GenVault`s Ambient-Temperature Technologies for Biospecimen

GenVault’s Ambient-Temperature
Technologies for Biospecimen Management
Michael Hogan
Chief Scientific Officer
GenVault Corporation
P3G Meeting
Luxembourg
September 30, 2009
© 2009 GenVault – Risk Free Sample Management
Slide -
1
The Core Physical Chemistry of GenVault Technologies
Oxidation
Rate
Dimension
Lipids
ROS
&
Heat
The Chemical Stability of Biomarkers
During Storage
Can be Approximated as a Plane
In “Oxidation, Hydrolysis” Space
Aromatic
Proteins
DNA
Non-aromatic
(0,0)
Proteins
Means
No oxidation
Or hydrolysis
H20, Catalysts & Heat
© 2009 GenVault – Risk Free Sample Management
RNA
Hydrolysis
Rate
Dimension
Slide -
2
Physical Chemistry of Cryogenics vs Ambient-Temp Preservation
• Traditional Cryogenics Solution:
“Collapse the plane” to the origin by cooling the
specimen, to slow oxidation & hydrolysis, based on the
temp dependence of the chemistry.
Use mechanical energy to “quiet” the sample by cooling,
keeping the underlying chemistry the same.
• The GenVault Ambient Temperature Solution:
“Collapse the plane” to the origin by removing water,
adding ROS scavengers, and eliminating O2 exposure.
Use chemistry to “quiet” the sample: -H2O, -ROS
keeping the temperature the same.
© 2009 GenVault – Risk Free Sample Management
Slide -
3
The Core Physical Chemistry of GenVault’s Technologies
Oxidation
Rate
Dimension
Lipids
ROS
&
Heat
The Chemical Stability of Biomarkers
During Storage
Approximated as a Plane
In “Oxidation, Hydrolysis” Space
Aromatic
Proteins
DNA
(0,0)
The
Specimen
Is at rest
Non-aromatic
Proteins
H20, Catalysts & Heat
© 2009 GenVault – Risk Free Sample Management
RNA
Hydrolysis
Rate
Dimension
Slide -
4
Cryogenics vs Ambient Temp Preservation
Practical advantages result from ambient temp stabilization:
Upon removing water and adding ROS scavengers:
The Specimen is Poised at Equilibrium,
in the absence of applied mechanical energy
-No need for energy to maintain -20C, -80C, -190C during storage
-No need for an energy-source backup to mitigate risk
(no generators, no LN reservoirs)
-No need for high-cost to maintain +4C or -20C during shipping
(no cryopacks)
-No need for high-cost, just-in-time, shipping protocols
(international book-rate instead)
Obviating cryogenics: in biobanking & sample sharing
© 2009 GenVault – Risk Free Sample Management
Slide -
5
The GenVault Technologies: Span the Biobanking Spectrum
PRESERVE
TRANSPORT
BANK
RECOVER
ANALYZE
GenPlates
GenTegra
GenSolve Automation
Dynamic Archive
Personal Archive
GenConnect
GenCode ID
© 2009 GenVault – Risk Free Sample Management
Slide -
6
Preserve = GenPlates -> Paper “Pop-Out” Hemispheres
GenPlate
• Treated macroporous Whatman filter paper
technology: exclusive license to GenVault
• H2O removed, ROS quenched by protein &
additives
• Proven stability, 20 years and counting
• High quality DNA recovery: with GenSolve
Enhancements
• Designed for a wide variety of ‘raw’ sample types
blood, buffy coat, homogenized tissue
• Contact inactivation of bacteria and viruses
• Proprietary biological barcode at aliquot level
GenCode ID = internal chemical bar code
© 2009 GenVault – Risk Free Sample Management
Slide -
7
“GenSolve “ Technology Enables DNA Recovery from
Dried Blood Spots on Very-Old Filter Paper Disks
•
•
•
•
•
•
•
•
Use 11-15 year old Blood Spot Cards
From California Birth Defects Cohort
Collaborators at March of Dimes & State of California
Two 3mm punches pooled per donor
From 1993-1998
Recover DNA with GenSolve, then Qiagen
>250ng of DNA from each
Illumina GWAS on each:
BMC Genetics 2009 Jul 22;10:38.
Whole genome microarray analysis, from neonatal blood cards.
Hardin J, Finnell RH, Wong D, Hogan ME, Horovitz J, Shu J, Shaw GM.
© 2009 GenVault – Risk Free Sample Management
Slide -
8
DNA Recovered from 11-15 year old Filter paper is >50kb
© 2009 GenVault – Risk Free Sample Management
Slide -
9
DNA from 11-15 Year Old Dried Blood -> high quality GWAS
Specimen
Name
No Calls
Calls
Total
Call Rate
(%)
MOD 881
1484
591008
592492
99.75
MOD 892
1581
590911
592492
99.73
MOD 919
1502
590990
592492
99.75
MOD 937
1607
590885
592492
99.73
MOD 920
2524
589968
592492
99.57
MOD 975
2840
589652
592492
99.52
MOD 986
2432
590060
592492
99.59
MOD 001
3240
589252
592492
99.45
Primary DNA
Isolate
© 2009 GenVault – Risk Free Sample Management
Slide - 10
Track Samples = GenConnect
•
•
•
•
Manages complex ambient-state and cryogenic storage media
And associated sample data, if needed, in a robust database
Enables Federated “virtual” biobanking among many sites
Enables a secured, multiple-user multiple-role collaborator group
Sample and Storage Data
Freezers
LN Tanks
GenConnect
Downstream Data
Clinical/ Patient Data
GenVault - Ambient Temperature Storage
Solutions
© 2009 GenVault – Risk Free Sample Management
Slide - 11
Bank & Transport Samples = GenTegra (DNA & RNA)
© 2009 GenVault – Risk Free Sample Management
Slide - 12
GenTegra-DNA
For purified DNA transport and banking
•
•
•
•
•
•
•
Mineral Matrix to dehydrate DNA
Antioxidants to scavenge ROS
Water soluble and inert to downstream analysis
For large (50ug) and small (50ng) DNA alloquots
No post-recovery cleanup
Just mix and air dry.
Microfuge Tube & 96 well plate formats
© 2009 GenVault – Risk Free Sample Management
GenTegra DNA
Slide - 13
ctrl
GenTegra DNA Performance at the Extremes of Temperature
RT
37°C
56°C
76°C
Affy 6.0
40
kb
Infinium
1M 40
kb
Affy 6.0
Infinium 1M
Affy 6.0
Concordance
Infinium 1M
Call Rate
Frozen
Control
99.50%
99.80%
GenTegra GenTegra GenTegra
RT
56°C
76°C
99.40%
98.70%
99.20%
99.70%
99.80%
99.70%
99.80%
97.70%
99.70%
99.90%
99.90%
99.90%
© 2009 GenVault – Risk Free Sample Management
Slide - 14
GenTegra-RNA: How to make RNA a Stable Biomarker
GenTegra™ RNA
• Inert, mineral matrix, plus antioxidants and Small-MW RNase inhibitors
• Stabilizes purified RNA upon contact in the fluid phase (days)
• Then long-term stabilization upon air-drying (years)
• Two-fold stabilization @ ambient temperature
• In the fluid-phase: during routine lab preparation and incubation,
without bench-top cryogenics
• In dry-state: during long term storage
without storage cryogenics
• Ideal for global RNA transport among biobanks
• Preserves RNA integrity during exposure to harsh shipping
temperature fluctuation (up to +60C)
• $(€) savings in resources and budget; eliminates high cost of
shipping wet/refrigerated samples
Slide - 15
GenTegra-RNA Development. One Month Dry, at 56C
15ug rat liver
RNA(Qiagen)
Dry ON at
RT
Matrix
RT
Rehydrate
33days at
RT / 56 C
Bioanalyzer
56 C
( ~264 day at RT)
+ Matrix
No Matrix
Frozen
Ctrl
Electropherogram showing RNA Integrity & Breakdown
28S:18S Ratio = ribosomal species 28S and 18S ratio; in part
linked to instability of the 28S rRNA structure relative to the
18S RNA. Decrease in the area of the 28S rRNA peak
reflects breakdown.
Slide - 16
GenTegra-RNA Development. 20 hrs in Solution, 37C with RNAse
Matrix
RNAse A/1
20hrs at 37 C
gel
+ Matrix
+ RNase 1
No matrix
No RNAse
+ Matrix
+ RNase A
+ RNase 1
+ RNase A
Frozen ctrl
1ug Hela RNA
(20ul)
Slide - 17
GenTegra-RNA Development . One Month Dry, at 56C, with RNAse
4 weeks at
RT / 37 C
Dry state storage :
Rehydrate
10min
90min 37 C
Gel
Frzn ctrl
Frzn ctrl
Dry ON at
RT
Frzn ctrl
Matrix
RNAse A/1
1ug Hela RNA
(20ul)
RT RT 37 37
+ Matrix
+ RNAse A/1
RT RT 37
+ Matrix
37
RT 37
+ RNAse A/1
Slide - 18
A Sample of Third PartyTesting: At a CLIA-Certified Lab
Representative GenTegra RNA Field Testing
DiagnoCure, Quebec City
Diagnostic Testing of the presence of Guanyl Cyclase C (GCC) as
a marker of occult metastases in FFPE pericolonic lymph nodes
from colon cancer patients
DiagnoCure Collaborators: Michel Houde, Guillaume Beaudry,
Genevieve Garon, Melanie Robitaille, Josianne Veer
Slide - 19
Testers Validate GenTegra at the Extremes of Temp Exposure
GenTegra™ RNA
• FedEx Shipping: can expose samples to up to +50C
• US Army Shipping: can expose samples to up to +70C
• Laboratory Medicine: fluid sample processing at 25C, 24 hrs
•
•
•
•
•
The Standard Test Plan:
Fluid phase RNA in GenTegra at 25C or 37C for 24 hrs
Dry State RNA in GenTegra at 25C, 37C, 56C for 2 weeks
Cells & high-value biopsies: fresh and FFPE
Use LoC Capillary Electrophoresis (Bioanalyser) = physical integrity
Use Q-rtPCR = biochemical integrity
Risk Free Sample Management Technologies
Slide - 20
GenTegra RNA Field Testing – FFPE and Frozen Tissues
Liquid Samples – 24h
• RNA tested (5ug):
– Colon (FFPE)
– Lymph Nodes (FFPE)
– T84 cells extracted with GCC
method
– T84 cells extracted with Trizol
– HelA cells extracted with Trizol
• Temperature tested:
– Room Temperature
– 37 C
Dry Samples – 2 weeks
• RNA tested (5ug):
– Colon (FFPE)
– Lymh Nodes (FFPE)
– T84 cells extracted with GCC
method
– T84 cells extracted with Trizol
– Liver (Fresh Frozen)
– Hela
• Temperature tested:
– Room Temperature
– 37 C
– 56 C
Slide - 21
Dry RNA Samples –T84 cells, Extracted with Trizol
2 weeks,
56°C
2 weeks,
Frozen
At -70°C
RNA degrades Over two weeks,
without GenTegra protection
Slide - 22
Liquid RNA Samples –T84 cells, Extracted with Trizol
24 hours in fluid solution at 25C or 37C
Slide - 23
T84 cell RNA: Q-rtPCR, after Two Weeks Dry, 56C
Two weeks dry at 56C
Actin Ct
Actin Copies
Sample ID
Mean Ct
RT
37°C
56°C
-70°C
StdDev Ct
Copies
(10+6)
StdDev
Copies
ratio
m1
14.69
0.78
3.91
2.2
63.4%
m2
14.55
0.24
3.95
0.62
63.9%
m0
13.98
0.26
5.84
0.99
94.4%
m1
13.36
0.43
9.10
2.57
147.2%
m2
13.87
0.07
6.22
2.82
100.7%
m0
14.27
0.20
4.79
0.65
77.4%
m1
13.82
0.32
6.54
1.44
105.8%
m2
13.95
0.33
5.98
1.25
96.8%
m0
14.00
0.40
5.84
1.49
94.6%
13.98
0.66
6.18
2.45
100.0%
Slide - 24
Lymph Node RNA: Q-rtPCR,
after Two Weeks Dry, 56C
Two weeks dry at 56C
Actin Ct
Sample Name
RT
37°C
56°C
-70°C
GCC Ct
GCC Copies
Copies ratio
Mean Ct
StdDev Ct
Mean
StdDev
Copies
StdDev
m1
24.64
0.10
23.25
0.30
18,305
3,560
82.2%
m2
24.28
0.09
23.01
0.19
21,495
2,807
96.5%
m0
23.54
0.07
23.05
0.23
20,946
3,439
94.0%
m1
24.91
0.04
23.13
0.31
19,900
4,343
89.3%
m2
24.61
0.13
23.10
0.25
20,243
3,610
90.9%
m0
23.38
0.08
23.06
0.26
20,806
3,806
93.4%
m1
25.51
0.09
23.27
0.10
17,844
1,178
80.1%
m2
24.88
0.03
23.09
0.19
20,260
2,549
91.0%
m0
23.74
0.12
23.12
0.21
19,950
2,900
89.6%
22.98
0.07
22.95
0.13
22,274
2,044
100.0%
Slide - 25
Technology in Development – Elastomer-Chaperone
• Self-contained sample acquisition, shipping & storage
– neonates, adult epidemiology, personalized medicine,
– DNA, most proteins, most small molecules
• 150uL elastomer matrix inside a Chaperone carrier
• Direct contact transfer from a pipet or heal, finger or ear-prick
The Elastomer-Chaperone Concept
Store blood on a 150uL Elastomer,
inside a protective tube with internal drying capacity &
2D bar code or RFID tag.
For ambient-temp acquisition, shipping and biobanking.
The Assembled Elastomer-Chaperone
The Cap can be touched to the skin or to a pipette.
The Cap is then screwed into the tube body.
Drying occurs over several hours, internally
Elastomer-Chaperone
Self-contained biospecimen preservation & carrier
2D Bar code or RFID tag = “Header”
© 2009 GenVault – Risk Free Sample Management
Slide - 26
Transport - Chaperone Performance - DNA
DNA Recovered from the Elastomer:
Whole blood, stored dry at RT or 56C for 34 Days
Native electrophoresis after Qiagen purification & picogreen quantitation
DNA Source:
Storage:
Condition:
Amt. on gel:
Blood
0days
Fresh
125ng
Blood
0days
Fresh
50ng
Elast.
15days
RT
125ng
Elast.
15days
56oC
125ng
Elast.
19days
RT
125ng
Elast.
19days
56oC
125ng
Elast.
34days
RT
125ng
Elast. Roche Roche
34days
56oC
125ng 50ng 250ng
% Recovery
100%
100%
77.2%
99.4% 102.7% 90.5%
111.2% 123.4%
DNA Yield:
2.4 g
2.4 g
1.9 g
2.4 g
2.7ug
2.5 g
2.2 g
NA
NA
At the DNA level, we have found that 100uL
of dried blood can be re-hydrated after up to
34 days of storage at RT or 56C (133F) by
addition of the same standard protease
solutions used to process fresh blood,
followed by standard protease treatment at
56C and then fluid release by a one minute
of centrifugation in a spin basket followed by
a standard Qiagen Mini prep column.
3.0 g
Bottom Line:
Picogreen quantitation shows ~100% recovery based on an expected DNA yield of 2.5+/-0.5µg from the same fresh
blood samples extracted prior to drying.
Gel band intensity shows that DNA remains >>40kb long over storage time
© 2009 GenVault – Risk Free Sample Management
Slide - 27
Transport - Chaperone Performance - Protein
Cancer markers, Acute Phase Reactants and other serum proteins - 1
Analytes were tested at Rules Based Medicine
(Austin TX) in a multiplexed fashion via the
Luminex-RBM bead immunoassay platform,
on 5 different specimen types: (1) serum
(SST), (2) EDTA-plasma (EDTA Pls), (3)
Heparin-plasma (Hep Pls), (4) Citrate-plasma
(Cit Pls) and (5) whole blood (WB) from 4
different healthy volunteers each. 100uL of
each sample was applied to 100uL Elastomer
elements, each with a different set of
chemical stabilizer treatment dried into the
Elastomer, and then air dried at RT for a day.
Upon drying, these specimens were sealed
and then stored at 25C for 28 days. The
specimens were rehydrated by adding 130uL
of water, incubation at RT for 30 minutes then
ejected from the Elastomer by spinning for
5min at 1000g in a microfuge spin basket
Data is presented as percent recovery for a fraction of those 114 nonzero protein analytes, comprising the multiplex panel of the most
abundant protein species
© 2009 GenVault – Risk Free Sample Management
Slide - 28
GenVault: P3G Summary
• Numerous ways to Introduce Ambient Temp Technology into Workflow
Replace Cryogenics for Nucleic Acid Transport Among Sites
Replace Cryogenics for Long-Term Nucleic Acid Storage
“The GenVault + Cryogenics Approach”
GenVault for the Nucleic Acids
(chemical stabilization of simple linear polymers like DNA & RNA)
Cryogenics for the rest
(cell viability & enzymatic activity is more than just a chemical problem)
For Example: at the April 2010 P3G Meeting, 2 hrs away in Chicoutimi
The CARTaGENE-Chicoutimi Biobank
-Automated GenVault “Dynamic Archive” for Blood Biobanking  DNA for Genomics
-Liquid Nitrogen & CryoStraws for Serum Biobanking  For Proteomics
-80C Freezers  For Flash Frozen Tissue
-All Storage Media Linked by GenConnect (Dry-Ambient, -190C, -80C)
© 2009 GenVault – Risk Free Sample Management
Slide - 29
CARTaGENE- Chicoutimi Biobank: GenVault + Cryogenics
GenVault’s Ambient-Temp Technology Coexists Nicely with Cryogenics.
GenConnect can Link it all Together
100,000
Plate
GenVault,
Fully
Automated
Archive
© 2009 GenVault – Risk Free Sample Management
Slide - 30