 8Univ of Pittsburgh, 9The Scripps Research
Institute, 10New York Univ
Cell sorting plays an important role in many in vitro and in vivo
studies. Sorted cells are often placed back into culture for
expansion and/or for in vitro experiments or adoptively transferred
into animals. In recent years, cell sorting has become an important
part of the workflow for many genomic/proteomic studies as well.
Thus, anything that the cells come in contact within the sorter has
the potential to affect the function (i.e. cytokine production,
proliferation), quality of cell products (i.e. presence of RNase may
degraded RNA,) and/or result in the presence of contaminating
foreign nucleic acids that could ultimately affect the experimental
results. Since every Flow Cytometry (FC) Shared Resource Lab (SRL)
has its own routine cleaning procedures for sorters, the ABRF Flow
Cytometry Research Group (FCRG) is interested in developing best
practices for maintaining a “clean” sorter. In order to develop best
practices, we first wanted to determine how good our standard lab
cleaning protocols were in keeping the instruments “clean”. In this
study, samples were collected from sorters located in 10 FC-SRL
across the country and sent to two labs for analysis. One lab
analyzed the samples for bacterial contamination and the other lab
analyzed the samples for endotoxin, mycoplasma and RNase
contamination. These contaminants were chosen for analysis
because they are known to alter cell growth, activation state and/or
adversely affect downstream genomic applications. Results from
this study are presented here.
Flow Cytometry Instrumentation (B104 –
B124)
241/B104
Expanding the MoFlo Legacy Capabilities with
Easy Laser Swapping
Janet Dow1, Sébastien Coquery2, Nancy Fisher3
1
Department of Microbiology and Immunology, Univ. of
North Carolina at Chapel Hill, Chapel Hill, NC, United
States, 2Department of Microbiology and Immunology,
Unversity of North Carolina at Chapel Hill, Chapel Hill,
NC, United States, 3Department of Microbiology and
Immunology, Univ of North Carolina, Chapel Hill, NC,
United States
Large expensive tunable lasers have become increasingly
impractical to replace on MoFlo Legacy and MoFlo XDP cell
sorters. New continuous wave solid-state lasers present a unique
opportunity to add versatility to the aging MoFlos by easily
swapping one or more lasers and by dramatically reducing the cost
of laser replacements. This upgrade expands the excitation
capabilities of these instruments to accommodate the detection of a
wider range of fluorophores, keeping these sorters relevant deep
into their golden years. Our goal was to replace a water-cooled
Coherent 1302C 1W tunable Krypton laser with an interchangeable
solid state laser system.
Our solution utilizes a 100mW Coherent OBIS 405 LX laser
attached to a kinematic magnetic base plate mounted on a heavyduty precision lab jack. Once affixed to the cytometer optical
bench, the lab jack provides a stable mounting platform for the
laser(s) and permits easy laser swapping with minimal re-alignment
necessary. We were also able to bypass the optical tower as the lab
jack/laser combination is small enough to fit near the opening of
the sort chamber, allowing a direct illumination of the stream and
therefore minimizing laser power losses.
The laser/heat sink assembly was bolted to the top mounting plates
of two 3”x3” kinematic bases (Thorlabs #KB3X3), while the bottom
base plates were bolted to the top of a 7”X4” heavy-duty Lab Jack
(Thorlabs #L490). In order to reach proper height (side opening of
the sort chamber), the lab jack was affixed to the optical bench
through stilts.
When an alternate laser line (i.e. 552nm) is desired, the 405nm
laser can be removed and be replaced with a different laser bolted
to two top mounting plates. Minimal laser alignment (beam-shaping
optic adjustment) is then performed.
This procedure does not require the assistance of a service engineer
which drastically reduces cost. However be sure to follow laser
safety guidelines.
242/B105
Accellix Automated Flow Cytometry
Julien Meissonnier, Harvey Kasdan, Micha Rosen,
Ephraim Carlebach, Allon Reiter
LeukoDx / Accellix, Jerusalem, Israel
Background: Medical flow cytometry (FC) provides diagnostic
answers by detecting the presence and concentration of cell
populations, and/or by measuring concentrations of cell surface
markers expressed on cells. Currently, FC is limited to high
complexity labs by time consuming pre-analytical steps, requiring
highly trained technologists. Inter-instrument and inter-operator
variability limit broad acceptance of IVD FC. Finally, interpretation
of FC results requires highly trained professionals typically available
only during business hours.
Index
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Scholars &
Emerging
Leaders
Poster
Session
Wednesday
15 June
Tuesday
14 June
Monday
13 June
Special
Lectures
Congress
Overview
Saturday
11 June
method, we have observed poor comprehension of key concepts
and stressful sessions at the instruments especially with novice
users. In recent years, research in education has suggested that
traditional lecture formats are ineffective. Active teaching
methodologies have been show to greatly improve information
retention and test scores. With this in mind we have revamped our
teaching methods and materials to incorporate active teaching
practices including; slogans and activities, workshop sections
during lecture, Think/Pair/Share exercises, hands-on activities, case
studies, models/samples and quizzes incorporated into lecture.
During our teaching we attempt to address multiple learning styles
(visual, auditory, read/write, kinesthetic) to provide a suitable
learning environment for all of our users. After implementation of
active learning we have observed greater information retention
resulting in less stressful sessions at the instrument and increased
user satisfaction and most importantly good data generation.
The Accellix compact table top multicolor flow cytometer
automates the 3 step process required for population identification
and/or cell surface marker measurement. Sample preparation and
reading are performed in a dedicated disposable cartridge.
192
Sepsis diagnosis and monitoring based on upregulated CD64
expression on neutrophils.
244/B107
Utilization of 3D Printing Technology in a Flow
Cytometry Shared Resource Facility
T cell subsets: proportion of T helper cells (CD4) to cytotoxic T cells
(CD8) compared with total T cells (CD3).
Alan Saluk, Matthew Haynes
Population analysis of cells: differentiating T cells, B cells, NK cells
and monocytes based on cell surface marker expression.
Index
193
Speaker/Author
The complexity of cellular interactions within tissues is not
physiologically replicated in conventional in vitro cell culture. Cells
grown in 2D lack crucial multifactorial metabolic gradients that
define and regulate the microenvironment in growing tumors, while
current 3D models cannot spatially resolve the dynamic interaction
between these gradients and pathophysiology. Improved methods
are needed for generating 3D models containing multiple cell types
integrated with multiplex chemical sensors for in situ assays of
chemical, metabolic and physiological gradients. We are
developing a new perfusion-based cell culture device that will
allow spatially correlated, mechanistic investigation of the
microenvironmental regulation of cellular pathophysiology. As a
first application of this new technology, we are investigating the
effects of extracellular pH and O2 gradients on the activation of
Poster Session
Abstracts
Results: for every measured LSP, the solution of ILS problem was
obtained for estimation of the following characteristics: volume,
Department of Chemical and Biological Engineering, Center
for Biomedical Engineering, The University of New Mexico,
Albuquerque, NM, United States
Oral Session
Abstracts
Methods: Blood samples were taken by venipuncture and collected
in a vacuum tube containing EDTA as anticoagulant. The sample
was 1000-fold diluted in 0.9% saline and measured with the SFC. A
Scanning Flow Cytometer fabricated by CytoNova Ltd.
(Novosibirsk, Russia, http://cyto.kinetics.nsc.ru/) which measures
the angle-resolved light-scattering profiles (LSPs) of individual cells
was used. A new optical model of erythrocyte with four parameters
(two thickness, the diameter and the spontaneous curvature) is
applied to solve the inverse light scattering (ILS) problem.
Jacqueline De Lora, Jason Velasquez, Catherine Sher,
James Freyer, Andrew Shreve
Commercial
Tutorials &
Exhibits
Background: Determination of red blood cell (RBC) morphology
and its functional properties allows one to assess blood status and
to reveal a number of diseases such as anemia, hereditary
spherocytosis, hereditary elliptocytosis, sickle cells disease, and
myeloproliferative disorders. In this case the quantity of clinical
significant parameter procures reliable information about healthy
state. Unfortunately, modern hematology analyzer cannot measure
such RBC characteristics as surface, sphericity index, anion
permeability and etc. In order to solve this problem we develop
scanning flow cytomerter and describe erythrocyte behavior with
advanced optical model of RBC.
245/B108
Comparing In Vitro Lung Adenocarcinoma Cell
Growth Using 2D and 3D Coculture Tissue Models
Scholars &
Emerging
Leaders
1
Cytometry and Biokinetics Laboratory, Voevodsky Institute
of Chemical Kinetics and Combustion SB RAS, Novosibirsk,
Russia, 2Novosibirsk State University, Novosibirsk, Russia
Poster
Session
Ekaterina Chernyshova1,2, Andrei Chernyshev1,
Konstantin Gilev1, Dmitry Strokotov1, Valeri Maltsev1,2
Wednesday
15 June
243/B106
Four Parameters of Red Blood Cells Population
Obtained by Scanning Flow Cytometer
Recent advances in consumer 3D printing technologies have
allowed for relatively inexpensive and easy to use bench top
fabrication systems to be utilized within the shared resource core.
The applications within the TSRI Flow Cytometry Core facility have
ranged from rapid prototyping to production of fully functional
parts for use within contemporary flow instrumentation. After
assessing various 3D printing technologies we opted for Fused
Deposition Modeling (FDM) style printer due to its relative low
cost, ease of use, and wider range of inexpensive consumable
fabrication materials. The facility purchased a MakerbotR
ReplcatorR Z18 printer and opted to use the free Computer Aided
Design (CAD) software 123D DesignR from AutodeskR for Mac
platforms. We will describe the design consideration and workflow
necessary to produce functional 3D printed objects that can be
utilized within the core facility with a minimum amount of postproduction processing. These objects will range from items
designed to more rapidly and cost effectively replace commonly
used objects to creation of instrument specific component designs
that allow for advanced upgrades to existing flow cytometry
instrumentation platforms. We will also address the limitations of
FDM style printing and suggest alternative styles based on the scope
and usage of the printed object. Finally, we will propose to utilize
ThingiverseR (www.thingiverse.com) for deposition of the .stl object
files that can be utilized by various different printer platforms with
the goal being a common resource for those wanting to utilize and
or share object designs.
Tuesday
14 June
These initial studies show that the cartridge-based Accellix system
can determine the presence and concentration of cell populations
as well as determine the concentration of cell surface markers.
Thus, implementation of a wide range of fully automatic IVD assays
with results in 30 minutes or less is possible using Accellix.
The Scripps Research Institute, La Jolla, CA, United States
Monday
13 June
Results and Conclusions: In a demonstration of cell surface marker
quantitation a comparison study of 118 blood samples showed a
correlation coefficient of 0.94 for Accellix determined neutrophil
CD64 compared to those determined using a FACS. In a study to
identify lymphocyte subsets a comparison study of 5 samples (run
in triplicates) showed a correlation coefficient of 0.99 for Accellix
determined T cell differentiation based on CD4/CD8 ratio, and
correlation coefficients of 0.94 and 0.99 for lymphocyte population
analysis to determine B cells (CD19/CD45) and NK cells
(CD56/CD3-/CD45) compared to FACS.
Sunday
12 June
Measuring sepsis induced immunosuppression via HLA-DR
expression on circulating monocytes.
Saturday
11 June
Applications implemented on Accellix:
Conclusion: We developed a method for advanced characterization
of RBC population (evaluating fifty four parameters) based on the
light scattering measurements of single red blood cells with SFC.
Such detailed characterization of RBCs is useful in particular in
clinical diagnostics to increase the quality of screening
examinations.
Special
Lectures
Accellix Cartridge: This disposable cartridge-based platform
provides 24/7 availability in a moderate complexity lab - ultimately
CLIA waved setting - by implementing sample preparation using
three reagent blisters. With different reagents in the blisters the
same cartridge structure can be used for multiple applications. The
three Accellix CD64 cartridge blisters contain staining cocktail of
conjugated monoclonal antibodies, lysis buffer, and reference
beads respectively. Once sample processing is complete, the
sample flows through a dedicated reading channel where data is
acquired.
surface area, diameter, thickness, refractive index, hemoglobin
concentration, and hemoglobin content. The derivative
characteristics: number of anionic exchangers on membrane,
elasticity and critical tension of membrane hemoglobin
concentration and content are also calculated. As the result, fifty
four parameters of the whole RBC population were evaluated
(including mean, standard deviation, skewness ). It was found that
values of volume, hemoglobin content measured by SFC are in
good agreement with ones measured by Sysmex XT-4000i.
Congress
Overview
Analytical data processing utilizing proprietary algorithms provides
answers directly to the user.
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Index
Abstracts
hypoxia-inducible factor (HIF-1Į), a critical transcription factor in
the cellular response to hypoxia. The use of A549 human lung
adenocarcinoma cells that are wild-type (WT) or knock-out (KO) for
HIF-1Į allows us to directly address the influence of HIF-1Į on
cellular response to microenvironmental stress.
As a first step, we have developed a high-throughput instrument
that builds upon the concepts of cell sorting technology to produce
uniform, cell-encapsulating microspheres using aqueous two-phase
separation chemistry. The instrument uses microfluidic devices with
defined orifice diameters, pressure driven flow of viscous solutions,
and acoustics tuned to produce uniform droplets in air. The cellencapsulating biomaterial consists of sodium alginate (ALG) and
polyethylene glycol (PEG) in buffer. The ALG/PEG droplets
crosslink upon interfacing with a receiving solution that contains
calcium ions, dextran and the interface stabilizer Pluronic F-127.
Varying the orifice diameter (70-200 ȝm), the flow velocity (1-10
mL/min) and piezoelectric frequency (1-40 kHz) controls the size of
the droplets in air (70-300 ȝm diameters at up to 30,000
droplets/second). In order to compare growth rates in mono- and
cocultures as well as to determine the initial cell concentration for
encapsulation, we measured the doubling times of A549-WT,
A549-KO and A549-CO (50:50 coculture) cells in monolayers. The
cells were stained with green (WT) and red (KO) membrane dyes,
followed by flow cytometry to identify the two cells in co-cultures,
allowing growth analysis by both differential cell counting and dye
dilution. The cells had doubling times of 15-18 hours in mono- and
co-cultures, as measured by either cell counts or dye dilution. We
are currently measuring the growth of encapsulated mono- and cocultures to compare growth rates between 2D and 3D methods.
These results are informing the design and implementation of a
droplet-packed perfusion cell culture system that will produce a
controllable and quantitative model of the chemical, metabolic and
physiological gradients produced in a tumor.
246/B109
Evaluation of Single Cell Plate Sorting in a Multiinstrument Core Facility
Michael Buonopane
Flow Cytometry, Dana Farber Cancer Institute, Boston, MA,
United States
The application of single cell sorting utilizing Flow Cytometry
technology has become an integral first step for further downstream
Genomic and Proteomic studies. Single cell biology dictates the
precise isolation of single cells into individual wells, which is
critical for the production of accurate data. Both the
instrumentation and operator are key elements in the successful
execution of single cell purification.
This has prompted me to plan experiments allowing me to ascertain
the optimal single cell sorting settings utilizing the Sony SH800Z
Cell Sorter and the BD FacsAria™ II.
The main objective was to compare cross platform instrumentation
set-up, ease of use, functional efficiency, and downstream assay
efficiency. Experiments were designed utilizing fluorescent bead
standards and cell lines, and the manufacturers’ recommended
single cell sort precision settings. Several nozzle sizes were used to
compare different sort droplets in each instrument, which were
collected in multiple types of microtiter plates.
In order to assess the single cell plating efficiency of the different
instruments, fluorescent microscopy will be used to verify the sort
efficiency baseline; cell culture will confirm cellular integrity,
viability, and sort-accuracy. PCR results will also validate sorting
efficiency.
Performance characteristics of each instrument-specific plate
calibration will be used to provide researchers a gauge to
accurately estimate the time required for single cell sort set-up and
plate alignment. It is desired that the data gathered will serve as a
guide, enabling the staff to better educate the researchers as to the
limitations and benefits of each instrument particular to various
experimental protocols and different cell types.
194
247/B110
Novel Ultrasensitive Blood Tests Using In Vivo Flow
Cytometry Platform
Vladimir Zharov
Arkansas Nanomedicine Center, University of Arkansas for
Medical Sciences, Little Rock, AR, United States
Cardiovascular diseases, cancers, and infections remain the main
causes of death in the U.S. and worldwide. The diagnosis of many
diseases begins with a common medical procedure: examination of
extracted blood samples. The sensitivity of current blood test is
limited by the small volume of blood collected, in which no less
than one disease-specific marker (e.g., tumor cell, clot, virus and
bacterium) can be detected. This method can miss many thousands
of abnormal cells in the whole blood volume (~5 liter in adults),
which can be sufficient for disease progression to difficult-to-treat if
not already incurable complications (e.g., metastasis, stroke, or
sepsis). We have developed novel concept of early disease
diagnosis (“in vivo reading written in blood”) by in vivo
photoacoustic (PA) detection of disease-associated circulating
markers using both natural (e.g., melanin in melanoma and
hemozoin in malaria) or artificial functionalized nanoparticles as
high contrast PA agents. Laser irradiation of markers circulating in
the blood vessels leads to the generation of PA waves that are
detected with a small ultrasound transducer attached to the skin. In
positive-contrast mode, when markers with higher absorption than
the blood background (e.g., malaria parasite-infected erythrocytes
with hemozoin) pass through the irradiated volume, localized
absorption transiently increases, resulting in a sharp positive PA
peak. In negative-contrast mode, when white clots consisting of
platelets or fibrin, with much lower absorption than the blood
background pass through the laser beam, a decrease in localized
absorption results in a negative PA peak. Unlike typical blood
sampling involving extraction of a volume of blood ranging from 10
μL (one drop) to a few milliliters, in vivo examination involves
nearly the entire volume of blood passing through 1–2-mmdiameter peripheral vessels over 0.5–1 h (a few minutes in larger
vessels) and thus will enable a dramatic increase in diagnostic
sensitivity, ultimately up to 103–104 times, reflecting the ratio of the
volume of blood sampled in vivo to that in vitro. In addition, the
integration of simultaneous diagnosis and therapy—theranostics—
can eradicate circulating abnormal cells, and thus can potentially
prevent, or at least inhibit deadly metastasis, sepsis or stroke. This
report summarizes recent advances of this platform with focus on
multicolor blood testing of melanoma, malaria, S. aureus –related
sepsis, and thrombosis. First clinical applications of this technology
for preventions of metastasis and stroke are discussed.
248/B111
Improving Flow Cytometer Performance by a Novel
Strategy for Optimizing Bandwidth Filters
Kai Witte
University Hospital Tuebingen, Tuebingen, Germany
Increasing usage of novel dyes conjugated to monoclonal
antibodies (mABs) as well as improved fluorescent proteins (FPs) as
reporters for gene expression have greatly improved the feasibility
of analyzing complex flow cytometric data with over 15 colors.
Obtaining a spillover spreading matrix (SSM) for all utilised
fluorochromes at optimized flow cytometer settings greatly aids
with developing staining panels for flow cytometry (Nguyen & al.
2013). However, for this approach it is necessary to acquire the
data with fixed voltage settings for all detectors. Perfetto & al.
(2014) proposed the panel specific separation index SIps as a
measure combining instrument performance and experimental
background.
However, most flow cytometers are being used to acquire a wide
array of experiments, especially in a Shared Resource Lab (SRL)
setting. Single stained controls or even standard beads (hard-dyed
with different fluorochromes than those being measured in
experiments) have traditionally been used to optimize performance
of flow cytometry hardware while the often overwelming effect of
The new BD FACSVia™ System is a small, easy to use flow
cytometer that features a compact optical design, fixed alignment,
and pre-optimized detector settings to simplify and improve
operational workflow with low cost of operation. It contains two
lasers and four fluorescence detectors for use with various
biological assays. The BD Leucocount™ clinical assay available on
the BD FACSVia System in Europe is designed to enumerate
residual white blood cells (rWBCs) in leucoreduced platelets (PLTs)
and red blood cells (RBCs). We evaluated multiple BD FACSVia
Systems to demonstrate robust performance using this assay.
Precision: A 21-day study was conducted on three systems by three
operators using custom-made controls (PLT and RBC). The stained
control cells were acquired twice per day to determine rWBC
absolute count (Abs Cnt).
Control
Mean (cells/μL)
CV% (95% UCL)
PLT High
15.11
6.12 (6.91)
Tuesday
14 June
Precision results
7.32
7.96 (8.99)
RBC High
16.50
6.81 (7.68)
RBC Low
7.58
9.03 (10.2)
Rico Bongaarts
Linearity: Eleven concentration levels of rWBCs were prepared in
PLT and RBC specimens and measured on one BD FACSVia System
to determine Abs Cnt of rWBCs. PLT results were linear in the range
of rWBC 0–453 cells/μL with a slope of 0.973, intercept -0.287,
and R2 0.998. RBC results were linear in the range of rWBC 0–436
cells/μL with a slope of 0.960, intercept 0.560, and R2 0.998.
Stability:PLT (n = 48) and RBC (n = 50) samples that were aged at 0,
24, and 48 hours were evaluated for sample stability and sample
staining stability on the BD FACSVia System. Results were
compared to time zero [T0 Age of Sample 0 hours, Age of Stain 0 min (A0hrs/S0min)] to
determine bias with a 95% CI in rWBC Abs Cnt at each time point.
Stability results
rWBC mean bias%
(95% CI)
rWBC mean bias%
(95% CI)
T1 (A24hrs/S0min)
1.2 (-2.4, 4.9)
3.8 (0,7.6)
T2
(A24hrs/S24hrs)
4.1 (1.2, 7.0)
4.5 (0.7, 8.4)
T3 (A48hrs/S0min)
0.8 (-2.2, 3.9)
6.4 (2.1, 10.8)
T4
(A48hrs/S60min)
-1.9 (-4.7, 0.9)
5.7 (1.5, 9.9)
Index
195
Speaker/Author
Time point
Poster Session
Abstracts
RBC
Oral Session
Abstracts
PLT
Loader Evaluation: PLT (n = 81) and RBC (n = 88) samples were
acquired on the BD FACSVia System manually and with a BD
FACSVia™ Loader. Absolute counts of rWBCs in the samples were
Commercial
Tutorials &
Exhibits
The availability of a high throughput technique, which is fully
automatable and is capable of selecting and sorting cell clusters is
Union Biometria’s large particle flow cytometry technology
(COPAS FP and BioSorter). Here we describe the COPAS FP
technology for the automated cell culture of human iPS cells.
Sample Carryover: Samples containing high (H) and low (L)
concentrations of rWBCs were prepared for the PLT and RBC
specimens. Percent sample carryover was determined on the BD
FACSVia System for PLT and RBC sample types: 100*((L1 – L3) /
(H3 – L3)). The rWBC average carryover was minimal: 0.23% in
RBCs and 0.16% in PLTs.
Scholars &
Emerging
Leaders
Biomedical methods based on stem cells are currently the most
dynamic areas of life science especially when it comes to cellular
test systems for pharmacological and toxicological screenings. Short
term goal is establishing new test systems to increase prediction of
screenings in the preclinical phase. This will lead to cost reduction
and higher efficiency. The development of reprogramming somatic
cells into human iPS cells opens unique perspectives for producing
human cell products in a tissue disease and patient specific
manner. To fully take advantage of the potential of this technology
there is a need to produce a high number of iPS cell lines using
high throughput techniques, to standardize the available protocols
and to deliver fully characterized cells. Recent examples of related
applications are the analysis and sorting of Organoids, Embryoid
Bodies, Cardiomyocytes and Adipocytes. Both COPAS FP,
equipped with a 500 or 1000 micron flow cell, and BioSorter
(flexible flow cell system) have been used for a diversity of cellular
applications.
Poster
Session
PLT Low
Wednesday
15 June
249/B112
Automated Analysis and Sorting of Human iPS Cell
Clusters by Large Particle Flow Cytometry
Union Biometrica Inc, Geel, Belgium
Monday
13 June
Perfetto & al. 2014. Q and B values are critical measurements
required for inter-instrument standardization and development of
multicolor flow cytometry staining panels. Cytometry A, 85: 1037–
1048.
BD Biosciences, San Jose, CA, United States, 2American
Red Cross Biomedical Services, Rockville, MD, United
States
Sunday
12 June
Nguyen & al. 2013. Quantifying spillover spreading for comparing
instrument performance and aiding in multicolor panel design.
Cytometry A, 83A: 306–315.
1
Saturday
11 June
Here I propose to utilise a similar approach to select optimal
bandwidth filters (and dichroic mirrors) for any detector/channel.
Resolution Q is measured with single stained controls of the
preferred fluorochrome(s); for estimating spillover spreading,
generic FMO controls are used to measure total Background: these
include a n-1 mix of all fluorochromes typically used for panel
design on a given instrument. An average separation index (SIav) is
calculated from the raw data and indicates the best filters to utilise
for an instrument. The SIav approach also helps to establish decent
reference settings for detector voltages (which can be improved by
optimizing voltage settings for specific staining panels of users if so
desired). This strategy does not take into account that spillover
might be avoided or at least decreased by prudent panel design.
However, complex staining panels with over 15 colors will almost
always result in considerable spillover spreading and it is these
experiments for which separation is often critical. Thus, utilising the
average separation index (SIav) when selecting optimized filters for
any multicolor flow cytometer configuration considerably improves
high-end applications.
Yang Zeng1, Maryam Saleminik1, Fred Mosqueda1, Julie
Nguyen1, Leonid Shatsman1, Anh Do Chau1, Rebecca
Hershock-Quintana1, Michelle Dabay2, Shalini
Seetharaman2, Anna Lin1, Kevin Judge1
Special
Lectures
It is widely acknowledged that fluorescence minus one controls
(FMOs) or suitable internal controls are needed to judge instrument
sensitivity and resolution, especially for dimly expressed markers,
being specific for any mAB staining panel (possibly also including
FPs and/or functional dyes).
250/B113
Analytical Performance of the BD FACSVia™ System
in the Enumeration of Residual Leucocytes from
Leucoreduced Blood Products
Congress
Overview
spillover spreading on background noise has not systematically
been evaluated.
Congress
Overview
Special
Lectures
Saturday
11 June
Sunday
12 June
Monday
13 June
Tuesday
14 June
Wednesday
15 June
Poster
Session
Scholars &
Emerging
Leaders
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Index
Abstracts
compared between manual loading and the BD FACSVia Loader
using Deming regression.
Loader results
Slope (95% CI)
Intercept (95% CI)
R2
PLT
0.95 (0.91, 0.99)
0.25 (-0.13, 0.62)
0.97
RBC
0.97 (0.92, 1.01)
-0.17 (-0.47, 0.12)
0.92
In this study we conclude that the new BD FACSVia System is an
easy to use instrument that simplifies operational workflow and
demonstrates precise and reliable performance with the BD
Leucocount assay. It has great potential to increase the world’s
access to flow cytometry technologies.
251/B114
Small Particle Flow Cytometry: Light Scatter
Performance for Extracellular Vesicle Studies
Oliver Kenyon
R&D, Apogee Flow Systems (UK), Castelldefels, Spain
In a typical extracellular vesicle sample the majority of vesicles are
smaller than 300nm7 in diameter and therefore difficult to measure
on a conventional flow cytometer. Flow cytometers are able to
measure vesicles of any size if labelled with sufficient fluorescent
molecules but in practise labelling is weak from such small
particles1and conventional flow cytometers struggle to resolve
300nm silica beads from optical noise by light scatter. However,
recent electronic and optical developments now allow the routine
measurement of particles which scatter orders of magnitude less
than 300nm silica beads and an order of magnitude less than
100nm latex beads which have often been used as a lower limit
reference. Such developments make light scattering an essential
tool for the reliable interpretation of extracellular vesicle data but in
future a 100nm silica bead is likely to be a far more suitable
reference particle due to its refractive index, size and stability. We
present data from 100nm silica beads analysed on a commercially
available flow cytometer, showing their relationship to 110nm latex
beads and demonstrating that particles which scatter 1000 times
less light than 300nm silica beads can now be measured routinely,
thus permitting the study of a greater proportion of a typical
extracellular vesicle population than has previously been possible.
252/B115
42CH Hyperspectral Detection System for Flow
Cytometry
Keegan Hernandez1, Masanobu Yamamoto1,2, J. Paul
Robinson1,2
1
Development Div., Miftek Corporation, West Lafayette, IN,
United States, 2Basic Medical Sciences, Purdue University,
West Lafayette, IN, United States
Introduction and Background: Hyperspectral detection in flow
cytometry is apowerful tool for over ten multi-color phenotypic
analysis withoutcompensation. There are many operating flow
cytometers in the market place withonly 4 color performance. An
objective of this work is to provide a universal
hyperspectraldetection system to analyze fluorescence signals from
a variety of flowcytometers. Using our prototype we were able to
evaluate a wide wavelengthrange (340-800nm) with 42channels
and 16 bit resolution.
Method and Results: In normal flow cytometry, fluorescence
andside scatter light from the flow cell is collected by a high NA
objective lensand collimated to detection optics including dichroic
mirrors, bandpass filtersand PMTs. Capturing the collimated
fluorescence light with an achromatic lensand optical fiber, it is
possible to analyze the entire visible light spectrum usinga
polychromator. We developed a custom polychromator
collaborated with partners,using an aberration corrected Toroidal
Mirror Grating and 42CH linear fiber array.The advantages of the
Toroidal mirror grating are 1) wide wavelength coveragefrom UV
340nm to IR 800nm, 2) high spectral resolution with equal
196
bandwidth, 3)high coupling efficiency using just one reflected flat
field. The focusedspectral line “image” is connected to a 42CH
fused silica fiber array with0.5mm core and NA0.22. Once
fluorescence light is distributed to the fiber, thesignal can be
detected by variety of sensors like APD, PMT or SiPM (Gaigermode
APD Array). For conventional flowcytometer application,
1mmʔAPDs (Hamamatsu) are applied. The amplified photocurrent
from the APD is converted to digital signal by 16bit current ADC.
Developedsystem was coupled to a conventional flow cytometer
FC500 (Beckman Coulter) andconfirmed fluorescence signal from
Rainbow beads. In combination with ȝPMT bySi-MEMS structure,
the highest sensitivity is possible to apply flow analysisin the
nanosecond domain. Due to smaller photocathode and dynode
area,ȝPMT andlow noise preamplifier with 70MHz bandwidth
measured very low dark counts lessthan 10 ( ~aW level) at room
temperature. It enable the analysis of the photonenergy spectrum in
nanoseconds, which we call “Photon Spectroscopy” flowcytometry.
Challenges include how to reduce background counts in detection
optics,but it may be possible to open new aspect in live cell
analysis.
Conclusion: A hyperspectraldetection system with toroidal mirror
grating, 42CH fiber array and advancedelectronics was developed.
It is possible to evaluate spectrum range of UV340nmto IR800nm
with 10nm resolution. In principal, any current
commercialinstrument can be updated with hyperspectral detection
system. In addition, Sibase photomultiplier and very high speed
electronics enables the analysis ofevery photons energy level.
Photon Spectroscopy for live cell analysis suggests“analog to
digital” paradigm shift in cytomics.
253/B116
Standardization and Monitoring of Multiple Flow
Cytometers for Global Flow Cytometric Testing
Brahmananda Chitteti1, Christele Gonneau, Nicolas
Anfossi2, Virginia Litwin3
1
Hematology, Covance Inc., Indianapolis, IN, United States,
Hematology, Covance Inc., Geneva, Switzerland,
3
Covance, Indianapolis
2
Multi-parametric flow cytometry has unquestionably become the
leading technology for cellular analysis as it allows the
simultaneous detection of numerous cellular characteristics on
individual cells. The information from these multiplex
measurements can provide a detailed understanding of the lineage,
developmental stage, activation state, or disease state of the tissue.
Therefore, the use of flow cytometry in drug development process
and in companion diagnostics is increasing dramatically. Global
testing of clinical samples on multiple instruments at various sites
requires the standardized instruments that perform very precisely
across investigation sites. In order to minimize the variability in the
quality of results, we have implemented instrument-to-instrument
standardization at all Covance Central Laboratory Services (CCLS)
sites globally using Application Settings based BD Cytometer Setup
and Tracking (CS&T) beads. Standardized PMT settings and
compensation are used across all these instruments. “Setup and
Performance Checks and Monitoring” is also carried out across all
the instruments globally on a daily basis. Standardization is
monitored using Cytocal® beads [Biotron Laboratories, Inc.] to
ensure accuracy and reproducibility within our standardized
instruments. In order to test the robustness and precision of our
standardized instruments, and to evaluate the efficacy of Cytocal®
beads based monitoring system, we conducted an additional
quality assurance program using fluorochrome specific fluorescent
beads instead of hard dyed beads. For this pilot project,
fluorochrome specific Fluorescein isothiocyanate (FITC),
Phycoerythrin (PE), V450 beads were run on eight FACSCanto II
standardized instruments, and monitored the median fluorescent
intensity (MFI) of each peak for a period of four weeks. Results
indicate that, within the single standardized instrument, the
coefficient of variation (CV) of MFI was less than 3% (Range: 0.382.9 %CV) for FITC channel, less than 4% (Range: 0.73-3.73 %CV)
for PE channel, and less than 4% (Range: 1.3-3.48 %CV) for V450
channel. When all eight standardized instruments were compared,
the grand mean MFI for all three channels was within the ±2
Chase ES, Hoffman RA. Cytometry. 1998 Oct 1;33(2):267-79.
2. Characterization of flow cytometer instrument sensitivity.
Hoffman RA, Wood JC. Curr Protoc Cytom. 2007 Apr;Chapter 1:
Unit1.20.
Tuesday
14 June
256/B119
Recent Advances in Instrumentation in Cambridge
Biomedical Research Centre Cell Phenotyping Hub
Wednesday
15 June
The lack of options in BD-produced water-cooled Sort Collection
Tube Holders for BD FACSAria was addressed by 3D-printing using
ProJet™ 3510 HDPlus printer (3D Systems, Rock Hill, SC) at the
Rockefeller University Precision Fabrication Resource Center.
Several new designs of tube holders were created and thoroughly
tested. Being relatively cheap and simple to produce, these holders
allow for greater versatility and convenience in sorting without
scarifying any quality of the experiment.
References:
1. Resolution of dimly fluorescent particles: a practical measure of
fluorescence sensitivity.
255/B118
A Semi-automated Approach to Tracking and
Maintaining Instrument Performance on a BD Influx
Flow Cytometer
A large number of cell-surface and intracellular markers have been
identified in recent years, allowing for more accentuated
differentiation and in-depth characterisation of cell subsets and
their functions. The need to employ an ever growing number of
simultaneous parameters in cell phenotyping has spurred
developments of new fluorescent dyes and the advent of the
generation of more sophisticated flow cytometers to accommodate
this demand.
1
Index
197
Speaker/Author
Three instruments in our shared resource laboratory (the Hub) have
been upgraded within the last 12 months to keep abreast with the
technological developments in reagent chemistry and
instrumentation (and enable first steps in deep phenotyping). Aria III
cell sorter, previously equipped with only 2 violet channels, has
been upgraded to 6 PMTs which can detect signals from 6 BV
Poster Session
Abstracts
Method: Here we describe a system that combines optics and
stream alignment using small picomotors, with performance
tracking using commercially available CS&T beads. The system is
The ultraviolet (UV) laser has triggered the development of a family
of new fluorescent dyes and pushed the boundaries for designing
truly polychromatic immunophenotyping panels. The brilliant
ultraviolet dyes (BUV395, BUV496, BUV737, and BUV805) have
recently been developed for use specifically with UV laser and
provide an excellent addition to the existing library of fluorescent
probes. The addition of a UV laser and the combination of UV dyes
with other fluorochromes such as BV dyes makes it possible to
analyse up to 18 fluorescence parameters in a single sample while
addressing some of the challenges of complex compensation
matrices.
Oral Session
Abstracts
Background: Software to track flow cytometric instrument
performance is becoming increasingly widespread and is now
commonly used in industrial, clinical and research fields.
Performance tracking requires a characterisation of the instrument,
generally expressed by how efficient each detector is at measuring
fluorescence emissions (Q), and what the background contributions
are to those measurements (B). Once the instrument has been
characterised, variations from those initial “baseline” values can be
recorded and compared over time in order to ensure some level of
reproducibility to the results that are obtained. To meet this need,
various software solutions (CS&T, QbSure etc) have been
developed that are based on the founding works of Hoffman and
Chase 1,2, with small modifications aimed at improving accuracy, a
necessity due to hardware related discrepancies that exist between
instruments.
Novel dyes excitable by the violet laser such as BV421, BV510,
BV570, BV605, BV650, BV711, and BV785 are spectrally
compatible with each other and can be used for simultaneous
labeling. They are characterised by the high brightness, which
makes them attractive for panel design involving identification of
rare cell subsets.
Commercial
Tutorials &
Exhibits
Flow Cytometry, Queensland Brain Institute, Brisbane,
Australia, 2Research, Flow Cytometry Consulting,
Viewbank, Australia, 3Flow Cytometry, Westmead Institute
of Medical Research, University of Sydney, Sydney,
Australia
Cambrdige, United Kingdom
Scholars &
Emerging
Leaders
Valeria Radjabova, Chris Bowman, Natalia Savinykh,
Anna Petrunkina
Poster
Session
Multiple designs of Custom Integrated Water-Cooled Sort
Collection Tube Holders for BD FACSAria (II-III) are currently used
at the Rockefeller University Flow Cytometry Resource Center and
have been published on "3D Print NIH" site (http://3dprint.nih.gov/)
in the format suitable for majority of 3D-printers.
Geoffrey Osborne1, Frank Battye2, Suat Dervish3
Monday
13 June
BD FACSAria (BD Biosciences, San Jose, CA) is an example of
expensive state-of-art instrumentation typically used at the Flow
Cytometry Shared Resource Laboratories (SRLs). Any bulk-sorting
experiment carried on BD FACSAria employs a tube holder, but the
variety of tube holders provided by the manufacturer (BD
Biosciences) is not enough to cover researcher's needs.
Results and Conclusions: Typical output generated by the system
when sub-optimally and optimally auto-aligned will be presented
and data showing typical and expected tracking performance will
be demonstrated. This system represents an improvement over
traditional jet-in-air cell sorting technology by removing the
subjective aspects of instrument alignment and then providing some
metrics for on-going quality assurance.
Sunday
12 June
1
Flow Cytometry Resource Center, Rockefeller University,
New York, NY, United States, 2Columbia College, Columbia
University, New York, NY, United States, 3The Fu
Foundation School of Engineering and Applied Science,
Columbia University, New York, NY, United States,
4
Precision Fabrication Resource Center, Rockefeller
University, New York, NY, United States
Saturday
11 June
Svetlana Mazel1, Benjamin Mazel1,2, Mark Mazel1,3,
James M. Petrillo4
Special
Lectures
254/B117
Custom Integrated Water-Cooled Sort Collection
Tube Holders for BD FACSAria
implemented on a BD Influx flow cytometer. The software interacts
with the picomotors (NewPort 8301 picomotor actuators) mounted
on the optics and focussing translators of the Influx. The system
then uses the data generated from the CS&T beads to determine
median, rCV and rSD values for each PMT and attempts a semiautomated optimisation of the optics and stream alignment using a
combination of live video feedback with live data streaming from
the cytometer. The process is iterative and aims to obtain and then
maintain the optimal stream position. The CS&T beads contain
“dim”, “mid” and “bright” populations based on their fluorescence
characteristics, that are linked to a known number of CD4
molecules via a bead lot file. Optimal PMT gains for each PMT are
set, based on the median position of the “dim” beads, then data
files are generated and stored to allow tracking of instrument
performance.
Congress
Overview
standard deviations (±2SD). These results supported the consistent
performance of our instruments in standardized manner. Our future
endeavors focus on using fluorochrome specific beads for all eight
channels on 22 instruments globally as a second layer of quality
assurance system for monitoring instrument-to-instrument
standardization.
Congress
Overview
Special
Lectures
Saturday
11 June
The current instrumental developments in the Hub offer exciting
opportunity to enhance our understanding of genotype-phenotype
associations by facilitating ‘Deep Phenotyping’ (extending
phenotypes into much greater detail).
Monday
13 June
These advances open wider horizons for our users and
collaborators. In a thought experiment, we have assembled a
potential 15+ colours panel compatible with both Fortessa analyser
and Aria Fusion sorter that provides a graphical illustration for the
research potential of the upgraded equipment. It needs to be
stipulated that for any specific project, an individualized panel
development and optimization must be pursued.
Sunday
12 June
dyes. Aria Fusion cell sorter has undergone configurational
transformation to be enabled with 5 pinholes, allowing to detect
fluorochromes excitable with spatially separated UV and violet
lasers simultaneously. LSR Fortessa analyser has been upgraded
with an additional UV laser. These cytometers are now fully
equipped for design, acquisition, analysis and sorting of cells with
complex phenotype. Moreover, the Hub does now house the new
Special Order Research Product, X30, a polychromatic analyser
capable to detect fluorescence signals in 28 detectors, including 6
UV channels.
257/B120
Acoustic Focusing for Single Cell Impedance
Spectroscopy
Daniel Kalb1, Jim Coons2, Claire K. Sanders3, Babetta
Marrone3
Index
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Scholars &
Emerging
Leaders
Poster
Session
Wednesday
15 June
Tuesday
14 June
1
Univ of New Mexico, Albuquerque, NM, United States,
CDC, Los Alamos National Lab, Los Alamos, NM, United
States, 3Los Alamos National Lab, Los Alamos
2
Background: Single cell electrical impedance spectroscopy (EIS)
uses multiple electric frequency tones, generally ~100kHz-20MHz,
to interrogate cells suspended in a conductive media flowing past
impedance sensing microelectrodes. Expanding upon the coulter
counter’s DC based measurement, the low frequency EIS
impedance parameters can still be used to probe the size of the
particle, while higher frequencies ranges have been shown to be
indicative of various physical features of the cell, including the
double layer capacitance, the membrane capacitance and the
cytoplasm resistance. This multiparameter analysis allows for a
label free classification of cells based upon their physical
characteristics. The signal to noise of an EIS system is related to the
ratio of the interrogating volume that the particle occupies and the
position of the particle within the electric field. To date, the chief
strategy to maximize this signal relative to the noise has been to
make small interrogating channels, just slightly larger than the cells
themselves ~20-40um large in each dimension. These small
channels are prone to clogging, generally requiring very dilute cell
populations and low volumetric throughputs which severely limit
overall event rates. The application of an acoustic standing wave
results in a tightly focused particle stream that will have a consistent
interaction with the interrogating field while maintaining channel
dimensions that are much less likely to cause clogging. Thus, the
use of acoustic focusing increases the throughput without
sacrificing the performance of the instrument.
Methods: The focusing acoustic field, driven by a 10MHz PZT, is
directly coupled into two commercial impedance spectroscopy
chips layouts with channel dimensions (W x L) of 75 x 75um for 2D
focusing and 75 x 25um for 1D focusing across with width. Two
cross-channel sets of electrodes pairs are continuously monitored
with IV converters leading into a high gain (100X) differential
amplifier. Impedance signals processed on the fly in LabVIEW and
parameters including the particles time of flight, the amplitude of
the signal and the phase shift are extracted.
Results: Initial results show good differentiation between both algal
particles and bead populations of various sizes with CVs of ~1015%. Additionally, higher frequency signals (~5-20MHz) are being
explored for non-size based biophysical changes. Micro-algal cell
lines (Chlorella vulgaris and Nannochloropsis salina) that
experience wide shifts in lipid content upon nitrogen deprivation
198
and being explored as model systems that experience a non-sized
based biophysical change.
Conclusions: EIS interrogation of cells has shown great promise as a
label free platform capable of differentiating cells based upon their
biophysical characteristics, however, the need for small
interrogation volumes on unfocused samples severely limits the
throughput of an EIS system. Tight focusing of the particles using
our acoustic focusing EIS system allows for higher event rates and
volumetric throughput while maintaining consistent interaction
with the interrogating EIS field.
258/B121
New Capabilities for Characterization of Expanded
Fluorescent Protein Reporter Cell Lines Using
Microcapillary Cytometry
Katherine Gillis, Mike Farbarik, Patrick de Borja, Vy
Nguyen, Sumeet Ahuja, Kamala Tyagarajan
Research and Development, EMD Millipore, Hayward, CA,
United States
The use of fluorescent protein reporters to study function,expression
and localization of proteins of interest has greatly expanded
inrecent years and now includes a wide variety of reporters such as
RFP, YFP,mCherry and dsRed in addition to the popularly used GFP
reporters. Co-expression studies have also become commonto
study the inter-relationship between multiple proteins
simultaneously.Common questions span transfection efficiency,
expression level, transcriptionand differentiation, or fundamental
cellular mechanisms; cell health, cellcycle and other related areas.
These studies need access to flexible yetsimplified cytometric
systems that are often equipped with multiple lasers withthe ability
to look at multiple fluorescent proteins and/or markers ofinterest to
allow for characterization of these populations. Traditional blue
laser based cytometricsystems have limited detection capabilities to
evaluate the range offluorescent proteins being studied. Here we
present data from the new models ofthe Guava® easyCyte
instrument portfolio to address the expanding needs of
fluorescentprotein researchers which now include models with
Blue/Green, Blue/Green/Violetand Blue/Green/Red lasers with 10,
12 and 14 channels of detection. These combinations now allow
for flexibilityin the use of fluorescent protein, capability for
multiplexed detection offluorescent proteins as well as evaluation
of GFP or other fluorescent proteinsin combination with
phenotyping or cell health markers in different channels. The
addition of the green (532nm) laser provides flexibility to
evaluatemCherry transfected cells

program book

Transcription

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