BAL

Transcription

BAL

ERS Annual Congress Amsterdam
26–30 September 2015
EDUCATIONAL MATERIAL
Educational Skills Workshop 20, 22, 24
Respiratory sampling techniques
Thank you for viewing this document.
We would like to remind you that this material is the
property of the author. It is provided to you by the ERS
for your personal use only, as submitted by the author.
©2015 by the author
Tuesday, 29 September 2015
ESW20 08:00–10:20
ESW22 10:40–13:00
ESW24 14:30–16:50
Room L002 RAI
You can access an electronic copy of these educational materials here:
http://www.ers-education.org/2015ew20
To access the educational materials on your tablet or smartphone please find below a list of apps to
access, annotate, store and share pdf documents.
iPhone / iPad
Adobe Reader - FREE
With the Adobe Reader app you can highlight, strikethrough, underline, draw (freehand), comment
(sticky notes) and add text to pdf documents using the typewriter tool. It can also be used to fill out
forms and electronically sign documents.
http://bit.ly/1sTSxn3
UPAD Lite - FREE
UPAD Lite is an advanced note-taking application with annotation features. You can handwrite notes,
highlight text, add sticky notes and reference images and export any type of document as a PDF or
PNG file by email or to cloud services.
http://bit.ly/1mQ1j0K
Noteability - $4.99
Noteability uses CloudServices to import and automatically backup your PDF files and allows you to
annotate and organise them (incl. special features such as adding a video file). On iPad, you can
bookmark pages of a note, filter a PDF by annotated pages, or search your note for a keyword.
http://bit.ly/TCrNad
Android
Adobe Reader - FREE
The Android version of Adobe Reader lets you view, annotate, comment, fill out, electronically sign
and share documents. It has all of the same features as the iOS app like freehand drawing,
highlighting, underlining, etc.
http://bit.ly/1deKmcL
iAnnotate PDF - FREE
You can open multiple PDFs using tabs, highlight the text and make comments via handwriting or
typewriter tools. iAnnotate PDF also supports Box OneCloud, which allows you to import and export
files directly from/to Box.
http://bit.ly/1p2SV00
ez PDF Reader - $3.99
With the ez PDF reader you can add text in text boxes and sticky notes; highlight, underline, or
strikethrough texts or add freehand drawings. Add memo & append images, change colour / thickness,
resize and move them around as you like.
http://bit.ly/1kdxZfT
Educational Skills Workshop 20, 22, 24
AIMS: Sampling the airways is a useful way of accessing “the core” of lung diseases. Since the
different techniques that are used to sample the airways can yield interesting immunological,
inflammatory, and clinical data, this workshop aims to provide practical skills for both clinical and
research purposes. The workshop does not require previous experience in this field. The workstations
will be organised to be interactive and to provide all the required skills needed to perform the
techniques.
TARGET AUDIENCE: Researchers, respiratory physicians interested in biological research,
biologists, pulmonologists, pathologists, technicians.
CHAIRS: C. M. Kaehler (Innsbruck, Austria), P.A. Schnabel (Homburg/Saar, Germany),
A. Spanevello (Tradate, Italy)
FACILITATORS: U. Costabel (Essen, Germany) , C. Kaehler (Innsbruck, Austria), P. Pignatti (Pavia,
Italy), H. Popper (Graz, Austria) , E. Schmutz (Heidelberg, Germany) , A. Spanevello (Tradate, Italy) ,
E. Thunnissen (Amsterdam, Netherlands), M. Wiebel (Heidelberg, Germany)
WORKSHOP PROGRAMME
PAGE
Four 30-minute workstations run simultaneously on the following topics:
Workstation 1 – Clinical cytology
TBNA and FNA – Cytology and R.O.S.E. rapid on-site evaluation
Workstation 2 – Histology
5
11
Workstation 3 – Sputum collection
Summary
Information of best practice
94
126
Workstation 4 – Analysis of BAL fluids
128
Additional resources
224
Faculty disclosures
225
Faculty contact information
226
Answers to evaluation questions
227
Regular physical activity
improves quality of life and
fitness in healthy individuals
and people living with
lung conditions.
In partnership with:
www.healthylungsforlife.org
Farbe/colour:
PANTONE 288 CV
ERS/ELF ER Journal 210x280 ad_v2 AW.indd 1
29/07/2015 16:22
TBNA and FNA – Cytology and R.O.S.E. rapid on-site evaluation
Dr Matthias Wiebel
Thoraxklinik at University of Heidelberg
Amalienstrasse 5
69126 Heidelberg
GERMANY
[email protected]
AIMS
Demonstration of practical aspects of ROSE with creating fast stained smears and highlighting
favoring aspects and problems
SUMMARY
Mediastinal structures like lymph nodes (LN) or tumor need to be assessed in the diagnostic evaluation
and staging of benign and malignant diseases. Invasive procedure like mediastinoscopy or thoracotomy
can be replace by transbronchial needle aspiration (TBNA) or fine needle aspiration (FNA). The
introduction of ultrasound guided fine needle aspiration using specially developed bronchoscopes and
gastroscopes have greatly improved the localization of mediastinal lymph nodes and the yield of fine
needle cell aspiration (FNA, TBNA).
The amount of cells collected in a sample depends on:
1. The size of LN, the puncture site and directabilty of the needle, 2. The underlying pathology (tumor,
necrosis, vascularization), 3. The number of needle movements inside the LN, 4. The number of
punctures (repeated sampling) in one LN position.
The result will also be influenced by the bronchoscopist:
1. Personal experience in EBUS and/or EUS,
2. Optimization of the endoscopic situation (appropriate sedation, application to the specific
procedure),
3. Evaluation of the specimen retrieved (blood, clot, lymph liquid, necrosis, pus),
4. Ability to produce a good smear (mainly monolayer, no or little clots, no or little crushing artifacts
due to the smearing process)
The further processing includes:
Smearing of specimens on glass slides and staining (May-Grünwald-Romanowsky / Giemsa with
airdrying fixation; Papanicolau with ethanol spray fixation and secondary glass covering of the slide),
and / or collecting the aspirated material in a container with a fixation medium (alcohol, formalin) for
cytoblock techniques.
Follows the cyto-pathological evaluation:
The time needed depends on the cell numbers displayed on the smears, the ability to detect the origin of
cells (lymphatic, tumor, necrosis, inflammatory). The additional amount of blood “dilutes” the cells
needed to produce a diagnostic statement. The time needed is also depending on the numbers of slides
to be evaluated. All in all the inspection for adequate material will reach from seconds to a couple of
minutes. In addition every slide needs to be marked for patient data and site of puncture. In cases with
less preserved vitality of cells time for evaluation will extend up to 15 minutes.
5
The results will be available within hours to days after the invasive diagnostic procedure.
The report will quote for:
1. Appropriate material: cells numbers, smearing quality (monolayer, undamaged cells) or
inappropriate material: blood, scarce cells, artifacts like crushing of cells, reduced vitality or
necrosis of cells.
2. Pathologic cells identified: tumor cells or aggregate, epithelioid cells or aggregates, macrophages
and materials phagotized, inflammatory cells.
3. Additional specific staining for tumor identification may follow on spare slides.
The report will summarize the findings including the patient history: no pathology detectable or
epithelial malignancy with differentiation into small cell cancer, non-small cell cancer (possibly
suggesting squamous cell cancer or adenocarcinoma, metastatic from other known malignancy),
malignant lymphoma, or epithelioid cells suggestive of granulomatous disease.
ROSE
The standard way of TBNA/FNA will produce situations with non-sufficient material for diagnosis. A
further/additional (repeat) diagnostic procedure will be necessary: this means a severe burden for the
patient due to the invasive procedure, a loss of time to start with therapy, additional costs. The same is
true for the medical system.
A possible solution is advocated with the rapid evaluation of material on site (ROSE) retrieved by
FNA/TBNA: Using a fast staining method (one to three minutes) and a microscope in the endoscopy
room with an experienced microscopist, slides can be evaluated as to sufficient cellularity (lymphocytes
or /and other cells in sufficient amount). Eventually diagnosis may be given as well (tumor,
granulomatosis etc.). The main aim will be to quote whether the material gained is sufficient for
diagnosis or an additional puncture is needed.
Structures needed for ROSE:
1. a microscope
2. rapid staining dyes, 90-second process
a. air-dried smear. Romanowsky Wright-Giemsa stain (a combination of Wright and Giemsa
stains), and the buffered Wright-Giemsa stain, (four steps: buffer, eosin Y, azure B, and
methylene blue) (e.g. Diff Quick). Technician for primary evaluation will be sufficient.
b. ethanol spray fixation of smear. Ultrafast Papanicolaou stain, achieves comparable visual
clarity in significantly shorter time (use 4% formaldehyde in 65% ethanol or 90% ethanol
fixative, Richard-Allan Hematoxylin-2 and Cyto-Stain. The presence of a cytopathologist is
needed.
Optimization of ROSE results:
Even though ROSE is intended to be fast in producing results time is needed for a reliable statement:
the materials have to be marked, the staining and microscopic evaluation need to be correct. Thus three
to five minutes per single ROSE slide series are appropriate and should be respected by the endoscopist.
An initial learning curb to get a standardized habitual process should be accepted by all involved.
6
In the recently published Guidelines of TBNA acquisition and preparation of specimens the following
questions are quoted (citation in italics):
Can Rose increase the diagnostic yield? No. Can Rose decrease the number of aspirations? No. But
additional punctures may be helpful in the individual setting.
Can Rose decrease the procedure time? No. Can Rose decrease the number of additional procedures?
Yes. Rose may help avoid an additional bronchoscopy.
Can Rose decrease the rate of complications? No. The procedure are mainly unchanged, but the time of
the procedure may be prolonged.
Is ROSE more cost-effective? There is no significant evidence for or against cost. But the time needed
by the cytopathologist or the technician may be important.
What is the concordance of ROSE with the final diagnosis? The concordance is reported to be high.
Is there an optimal staining method? No, but Diff Quick seems to be the most applied staining set.
Who needs to perform ROSE? Cytopathologists will be able to evaluate specimens for adequacy and
put forward a probable or even definitive diagnosis. Technicians and seemingly trained
pulmonologists may at least decide on sufficiency of specimens.
TBNA and FNA add importantly to the armamentarium of diagnostic procedures in pulmonology. The
addition of Rose can improve and speed up the diagnostic process. The successful implementation of
ROSE requires dedicated endoscopists and cytopathologists.
REFERENCES
1. E.H.F.M. van der Heijden et al. World Association for Bronchology and Interventional
Pulmonology. Guideline for the acquisition and preparation of conventional and endobronchial
ultrasound-guides transbronchial needle aspiration specimens for the diagnosis and molecular
testing of patients with known or suspected lung cancer. Respiration 2014;88:500-517
7
TBNA droplet and smearing procedure
Slide smearing. Left: clot dispersion by dragging the material similar to dab smear in solid biopsy.
Right: saving spilt over material
Left: Fast staining Hemacolor (like DiffQuick). Right: spray fixation for rapid Papanicolau staining
8
Smear slides: (left to right) almost empty; broad droplet smear slightly uneven with fine aggregates at
the end; drag smear of droplet with diminishing thickness; droplet with local coagulation ring and thin
smearing
Smear slides: left thick droplet thin smear, right coarse spreading
Micoscope workplace with electronic camera and screen
9
Microscope screenshot:left: mainly monolayer activated lymphocytes; middle left: monolayer
anisokaryosis and moulding in SCLC; middle right: squamous cell cancer and necrosis; right:
anisokaryosis and preserved cytoplasm
Smear slide: left: anthracosis with damages macrophages; middle lymphocytes and macrophages;
right: epithelioid granuloma and lymphocytes
Copyright for all microscopic images by Dr. M. Wiebel, Heidelberg, Germany
10
Prof. Helmut H. Popper
Pathology
Medical University Graz
Auenbruggerplatz 25
8036 Graz
AUSTRIA
[email protected]
Dr Erik Thunnissen
Pathologie
VU University Medical Center
De Boelelaan 1117
1081 HV Amsterdam
NETHERLANDS
[email protected]
AIMS
To understand:
 different respiratory sampling techniques, and
 interaction required with pathologists to obtain as much analysis as possible.
SUMMARY
Success in the cyto-histological diagnosis of lung cancer depends upon, amongst other things, the
correct sampling and processing of tissue. Summarised by a working group.[1]
Sampling techniques
Bronchial washing can be taken before or after biopsy sampling. Recommended is to recover as much
as possible washing fluid after instilling 20-30 ml of isotonic saline.
BAL increases the diagnostic yield in pulmonary peripheral lesions and is primarily used for diagnosing
infections and interstitial lung diseases.
Bronchial brushing is very useful in endoscopically visible tumours. Care should be taken to avoid
withdrawing the unsheathed brush through the working channel of the bronchoscope. The area of
suspected malignancy should be brushed two or three times.
Sputum is not recommend sputum as suitable material for diagnosis of lung cancer, due to its low
sensitivity. However, in patients, who are unable to have a bronchoscopy or other minimal invasive
procedure and where only diagnosis of malignancy is needed, sputum analysis may be helpful. Repeated
sputum sampling over several days increases the sensitivity. Shedding of tumor cells can also be
provoked by inhalation of prewarmed isotonic sodium-chloride solution prior to sputum collection.
Transbronchial needle aspiration cytology is frequently used. The diagnostic yield of conventional
(non/ultrasound guided) transbronchial needle aspiration (TBNA = without endobronchial ultrasound,
synonymous “EBNAB”) is high for endoscopically visible bronchial abnormalities, suggesting
neoplasia, particularly when the lesion is due to extrinsic compression, submucosal infiltration, or an
exophytic mass.
11
Linear Endobronchial Ultrasound (EBUS)-TBNA has been shown to have a high-pooled sensitivity of
93% and specificity of 100% for the confirmation of malignancy.
Endoscopic ultrasound (EUS) is especially useful for sampling posterior mediastinal and
paraoesophageal lymph nodes (stations 4L, 7,8 and 9). In addition the left adrenal can be accessed and
identified in 97% of cases. Combined EBUS and EUS is as sensitive as surgery for staging the
mediastinum in lung cancer.
Biopsies of endobronchial visible tumours have a yield of 75-95% for a histological diagnosis of
malignancy, after taking at least three forceps biopsy samples.
In patients with solitary peripheral pulmonary nodules, the endoscopic diagnostic procedure is usually
performed as transbronchial lung biopsy (TBBx) under fluoroscopic or stereologic guidance. However,
for peripheral lesions transthoracic needle biopsy (core needle or needle aspiration) is frequently used,
because of the larger sample size.
Rapid On Site Examination (ROSE) is a quick cytological examination for the presence of tumour or
lymphoid cells by pathologist or trained person. Initially, ROSE was set up for conventional (nonultrasound guided) TBNA, for confirming the representativity of the sample. However, with the aim of
obtaining as much tumour material as possible to allow more biomarker testing, the original goal is
redundant, and additional needle passes may be required to obtain further tissue for molecular testing.
Video assisted thoracoscopic (VATS) or surgical open lung biopsies re used for diagnosis of interstitial
lung diseases or lung tumors.
Resection (bi-lobectomy, pneumectomy) is usually performed as treatment of lung cancer. Each
procedure involving cytology and histology has their own artifacts at microscopic level.
Preservation of tissue architecture can be achieved by either instillation of freezing medium diluted 1:2
by PBS (frozen section diagnostics), or formalin. Lung tissue can thus be expanded and artificial
collapse can be counteracted.
Pathology
The best strategy for the handling of small samples in suspected lung cancer is always based on accurate
and relevant clinical information 1) sample site(s), 2) Whether the clinical suspicion is on primary
tumour / metastasis; 3) Question diagnosis? If appropriate: also predictive test (s)?; 4) Previous relevant
surgical, oncological, radiotherapeutic treatment, if any; 5) Smoking history (never, ex (pack years,
years since quitting), current smoker (pack years, years of smoking).
This information will be used by the pathologist to i) determine priorities of the diagnostic approach
(see below), ii) determine how specific a diagnosis is required, iii) plan the necessary investigations and
anticipate the use of IHC and molecular biological tests, and iv) prevent unnecessary usage of tissue on
tests which are not required or irrelevant given the clinical picture. Thereby, preserving scarce tissue
for the most important tests required in each particular case.
Cytology is a powerful tool in the diagnosis of lung cancer. The advantages of diagnostic cytology are
that it is a minimally-invasive, simple procedure, helps in faster reporting, and it is relatively
inexpensive. The accuracy of the cytological examination depends greatly on the quality of collection,
preparation, staining and interpretation of the material. However due to small sample size possibility
for predictive analysis is limited.
12
Many cases can be adequately diagnosed (identified and typed), within the limits imposed by the nature
of the specimen, using standard morphological stains (H&E, mucin stain or cytological stain(s) of
choice).
The first step is the identification of malignancy and in clinical suspicion of lung cancer, the positive
identification of carcinoma, as opposed to alternatives: i) lymphoma, sarcoma or other malignancy,
ii) where relevant, in situ, non invasive, pre-invasive or reactive changes (hyperplasia, dysplasia etc),
iii) non-neoplastic pathological processes (inflammation, benign tumours, etc) and iv) artefacts which
may mimic neoplasia.
If carcinoma is identified, it may be assumed in many centres that the default diagnosis is primary lung
cancer, but an adequate clinical history is essential to indicate if there is any suspicion or possibility
that the lung tumour under investigation could be metastatic disease. The pathologist must always be
alert to the possibility of metastatic disease, however primary lung cancer has a wide range of
appearances. In the absence of a suggestive history, metastatic disease may be suspected from the
morphological appearance of the carcinoma. The unnecessary or inappropriate pursuit with
immunohistochemistry (IHC) or exclusion of a diagnosis of extra-thoracic carcinoma, metastatic to lung
can potentially waste a great deal of time, laboratory resources and valuable tissue, since many options
are possible and specificity is notoriously low.
After accepting the tumour as primary lung carcinoma, the next step is to determine whether the tumour
represents small cell lung cancer (SCLC) or non-SCLC (NSCLC). Up to around 40% of NSCLC cases,
it will not be possible to ascribe a NSCLC subtype by morphology alone (NSCLC not otherwise
specified, NSCLC-NOS). Using IHC the percentage of unclassifiable NSCLC, NOS is reduced to
around 4%. Such cases should NOT be diagnosed as large cell carcinoma, but remain NSCLC-NOS.
Proactive block cutting (based on clinical information) and reflex testing prevents wasting by avoiding
recutting the block, but at the same time also holds a risk of losing quality of epitopes and DNA when
not used shortly after cutting. Additional sections may be taken from a small sample tissue blocks at
the time of initial sectioning for morphological diagnosis, in anticipation of IHC and predictive analysis.
REFERENCES
1. Thunnissen E, Kerr KM, Herth FJ, Lantuejoul S, Papotti M, Rintoul RC, Rossi G, Skov BG,
Weynand B, Bubendorf L, Grunberg K, Johansson L, Lopez-Rios F, Ninane V, Olszewski W,
Popper H, Jaume S, Schnabel P, Thiberville L, Laenger F. The challenge of NSCLC diagnosis and
predictive analysis on small samples. Practical approach of a working group. Lung Cancer
[Internet] 2011/12/06 ed. 2012; 76: 1–18
Available from: http://www.ncbi.nlm.nih.gov/pubmed/22138001.
EVALUATION
1. Rapid on site evaluation of during cytology sampling for lung cancer is performed with EBUS/
EUS /TTBA. What is appropriate for predictive analysis?
a. When a node is negative continue sampling.
b. When node is positive continue sampling same node.
c. When node is negative go to next enlarged node.
d. When node is positive go to next enlarged node.
13
2. If a transbronchial biopsy did not yield in diagnostic tissue in lung cancer or an interstitial
disease, what would you do next?
a. repeat TBA
b. proceed to VATS.
c. perform EBUS TBNA
d. perform BAL
3. Can an UIP diagnosis we made on cryobiopsies?
a. no
b. yes
c. yes, but a differential diagnosis of underlying diseases is most often not possible
d. no, the material is not sufficient
4. On cytologic specimen the diagnosis of mucin negative NSCLC NOS was made. What should be
done next?
a. Immunohistochemistry for TTF1 and p40
b. Immunohistochemistry for p63 and cytokeratin 5/6
c. Immunohistochemsirty for Surfactant Apoprotein A and cytokeratin 7
d. Immunohistochemsirty for cytokeratin 7 and napsinA
5. Does it matter, if a lung tissue specimen is placed in a large container with formalin, or is any
container suitable?
a. does not matter
b. does matter, but I don’t know why
c. does matter, because of fixation artifacts induced by squeezing the tissue
d. does matter, because the tissue can stay therein for many hours
6. Can molecular testing be done on bone biopsies?
a. yes, bone metastasis is equal to any other metastasis specimen
b. no, bone metastasis shows less genetic changes than other metastasis
c. no, DNA is degraded by acidic solutions used for decalcification
d. yes, by decalcification normal tissue is destroyed and cancer tissue remains
14
RESPIRATORY SAMPLING TECHNIQUES
Helmut Popper
Erik Thunnissen
15
Conflict of interest disclosure Popper
 I have no, real or perceived, direct or indirect conflicts of interest that relate to
this presentation.
 I have the following, real or perceived direct or indirect conflicts of interest that
relate to this presentation:
Affiliation / financial interest
Nature of conflict / commercial company name
Tobacco-industry and tobacco corporate affiliate related
conflict of interest
NO
Grants/research support (to myself, my institution or
department):
Unrestricted research grants from
Eli Lilly (IALT-BIO), Astra Zeneca, Pfizer, Hofman La Roche,
Boehringer-Ingelheim
Honoraria or consultation fees:
Advisory board honoraria from
Hofman La Roche, Eli Lilly, Boehringer-Ingelheim, Novartis, Pfizer
Participation in a company sponsored bureau:
NO
Stock shareholder:
NO
Spouse/partner:
NO
Other support or other potential conflict of interest:
NO
This event is accredited for CME credits by EBAP and speakers are required to disclose their potential conflict of interest going
back 3 years prior to this presentation. The intent of this disclosure is not to prevent a speaker with a conflict of interest (any
significant financial relationship a speaker has with manufacturers or providers of any commercial products or services relevant to
the talk) from making a presentation, but rather to provide listeners with information on which they can make their own judgment.
It remains for audience members to determine whether the speaker’s interests or relationships may influence the presentation.
Drug or device advertisement is strictly forbidden.
16
Conflict of interest disclosure Thunnissen
 I have no, real or perceived, direct or indirect conflicts of interest that relate to
this presentation.
 I have the following, real or perceived direct or indirect conflicts of interest that
relate to this presentation:
Affiliation / financial interest
Nature of conflict / commercial company name
Tobacco-industry and tobacco corporate affiliate related
conflict of interest
Na
Grants/research support (to myself, my institution or
department):
Na
Honoraria or consultation fees:
Eli Lilly support meeting European experts 2010
Participation in a company sponsored bureau:
Na
Stock shareholder:
Na
Spouse/partner:
Na
Other support or other potential conflict of interest:
Na
This event is accredited for CME credits by EBAP and speakers are required to disclose their potential conflict of interest going
back 3 years prior to this presentation. The intent of this disclosure is not to prevent a speaker with a conflict of interest (any
significant financial relationship a speaker has with manufacturers or providers of any commercial products or services relevant to
the talk) from making a presentation, but rather to provide listeners with information on which they can make their own judgment.
It remains for audience members to determine whether the speaker’s interests or relationships may influence the presentation.
17
Introduction
AIMS
• Understand different sampling techniques
• Understand interaction required to obtain optimal diagnostic
and predictive analysis
18
Five items with essential clinical information for pathology diagnosis
1 Sample site(s)
2 Whether the clinical suspicion is on primary tumour / metastasis
3 Question diagnosis? If appropriate: also predictive test (s)? (D+P?)
4 Previous relevant surgical, oncological, radiotherapeutic treatment, if any
5 Smoking history (never, ex (pack years, years since quitting), current
smoker (pack years, years of smoking)
19
20
HISTOLOGY LUNG CANCER
• Small cell lung cancer (SCLC)
• Squamous cell carcinoma (SqCC)
• Adenocarcinoma (AdC)
21
NSCLC – NOT OTHERWISE SPECIFIED (NOS)
Resection specimen
Biopsy / Cytology
Large cell carcinoma
1%
NOS
34%
Travis et al. J Thorac Oncol 2011;6:244–285; Thunnissen et al Virchows Arch. 2012;461:629–638
22
HE
HE
p63
PAS-D
TTF-1
Thunnissen et al. Virchows Arch 2012;461:629–638
NSCLC Favors
adenocarcinoma
23
24
Adenocarcinoma
´NOS´
25
NSCLC: ADENOCARCINOMA VS SQUAMOUS
CELL CARCINOMA
Histology
P63/40
TTF1
Mucin
Squamous cell carcinoma
+++
–
–
Adenocarcinoma
–/+
–/+
–/+
+
–
+
–
+
+
NOS
–/+
+++
–
+
–
+
• 85–90% favour adenocarcinoma or squamous cell
carcinoma
• Remaining:
– Bx NOS (not otherwise specified)
– Rx large cell/adenosquamous carcinoma
26
NSCLC: H&E + IMMUNO TTF1, P63
Resection specimen
Large cell carcinoma
0,2%
Biopsy / Cytology
NOS
4%
27
WHAT IS AN ACCEPTABLE RATE FOR NSCLC
NOT OTHERWISE SPECIFIED (NOS)?
• A diagnosis of NSCLC-NOS should be given in <10% of cases.
[This figure is achieved with the judicious use of
immunohistochemistry in morphologically indeterminate
cases]
• A recommended approach should include TTF-1 to predict
adenocarcinoma. For predicting squamous cell carcinoma, p63
or p40 and CK5/6 testing are useful
Kerr et al. Ann Oncol 2014;25:1681–1690
28
MODEL QUANTITATION IMMUNOHISTOCHEMISTRY
INDIRECT TYRAMIDE/POLYMER IHC VS. INDIRECT SABC IHC
Fluorescence intensities of immunohistochemical versus direct labeling
DI G
Cy3
= indirect polymer IHC
*
( Thousands)
Fluor escence I nt ensit y ( AU)
20
= indirect SABC IHC
*
*
++
10
Absorption
*
0
*
1
*
10
80
-
Spot=t ed
DNA concent
r at ion ( ng/ ul)
DNA concentration
epitope
concentration
Semi Q: -
-
-
+/-
+
++
++
Absorption
Modified from Prinsen CF, et al. Appl Immunohistochem Mol Morphol. 2003 ;11:168-73. 29
Linear dynamic range = quantitative range,
more signal enhancement:
closer to black and white; positive/ negative
*
= indirect SABC IHC
*
*
*
*
*
++
Absorption
-
Difference between positive and negative: factor 2 in concentration
MODEL QUANTITATION IMMUNOHISTOCHEMISTRY
INDIRECT TYRAMIDE/POLYMER IHC VS. INDIRECT SABC IHC
Linear dynamic range = quantitative range,
More signal enhancement: closer to black and white; positive/ negative
*
= indirect SABC IHC
tyramide
*
t
*
*
*
*
++
Absorption
-
negative positive
Difference between positive and negative: factor 2 in concentration
IMMUNOHISTOCHEMISTRY (IHC) FOR ALK IN
LUNG CANCER
•
•
•
•
Two antibodies (5A4, D5F3) work equally well
Testing can be quick
IHC used for screening will detect most ALK positive cancers
Confirm ALK IHC+ cases with FISH
32
Marchetti et al. J Thorac Oncol 2013; 8:352–358; Kerr et al. Ann Oncol 2014;25:1681–1690;
Thunnissen, et al. Virchows Arch. 2012;461:245-57
33
PATHOLOGIST PRACTICE
34
CELL AND TISSUE ACQUISITION
•
•
•
Sputum
Biopsy from
Scopy ~ 1 mm
– Bronchus
– Peripheral
– Lymph node small needle cytology
•
Puncture through chest wall
– Pleural fluids
– Lung trans-thoracic needle biopsy 1 cm x 0.8 mm
•
Metastatic sites: lymph node 1 x 0.5 cm, brain, bone (decalcify
EDTA)
•
Autopsy 2 m
35
36
37
PATHOLOGY FORM
Where is material sampled?
Fixative, time start fixation?
Clinical info:
- Tumor
- Suspicious for primary/
metastasis
- Predictive analysis?
38
DIAGNOSIS BY
CYTOLOGY AND
HISTOLOGY
39
TRANSBRONCHIAL
BIOPSY
The yield is usually low
The reason might be that there is often
a peritumoral inflammatory or scarring,
which prohibits the biopsy forceps to
reach the tumor
Other problems are insufficient tissue,
tinny pieces from the periphery of the
tumor
40
TRANSBRONCHIAL
BIOPSY
BAL
Can be diagnostic in some diseases
Usually TBX is not very useful in ILD
unless some lesions are located in more
central locations
Follow patterns seen in HRCT: If there
are nodular lesions TBX might sample
diagnostic tissues – take a minimum of
4 biopsies
41
TRANSBRONCHIAL
BIOPSY
BAL
Can be diagnostic in some diseases
Usually TBX is not very useful in ILD
unless some lesions are located in more
central locations
Follow patterns seen in HRCT: If there
are nodular lesions TBX might sample
diagnostic tissues – take a minimum of
4 biopsies
42
TRANSTHORACIC NEEDLE BIOPSY
43
TRANSTHORACIC NEEDLE BIOPSY
44
45
46
47
48
49
50
51
ANALYSIS BY RESECTION
MARGIN FROZEN
SECTIONS
➠
52
TUMOR CAN ALSO BE
ANALYSED THIS WAY
53
Fixation
Dehydration in alcohol
Xylol
Paraffin (56’C)
54
55
56
57
4 μm coupes
58
59
60
Automatic stainer for H&E
61
Inside automatic stainer
62
example
63
TISSUE MANAGEMENT
• Many doctor’s responsibility
• Lung cancer ‘hidden’ organ
• Small samples
64
PATHOLOGY: OLD FASHION
Pulmonologist: Clinical information
Question: diagnosis?
Pathology:
Cut many sections
65
NEW FASHION: TISSUE MANAGEMENT
Pulmonologist: Clinical information
Questions: diagnosis, prediction “D+P”, if malignant
Pathology “D+P” code: specific handling in contrast to regular
D+P
D+P
NOT performing these additional cuts saves material...
Adequate clinical information is essential
66
HE first
Diagnostic stain TTF1
Diagnostic stain mucin
Diagnostic stain P63/p40
Predictive stain ALK IHC
Predictive stain ALK FISH
DNA isolation EGFR
DNA isolation
DNA isolation
D+P
MORE ANALYSIS FROM ONE BIOPSY?
HE last
67
“1st”
“last”
68
TISSUE MANAGEMENT = INTERACTION BETWEEN
PULMONOLOGIST/ONCOLOGIST/
SURGEON/RADIOLOGIST AND PATHOLOGIST
Sample collector:
Pathology :
Sampling more (≥4)
biopsies/ tumor tissue
Distribute samples over
>1 block
Clinical request for
diagnosis + prediction?
Careful initial cut
Clinical suspicion of
metastases y/n?
Spare section for reflex
analysis
Focused diagnostic
analysis
69
CANCER IN THE LUNG: PRIMARY VS
METASTASES
•
•
•
•
•
•
•
•
•
•
•
Clinical information / PALGA* essential:
Primary pulmonary adenocarcinoma: Surfactant prot A, Napsin A, TTF1
Metastases:
Colorectal: CK7, CK20, CDX2
Prostate: PSA, PAP
Breast: ER, PR, GCDFP15, GATA3
Germ cell: PLAP, AFPHcG, CD30, OCT3/4, Sox2, Sox17
Melanocyte: Melan A, HMB45, Sox 10, MITF
Mesothelium: Calretinin, CK5/6, D2-40, WT-1
Kidney: RCC, CD10, Pax2, Pax8
Ovary: CA125, Pax5, Pax 8
• Pathological anatomy national automated archive –
a nationwide histopathology
and cytopathology network in the Netherlands
Nonaka et al. Diagnostic Histopathology 2010;16:581–592
70
CANCER IN THE LUNG: PRIMARY VS
METASTASES
•
•
•
•
•
•
•
•
•
•
•
Clinical information / PALGA* essential:
Primary pulmonary adenocarcinoma: Surfactant prot A, Napsin A, TTF1
Metastases:
Colorectal: CK7, CK20, CDX2
Prostate: PSA, PAP
Breast: ER, PR, GCDFP15, GATA3
Germ cell: PLAP, AFPHcG, CD30, OCT3/4, Sox2, Sox17
Melanocyte: NOT
Melanperforming
A, HMB45, Sox
10,stains
MITF saves material..
these
Mesothelium: Calretinin, CK5/6, D2-40, WT-1
Adequate clinical information is essential
Kidney: RCC, CD10, Pax2, Pax8
FORPax
optimal
TISSUE MANAGEMENT
Ovary: CA125, Pax5,
8
• Pathological anatomy national automated archive –
a nationwide histopathology
and cytopathology network in the Netherlands
Nonaka et al. Diagnostic Histopathology 2010;16:581–592
71
GUIDANCE ON TISSUE HANDLING: SPECIMEN
PROCESSING
• Standard fixation using 10% neutral buffered formalin (4%
formaldehyde) is recommended [V, A]
• Fixation time should be no less than 6 h, and no greater than 48
h [IV,A] Record start of fixation time on clinical form
• Sections for biomarker testing should ideally be cut
immediately before analysis [IV, A]
72
CYTOLOGY HANDLING
• Cytology samples (cell blocks, stained direct smears or liquidbased preparations) can be used reliably to detect EGFR
mutations and ALK rearrangements [III, A]. At this time, a cell
block is the most widely applicable cell source
73
STATION 7 ENLARGED PET POSITIVE
74
FNA/
EBUS/ EUS
Cytology
ROSE
Rapid
On
Site
Evaluation
Bulman et al. Am J Respir Crit Care Med 2012;185:606–611; Thunnissen et al. Lung cancer 2012,76:1–18
75
ROSE function changed:
Sample more from positive site
Repeat more passes in tumor positive nodes
Make
2 smears (PAP MGG)
cell block
Bulman et al. Am J Respir Crit Care Med 2012;185:606–611; Thunnissen et al. Lung cancer 2012;76:1–18
76
CYTOBLOCK PROTOCOL
•
•
•
•
•
Thinprep™ Cytolyt solution (Hologic)
Postfix with formaldehyde 4 hrs RT
Centrifuge; pellet
Shandon™ Cytoblock™ (J Clin Pathol 1992;45:1122-1123;Thermo Scientific)
Transfer in alcohol 70% to cassette, use 2 sponges/ transfer
casette in alcohol 70% with eosin/ VIP
Kulkarni et al. Diagn Cytopath 2009;37:86–90
77
Cytology
Tumor cellularity
Sufficient
for
Diagnostic/
predictive
testing ?
no
yes
78
CYTOLOGY ALK FISH PROTOCOL
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Online Resource 2
Basel (Switzerland) protocol for FISH on cytological specimens (Papanicolaou-stained smears or cytospins); Vysis ALK breakapart assay
Day 1:
- Place the uncovered slide(s) for 2 minutes in 2xSSC at 73°C in a microwave
- Place slide(s) for 30 minutes in 0.5mg/lm Pepsin in 0.01N HCL at 37°C in a
microwave
- Place slide(s) for 5 minutes in formalin 1% / 100X MgCl2 (25ml 10% formalin,
74ml PBS and 1ml 2M MgCl2) at room temperature (RT)
- Place slide(s) for 5 minutes in PBS at RT
- Place slide(s) for 1 minute in 70% ethanol at RT
- Let dry slide(s) for 10 minutes at RT
- Warm up heating plate to 45°C
- Place slide(s) for 2 minutes on the heating plate
- Apply 10μl ALK probe “ready to use” to the target area of the slide
- Cover area with coverslip (20x20mm)
- Seal slide(s) with rubber cement or parafilm
- Place slide(s) on the surface of the HYBriteTM (or ThermoBriteTM)
denaturation/hybridization system. Fill in empty slots with blank glass slides
- Close the lid of the HYBriteTM and start the following program:
o Co-denaturation: 8 minutes at 74°C o Hybridization: overnight at 37°C
Day 2:
- Heat up water bath to 73°C together with a coplin jar containing 2xSSC/0.3%
NP40 wash solution
- Remove the rubber cement or parafilm and the coverslip from the slide(s)
- Place slide(s) in the 2x SSC/0.3% NP40 at 73°C. When all slides are in the coplin
jar incubate for 2 minutes
- Remove the slide(s) from the wash solution and place them in a coplin jar containing 2x SSC/0.1% NP40 and incubate the
slides for 1 minute at RT
- Remove the slide(s) from the wash solution and place them vertically in the dark on a paper towel for drying
79
Experienced cytopathologist
• FISH is a robust technology applicable to almost all
types and formats of cytological specimens,
including conventional smears, cytospins, or liquidbased preparations.
• Use adhesive-coated and positively-charged slides
• FISH works equally well on unstained specimens
as well as those stained with Papanicolaou, H&E or
May-Grünwald-Giemsa
• Precise relocation of tumour cells using an
automated stage greatly facilitates FISH scoring
and review, especially in cases with a low
proportion of tumour cells on the slide.
80
CYTOLOGY ALK FISH PROTOCOL
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Online Resource 2
Basel (Switzerland) protocol for FISH on cytological specimens (Papanicolaou-stained smears or cytospins); Vysis ALK breakapart assay
Day 1:
- Place the uncovered slide(s) for 2 minutes in 2xSSC at 73°C in a microwave
- Place slide(s) for 30 minutes in 0.5mg/lm Pepsin in 0.01N HCL at 37°C in a
microwave
- Place slide(s) for 5 minutes in formalin 1% / 100X MgCl2 (25ml 10% formalin,
74ml PBS and 1ml 2M MgCl2) at room temperature (RT)
- Place slide(s) for 5 minutes in PBS at RT
- Let dry slide(s) for 10 minutes at RT
- Place slide(s) for 2 minutes on the heating plate
- Apply 10μl ALK probe “ready to use” to the target area of the slide
- Cover area with coverslip (20x20mm)
- Seal slide(s) with rubber cement or parafilm
- Place slide(s) on the surface of the HYBriteTM (or ThermoBriteTM)
denaturation/hybridization system. Fill in empty slots with blank glass slides
- Close the lid of the HYBriteTM and start the following program:
o Co-denaturation: 8 minutes at 74°C o Hybridization: overnight at 37°C
Day 2:
- Heat up water bath to 73°C together with a coplin jar containing 2xSSC/0.3%
NP40 wash solution
- Remove the rubber cement or parafilm and the coverslip from the slide(s)
- Place slide(s) in the 2x SSC/0.3% NP40 at 73°C. When all slides are in the coplin
jar incubate for 2 minutes
- Remove the slide(s) from the wash solution and place them in a coplin jar containing 2x SSC/0.1% NP40 and incubate the
slides for 1 minute at RT
- Remove the slide(s) from the wash solution and place them vertically in the dark on a paper towel for drying
ON LINE SUPPLEMENT
Thunnissen et al., Virch. Arch. 2012, ALK review
Responsible author is Lukas Bubendorf:
expert on cytology FISH
e-mail: [email protected]
81
INTERACTION BETWEEN PATHOLOGIST AND
PULMONOLOGIST/ONCOLOGIST/SURGEON/RADIOLOGIST
•
Pathologists knows diagnosis (D) and implications for personalised
medicine (Prediction = P) agreement on action
•
Inform pathologist possible clinical reasons for metastases
•
Allows reflex testing when needed, due to previous communication,
also reimbursements, timely molecular analysis
•
Turn around Time (TAT) EGFR and ALK 1 week. Max 10 working days.
Extramural within 3 days, intramural within 24 hours
•
Practice in Amsterdam Vumc: reflex for “D+P” NGS/EGFR, KRAS,
BRAF, HER2, analysis and IHC ALK, ROS1
•
Practice in Graz MUG: reflex for EGFR, EML4-ALK1 (IHC pre-test),
ROS1 (IHC pre-test)
Lindeman et al. J Mol Diagn 2013;15:415–53; Kerr et al. Ann Oncol 2014; 9:1681–1690
82
• Requirement of the assay
• Sufficient amount of DNA
for HRM/ seq
• 1~30% tumour cells
• NGS Miseq: 50 ng
• Ion Torrent 10 ng
83
TTF1
p63
84
Lymphangitis
carcinomatosa,
Sufficient for diagnosis
Not enough for prediction
TTF1
p63
85
• Sufficient DNA for HRM/ seq/
NGS
• ~30% tumour cells
• Not enough tumour DNA
• <(0.)1% tumour cells
86
• Sufficient DNA for HRM/ seq/
NGS
• ~30% tumour cells
• Not enough tumour DNA
• <1% tumour cells
• Cytology (block):
same requirements
• Glass slide: once
87
“1st”
“last”
88
TISSUE MANAGEMENT = INTERACTION
Sample collector:
Pathology :
Sampling more (≥4)
biopsies/ tumor tissue
Distribute samples over
>1 block
Clinical request for
diagnosis + prediction?
Careful initial cut
Clinical suspicion of
metastases y/n?
Spare section for reflex
analysis
Focused diagnostic
analysis
89
BIOMARKER ANALYSIS
• The same pathologist should, if possible, review all available
tumour material from the same patient including biopsies and
cytology specimens to select the most suitable for biomarker
analyses [IV, A]
• A pathologist should be involved in sample preparation for
DNA extraction [V, A]
• Enrichment of samples by micro- or macrodissection to
maximise tumour cell content before DNA extraction is
recommended [III, A]
• Turn around time 10 days
Kerr et al. Ann Oncol 2014; 9:1681–1690; Lindeman et al. J Mol Diagn 2013;15:415–53;
90
FOR PRECISION MEDICINE: TUMOR SAMPLE
• Sample more tumor
• Cervical lymph node: excision preferred over
biopsy
• More biopsies in different tubes/ blocks
• Biopsy preferred over cytology
• Cytology: more passes with `ROSE`
• Smear work also for FISH, sequential testing,
but limited options
91
CONCLUSIONS
• Interaction between doctors involved in the patient
management is essential for optimal diagnostic and predictive
analysis
• Histology samples preferred, but cytology works well too (for
limited number of predictive tests)
• ROSE: more passes when tumor positive: cell block
92
WHAT TYPES OF TISSUES FOR RESEARCH
• EBUS FNA and Cytoblock can be used in tumor research
• BAL useful for research in non-tumor diseases; also analysis of
proteins from fluid
• Transbronchial biopsy; low yield, less useful
• Transthoracic biopsy useful in tumor research, but also in
research on certain non-tumor conditions
• VATS biopsy most useful; if preevaluated by frozen section,
samples can be stored in liquid nitrogen and used fresh for all
types of molecular analysis
• Animal lung (rodents); most useful, but needs knowledge in
anatomy and histology of mammals
93
Sputum collection
Dr Patrizia Pignatti
Allergy and Immunology Unit
Fondazione Salvatore Maugeri
Via Salvatore Maugeri 10
27100 Pavia
ITALY
[email protected]
Prof. Antonio Spanevello
Division of Pneumology
Salvatore Maugeri Foundation IRCCS
Via Roncaccio 16
21049 Tradate
ITALY
Department of Clinical and Experimental Medicine
University of Insubria
Via Guicciardini 9
21100 Varese
ITALY
[email protected]
AIMS
Among respiratory sampling techniques, sputum collection represents a non invasive way to assess
airway inflammation. The aims of this workstation are:




To highlight standardized procedures of sputum collection and processing
To evaluate the pros and cons of this methodology
To show difficulties and drawbacks
To discuss clinical utility.
SUMMARY
Sputum analysis is a useful method to evaluate airway inflammation in asthma, chronic obstructive
pulmonary disease (COPD) and chronic cough both in research and in clinical practice. Induced
sputum is needed when patients cannot spontaneously produce sputum allowing sampling of low
airway secretions also in these subjects.
Sputum induction is performed through the inhalation of ultrasonically nebulised saline solution
(isotonic or hypertonic) over different time periods. The patient is invited to cough and to collect
secretions. The subject is asked to inhale 200 mg of salbutamol before induction, and forced
expiratory volume in 1 s (FEV1) is monitored before and after each inhalation to either prevent or
detect possible bronchoconstriction 1.
Sputum induction is a simple, safe and well-tolerated procedure even in patients with severe lung
diseases and exacerbations. It is recommended that experienced staff applies standard operating
procedures taking into consideration the degree of airway obstruction, use a modified protocol for
subjects with severe airway obstruction, and assess lung function and symptoms during the procedure
94
Sputum induction is considered to be safe if the fall in FEV1 is within 5% of baseline after waiting 15
min. If a FEV1 fall > 20% compared to baseline occurs, the inhalation must be stopped. This adverse
effect can affect 11% of asthmatics and patients with COPD 2.
After collection, sputum sample is processed within 2 hours according to a standardised method with
mucolitic agents (dithiothreitol). Centrifugation is required to separate sputum cells from the fluid
phase which is stored at -80°C for soluble mediator evaluation. If the soluble mediators are affected
by mucolitic agents, sputum should be processed with phosphate buffer alone 3-4.
Sputum from a healthy subject is rich in macrophages and neutrophils and poor in eosinophils,
lymphocytes and epithelial cells 5. Cut-off for sputum eosinophils varies from >2 or >3% according
to different authors and ERS guidelines.
Asthma is characterised by sputum eosinophilia, which predicts a favourable response to
corticosteroids. However, non-eosinopilic asthma accounts for 25–55% of steroid-naive asthmatics
and is associated with a poor response to corticosteroids 6.
In up to 40% of subjects with chronic cough, a sputum eosinophil count >3% is shown. These subjects
with cough, sputum eosinophilia and no lung function alterations receive the diagnosis of eosinophilic
bronchitis, and have an objective response to corticosteroid treatment.
In COPD, neutrophils are usually increased and they are associated with reduced FEV1, suggesting
that neutrophilic airway inflammation is functionally relevant. A cut-off for sputum neutrophilia
should take into account age, since neutrophilis accumulate in the airways with aging. Sputum
eosinophilia could be present in subjects with COPD and usually predicts a response to corticosteroid
therapy 7. Sputum analysis can be useful also at exacerbation to classify the type of exacerbation
8.
This methodology has many pros: it is relatively non invasive, it’s quite rapid if performed by trained
staff, it can be repeated during follow-up to monitor adhesion to therapy and change it if inflammation
is not under control. Furthermore, from one single rapid and not expensive test you can get many data
with different meanings: total cell count, cell viability, eosinophilic, neutrophilic or mixed
inflammation.
The cons are: despite the induction procedure, few subjects cannot produce sputum; you cannot repeat
the induction within 24 h in order to avoid neutrophilic inflammation due to the inhalation of the
hypertonic saline solution, you need to have laboratory facility.
Many attempt to substitute sputum eosinophil data with surrogate markers of airway inflammation
such as FeNO and blood eosinophils still did not reach the same sensitivity and specificity of sputum
analysis 9. Furthermore, to use blood eosinophils as surrogate of sputum eosinophils to evaluate
aireway inflammation could be misleading since some asthmatic patients present only local
inflammation 10.
In conclusion, non-invasive methods such as induced sputum have been successfully introduced in
clinical practice and research to study airway inflammation involved in the pathogenesis of respiratory
diseases.
REFERENCES
1. Pizzichini E et al . Safety of sputum induction. Eur Respir J Suppl. 2002; 37:9s-18s
2. Paggiaro PL, et al. Sputum induction. Eur Respir J Suppl. 2002 Sep;37:3s-8s
3. Efthimiadis A, et al. Eur Respir J Suppl. 2002 Sep;37:19s-23s
95
4. Spanevello A, et al. Induced sputum to assess airway inflammation: a study of
reproducibility. Clin Exp Allergy 1997; 27: 1138-1144
5. Spanevello A, et al. Induced sputum cellularity. Reference values and distribution in normal
volunteers. Am J Respir Crit Care Med 2000; 62: 1172-1174.
6. Kips JC, et al. The use of induced sputum in clinical trials. Eur Respir J Suppl. 2002 Sep; 37:47s50s.
7. Balzano G, et al. Eosinophilic inflammation in stable chronic obstructive pulmonary disease.
Relationship with neutrophils and airway function. Am J Respir Crit Care Med. 1999; 160:148692
8. Bafadhel M, et al. Acute exacerbations of chronic obstructive pulmonary disease: identification
of biologic clusters and their biomarkers. Am J Respir Crit Care Med. 2011; 184:662-71
9. Korevaar DA, et al. Diagnostic accuracy of minimally invasive markers for detection of
airway eosinophilia in asthma: a systematic review and meta-analysis. Lancet Respir
Med. 2015; 3:290-300.
10. Schleich FN, et al. Importance of concomitant local and systemic eosinophilia in uncontrolled
asthma. Eur Respir J. 2014; 44:97-108.
EVALUATION
1. A pre-treatment with short acting 2 agonist
a. Reduces the bronchoconstriction caused by the inhalation of hypertonic saline solution
b. Increases the amount of sputum collection
c. Avoids contamination of the collected sample
d. It is useless
2. You can always perform the induction independently of patient’s FEV1
a. Yes
b. No
c. It depends on the smoking history of the patient
d. None of the previous response
3. The amount of total sputum cells should be expressed as
a. Cells/ml
b. Cells/mg
c. Both the previous response
4. Sputum sample of a healthy subject is mainly composed by:
a. macrophages
b. neutrophils
c. eosinophils
d. epithelial cells
5. Sputum analysis in COPD patients is mainly useful to:
a. Predict reduction in FEV1
b. Evaluate severity of the disease
c. Evaluate the effects of smoking
d. Evaluate eosinophilic inflammation and predict response to corticosteroid therapy
96
EDUCATIONAL SKILLS WORKSHOP
RESPIRATORY SAMPLING TECHNIQUES
WORKSTATION 3: SPUTUM COLLECTION
Patrizia Pignatti 1 – Antonio Spanevello 2-3
1
Allergy and Immunology Unit, Fondazione Salvatore Maugeri, IRCCS Pavia, Italy
Division of Pneumology, , Salvatore Maugeri Foundation IRCCS, Tradate, Italy,
3 Department of Clinical and Experimental Medicine, University of Insubria, Varese, Italy
2
97
Conflict of interest disclosure
 We have no, real or perceived, direct or indirect conflicts of interest that relate to this
presentation.
This event is accredited for CME credits by EBAP and speakers are required to disclose their potential conflict of interest going back 3 years
prior to this presentation. The intent of this disclosure is not to prevent a speaker with a conflict of interest (any significant financial relationship
a speaker has with manufacturers or providers of any commercial products or services relevant to the talk) from making a presentation, but
rather to provide listeners with information on which they can make their own judgment. It remains for audience members to determine whether
the speaker’s interests or relationships may influence the presentation.
98
Introduction
AIMS
• Aim 1: Present induction and processing method
• Aim 2: Discuss pros and cons of this methodology
• Aim 3: Show practical application cases
99
•Give the patient detailed information
•Check safety of the equipment ultrasonic
nebulizer (output ~ 1ml/min)
•Measure pre-bronchodilator FEV1
•Pre-treat with 200 mg of salbutamol
•After 10 min measure FEV1
•Start inhalation with a fixed concentration of hypertonic
solution (3% or 4.5%) or use increasing concentrations (3, 4
5%).
•Measure FEV1 after each inhalation period. STOP if
there is a FEV1 fall ≥ 20% of post-bronchodilator value
•Ask the patient to cough and spit after 5, 10, 15 and 20
min or whenever they have the urge to do so
100
ALTERNATIVE METHOD OF SPUTUM INDUCTION
•Start inhalation with 0.9% saline solution induction for 30 sec, 1 and 5 min measuring
FEV1 after each period
•If no sputum production and no bronchoconstriction increase to 3-4.5% hypertonic
solution
Additional recommendations
•Maintain the same protocol in a given study
•Do not repeat induction within 48h of the first induction
•Keep strictly to safety recommendations
101
ERS GUIDELINE RECOMMENDATIONS FOR
SAFETY
•
Sputum induction should be performed by adequately trained technicians
•
A physician should be present
•
Hygiene and sterility should be assured
•
Pre-treatment with short acting b 2 agonist
•
Accepted both standard concentration of hypertonic saline (3 or 4,5%) 15
or 20 min. or increasing concentrations (from 3 to 4 or 5%). Modified
protocol0.9% saline
•
No data on safety of sputum induction in subjects with FEV1 < 1L or <50%
pred.
•
Maintain the same protocol of induction (hypertonic and duration) for the
same subject for all the subsequent evaluations
•
FEV1 checked after each inhalation period
102
Methods of sputum processing for cell counts, immunocytochemistry and in situ
hybridisation.
Efthimiadis A1, Spanevello A, Hamid Q, Kelly MM, Linden M, Louis R, Pizzichini MM, Pizzichini E,
Ronchi C, Van Overvel F, Djukanović R.
Whole sputum sample
Plug selection
103
Selection with
the use of
inverted
microscope
104
ERS GUIDELINES - PROCESSING: KEY POINTS
1. Complete solubilization of the sample
2. Filter the sample to remove debris
3. Perform manual total cell count prior to centrifugation
4. Count the entire volume of the counting chamber
5. Prepare cytospin with adequate cell amount
6. Optimize buffer and stains
7. Perform a 400-nonsquamous cell differential count
8. Report squamous cells separately
9. Include negative and positive controls for special stains
10. Implement a regular quality control system
11. Use standard operating procedures
105
ERS GUIDELINES RECOMMENDATIONS FOR
DIFFERENTIAL CELL COUNT
 Total cell count and viability
 Differential cell count: count at least 500 non
squamous cells
 Calculate the percentage of each cellular component
(macrophages, neutrophils, eosinophils, lymphocytes
and epithelial cells) with respect to the total cell
count. Squamous cells must be counted separately.
Squamous cell
 Calculate the percentage of squamous cells with
respect to the total cell count Saliva
contamination
106
PROCESSING SPUTUM SAMPLES
Smearing of sputum sample
Cytospin of processed sputum sample
107
CELL STAINING AND ANALYSIS
►Diff-Quick
►May Grunwald-Geimsa
Metachromatic
cells
►Toluidine blue
http://pathology.mc.duke.edu/research
►Oil red
►PERLS
108
DIFFERENTIAL CELL COUNT
NORMAL VALUES
 Macrophages 69.2 ± 13.0%
 Neutrophils 27.3 ± 13.0%
 Eosinophils 0.6 ± 0.8%
 Lymphocytes 1.0 ± 1.2%
 Epit. cells 1.5 ± 1.8%
Spanevello A et al. Am J Respir Crit Care Med 2000
109
INFORMATION COMING FROM TOTAL CELL COUNT
AND VIABILITY
 High total cell count  Possible viral or bacterial infection
High viability -> Recent and rapid cell recruitment
Low viability  -“old” material
-Bronchial epithelial cell-rich material
-Patient treated with corticosteroids (for eosinophils)
110
Salive contamination of the sample
When the sample is non-idoneous?
>20-30% squamous cells (plug selection method)
>70% squamous cells (in toto method)
111
EFFECT OF THE SMOKING HABIT ON
SPUTUM CELLS (I)
Antracotic inclusion in
macrophage cytoplasm
This type of deposits remains
for long time in the cytoplasm of
macrophages even after smoking
quitting
112
negative
ANTRACOTIC INCLUSIONS
PERLS
Staining
positive
113
EFFECT OF THE SMOKING HABIT ON
SPUTUM CELLS (II)
Increase in sputum neutrophils
Sputum neutrophilia
usually reduces 6 weeks
after smoking quitting
Hargreave FE
114
ASTHMATIC SUBJECT
Eosinophils
Apoptotic eosinophils
115
CHARCOT LEYDEN CRYSTALS
116
Eosinophils
Asthma
- onset
- follow up (corticosteroid therapy)
- exposure to the sensitizing agent
- exacerbations (also after viral
infections)
Eosinophilic bronchitis
Neutrophils
Airway infections
Severe asthma – resistent to steroids
COPD
Pollution exposure
Smoking, etc..
117
SCARCE SPUTUM SAMPLES
The International Guidelines for sputum processing, as well as other
published studies, have left the question open as to whether there
should be a weight threshold for the portions collected, below which the
analysis loses significance and reproducibility.
Balbi B, Pignatti P, Corradi M, et al
Sputum samples with low viability
Low viability of the sample could affect total and differential cell count
Use of inhaled corticosteroids
particularly decrease eosinophil viability
Apoptotic or damaged eosinophils
are much more recognizable than
apoptotic neutrophils
118
WHAT PERCENTAGE OF SPUTUM EOSINOPHILS
SHOULD WE CONSIDER SIGNIFICANT FOR
AIRWAY EOSINOPHILIA?
The cut-off value for sputum eosinophilia varies from 2-3% according to different
authors and to ERS sputum Guidelines
Assessment and reproducibility of non-eosinophilic asthma using induced sputum
Simpson JL, et al. Respiration 2010
All cut-points greater than 2% eos were reproducible
3% cut-point resulted in the highest agreement to distinguish eosinophilic from
non-eosinophilic airway inflammation in asthma
119
APPLICATION OF INDUCED SPUTUM (I)
Stability of airway inflammation phenotypes:
• Repeat evaluation of airway inflammation after 6mts/1year
• In stable condition reduction of airway inflammation
• Exacerbation  increase airway inflammation
• Viral or bacterial infection partly responsible for the change in
inflammatory pattern
120
APPLICATION OF INDUCED SPUTUM (II)
Bronchial inflammation can mirror nasal inflammation:
Consider nasal inflammation
United airway disease
NASAL INFLAMMATION
vs
BRONCHIAL INFLAMMATION
121
APPLICATION OF INDUCED SPUTUM (III)
Bronchial inflammation pre and post specific inhalation
challenge:
When is it important to precisely determine sputum cells count?
PRE specific inhalation challenge (SIC)
POST specific inhalation challenge (SIC)
Eosinophils
122
APPLICATION OF INDUCED SPUTUM (IV)
Identification of Eosinophilic Bronchitis
Asthma
Eosinophilic bronchitis
Respiratory
symptoms
Dyspnea, cough, wheezing, chest
thigtness
Cough
Pulmonary function
test (FEV1)
Normal or reduced
Normal
Reversibility test
Frequently positive (>12%)
Negative
Methacholine test
Frequently positive (PD20<1000 mg)
Negative
Chest X ray
Frequently normal
Normal
Atopy
Present/Absent
Present/Absent
Sputum eosinophils
Frequently present
Present as unique
alteration
123
APPLICATION OF INDUCED SPUTUM (IV)
Eosinophilic airway inflammation in COPD patients
31 patients with stable mild-to-moderate COPD
Zanini A, et al. Int J COPD 2015
124
Conclusion
•Sputum collection and evaluation is an useful method
to assess airway inflammation.
•It can be easly done by trained personal
125
Sputum collection
Information of best practice
27100 Pavia
ITALY
[email protected]
Via Roncaccio 16
21049 Tradate
ITALY
Department of Clinical and Experimental Medicine
Via Guicciardini 9
21100 Varese
ITALY
[email protected]
Keep strict to International Guidelines both for induction and processing
Induction:
1.
2.
3.
4.
5.
Sputum induction should be performed by adequately trained technicians
A physician should be present
Hygiene and sterility should be assured
PreAccepted both standard concentration of hypertonic saline (3 or 4,5%) 15 or 20 min. or increasing
concentrations (from 3 to 4 or 5%). Modified protocol0.9% saline
6. No data on safety of sputum induction in subjects with FEV1 < 1L or <50% pred.
7. Maintain the same protocol of induction (hypertonic and duration) for the same subject for all the
subsequent evaluations
8. FEV1 checked after each inhalation period
Processing:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Complete solubilization of the sample
Filter the sample to remove debris
Perform manual total cell count prior to centrifugation
Prepare cytospin with adequate cell amount
Optimize buffer and stains
Perform a 400-nonsquamous cell differential count
Report squamous cells separately
Include negative and positive controls for special stains
Implement a regular quality control system
Use standard operating procedures
126
Handling sputum induction with other functional and/or inflammatory evaluations
1. Methacholine challenge can be performed 1h before sputum induction without interference in
inflammatory sputum cells (Spanevello A et al. Thorax 1999)
2. Sputum induction with hypertonic, but not isotonic, saline led to a marked decrease in FeNO not
related to a fall in FEV1 following sputum induction (Beier J et al. ERJ 2003)
3. Hypertonic saline solution affects the evaluation of some cytokines in exhaled breath condensate
(Carpagnano GE, et al. Chest 2005)
127
Analysis of bronchoalveolar lavage (BAL) fluid
Prof. Dr Ulrich Costabel
Ruhrlandklinik
Dept Pneumology/Allergy
Tueschener Weg 40
45239 Essen
GERMANY
[email protected]
AIMS



To describe the technique and laboratory processing of bronchoalveolar lavage (BAL)
in the diagnostic work-up of diffuse lung disease
To explain how the results of BAL cytology should be interpreted in accordance with
clinical information
To provide the basis for understanding the sensitivity and specificity of BAL findings
for major diffuse lung disease: when is BAL alone sufficient, when is biopsy needed?
SUMMARY
Since its first application in the 1970s as a valuable research tool to study local immune and
inflammatory mechanisms, bronchoalveolar lavage (BAL) has gained widespread acceptance
as a powerful investigative tool in pulmonary medicine and has become a standard diagnostic
procedure in patients with interstitial lung disease (ILD). BAL is used to collect cells, inhaled
particles, infectious organisms and solutes from the lower respiratory tract and, in particular,
from the alveolar spaces of the lung.
Diagnostic BAL serves as a "window to the lung". BAL is regarded as providing
complimentary information to histopathology from biopsies, but nevertheless has several
advantages over biopsy procedures. It is safe and associated with virtually no morbidity.
Thus, it can be used repeatedly to investigate serial changes. In addition, BAL collects
samples from a much larger area of the lungs than can be obtained by the small tissue
fragments of transbronchial biopsy or even by surgical biopsy specimen, therefore giving a
more representative view of inflammatory and immunological changes.
Technical aspects
Guidelines and recommendations for a standardised approach to the performance of BAL, and
the laboratory processing and the analysis of recovered fluid have been published (1-6). When
the guidelines are followed, the results of lavage are sufficient for practical diagnostic
purposes.
Bronchoscopic procedure
BAL is usually performed during fibreoptic bronchoscopy (FOB) with topical anaesthesia,
following general inspection of the tracheobronchial tree. The fibreoptic bronchoscope is
gently introduced until impacted, or "wedged", in a segmental or subsegmental bronchus. In
diffuse lung disease, the middle or lingular lobe is recommended as a standard site of BAL.
Localised disease naturally requires lavage of the radiographically involved area.
128
Commonly, sterile, unbuffered isotonic saline (0.9 % NaCl solution) is used as the instillate.
The fluid is instilled with syringes through the biopsy channel of the bronchoscope, using
multiple aliquots of 20–60 mL (usually four to five aliquots), up to a total volume of 100–300
mL. Smaller instilled volumes (<100 mL) increase the likelihood of contamination of lavage
fluid by the bronchial spaces, including inflammatory cells from this compartment, which
may influence the differential cell counts. After each instillation, the aliquot is immediately
recovered by gentle hand suction into the syringe or gentle wall suction into a fluid trap.
Usually, 40–70 % of the instilled volume is recovered. The total volume retrieved should be
greater than 30% of the instilled volume in order to avoid a misleading cell differential.
BAL is generally well tolerated. Side-effects are more or less those of routine FOB under
local anaesthesia. There is practically no mortality, and the complication rate is low, ranging
0–2.3% compared to 7 % with transbronchial biopsy and 13 % with surgical lung biopsy (710). There are no absolute contraindications for performance of BAL beyond those noted for
bronchoscopy.
Laboratory processing
The total recovered fluid should be transferred to the laboratory as quickly as possible, since
the cells are not well preserved in the saline solution. The total number of cells are counted in
a haemocytometer, either in a sample of the pooled native fluid or in a re-suspension of the
cells after the first centrifugation. Cell viability is assessed by trypan blue exclusion and
should range 80–95%.
For the enumeration of cell differentials, 600 cells are counted after staining with MayGrünwald-Giemsa stain. A high percentage of epithelial cells (>5%) is indicative of
contamination of the alveolar samples by bronchial cells. Such BAL probes may not be
representative for the diagnosis of diffuse parenchymal lung disease. A number of soluble
components have been measured in BAL fluid. None of them has proven to be useful in
clinical settings (4, 5)
Normal findings in healthy adults without lung disease
The BAL fluid obtained from healthy, non-smoking adults without lung disease contains only
small percentages of lymphocytes, neutrophils and other inflammatory cells. Alveolar
macrophages are the predominant cell population.
Cigarette smoking is a strong confounding factor with significant effects on BAL samples.
The alveolar macrophages from smokers are much larger than those in non-smokers, and
contain cytoplasmic inclusion bodies (smoker´s inclusion bodies). On gross examination, the
recovered BAL fluid has a light to dark brown and turbid appearance caused by the colour of
the tar-laden macrophages. The total cell yield is three to five fold higher in smokers, due to a
three to five fold numerical increase in the number of macrophages, leading to a relative
decrease of the percentage of lymphocytes.
The normal values of differential cytology following BAL that have been proposed in the
literature are somewhat variable. In most of the published studies there are only small
numbers of normal patients/persons (mostly volunteers) available for comparison. For
practical reasons, the following percentages can be expected as normal in nonsmokers (2):
 macrophages >80%
 lymphocytes 15%
 neutrophils 3%
 eosinophils 0.5%
 mast cells 0.5 %
129
BAL as a clinical diagnostic tool
BAL is broadly indicated in every patient with unclear ILD or unclear pulmonary shadowing,
no matter what cause is suspected. The underlying disorders may be of infectious, noninfectious, immunological or malignant aetiology. BAL may also be indicated in patients with
normal chest radiograms when clinical and lung function tests are abnormal and point toward
a diffuse lung disease, or in patients with unexplained pulmonary symptoms in whom a
normal BAL finding may allow the exclusion of significant, active ILD.
Interpretation of BAL cytology
Changes in the profile of the cell differentials of BAL fluid have been described in various
ILDs. Clearly, an abnormal BAL cell differential count does not allow a specific diagnosis,
but BAL studies should not be limited to counting cell differentials. It is at least as important
to note the morphological appearances of cells and particles. Furthermore, BAL cell
differentials should not be used as an isolated finding for making a diagnosis, but should
always be interpreted in the context of disease history, and clinical, laboratory and
radiological findings.
BAL in differential diagnosis of ILD
BAL has improved diagnostic work-up in ILD, and some investigators use BAL more
frequently than transbronchial lung biopsy. In two international statements on the major
interstitial lung diseases, BAL was considered helpful in strengthening the diagnosis in
patients with sarcoidosis in the absence of biopsy, and BAL and/or transbronchial biopsy
were considered requirements for the exclusion of other diseases in a patient with IPF who
did not undergo surgical biopsy (one of the four major criteria for making a clinical diagnosis
of the disease) (11, 12). Recommendations for BAL were also included in the interstitial lung
disease guideline of the British Thoracic Society (13).
BAL findings may be, on occasion, very specific, can directly confirm a particular diagnosis
and then replace lung biopsy (table 1). It is of interest to note that many of these disorders are
included in the group of the alveolar filling syndromes. Obviously, the abnormal material
which accumulates in the alveolar spaces in these syndromes can easily be washed out by
lavage and is so characteristic that a specific BAL diagnosis is possible.
In other selected lung diseases, BAL findings are not diagnostic, but may help narrow the
differential diagnosis in the appropriate clinical setting (14). The list of diseases with a
lymphocytic, neutrophilic, eosinophilic or a mixed cellular pattern is long (table 2). Diseases
with an increase in lymphocytes can be further differentiated into those with an elevated,
normal or decreased CD4/ CD8 ratio (table 3). However, the importance of the CD4/CD8
ratio has been debated because of the high variability in several diseases (14). Only 50-60%
of sarcoidosis patients show an increased CD4/CD8 ratio, and in 15% the ratio is even
decreased. In extrinsic allergic alveolitis, the ratio is not always decreased, as was seen in
early BAL studies, but may also be normal or increased.
Some of the ILDs almost always show an abnormal BAL (high sensitivity), although the
specificity is low; however, in combination with clinical and HR-CT features, the diagnosis
may be possible without a biopsy (Table 4). For example, if the HR-CT scan shows a patchy
ground glass pattern, BAL may be able to reveal that this patient suffers from extrinsic
allergic alveolitis (high lymphocyte count), or a smoking related respiratory
bronchiolitis/interstitial lung disease (high smoker macrophage count and normal cell
differential), or alveolar hemorrhage (high count of hemosiderin laden macrophages). In druginduced lung disease, BAL findings are not specific but can contribute to the expected
130
clinicopathologic pattern of a given drug-induced lung disease and can help to exclude other
conditions such as infection or lung involvement by the underlying disease (15). Sometimes,
even a normal lavage may be useful to exclude some disorders with high probability (e. g.
extrinsic allergic alveolitis, eosinophilic pneumonia, alveolar hemorrhage) and to focus
attention on other direction.
Assessment of disease activity and prognosis
It is not still established whether BAL is clinically useful for assessing the activity of disease
processes and to provide prognostic information. Nevertheless, in IPF a recent retrospective
analysis revealed that an increased baseline BAL neutrophil percentage is an independent
predictor of early mortality whereas lymphocyte and eosinophil percentages are not (16). In
idiopathic NSIP, the BAL lymphocyte count is higher and a BAL lymphocytosis is more
frequently observed than in IPF, and this has been found to be associated with a longer
survival (17).
It has not been proved that BAL is better than other parameters to guide therapy. In this
regard, serial BAL to monitor the course of disease cannot be routinely recommended at
present.
REFERENCES
1. Klech H, Hutter C, eds. Clinical guidelines and indications for bronchoalveolar lavage
(BAL): report of the European Society of Pneumology Task Force on BAL. Eur Respir J
1990; 3: 937 – 974
2. Klech H, Pohl W, eds. Technical recommendations and guidelines for bronchoalveolar
lavage (BAL). Report of the ERS Task Group. Eur Respir J 1989; 2: 561 - 585
3. BAL Cooperative Steering Group. Bronchoalveolar lavage constituents in healthy
individuals, idiopathic pulmonary fibrosis, and selected comparison groups. Am Rev
Respir Dis 1990; 141: S169 - S202
4. Haslam PL, Baughman RP, eds. Report of European Respiratory Society (ERS) Task
Force: guidelines for measurement of acellular components and recommmendations for
standardization of bronchoalveolar lavage (BAL). Eur Respir Rev 1999; 9: 25 – 157
5. Haslam PL, Baughman RP. Report of ERS Task Force: guidelines for measurement of
acellular components and standardization of BAL. Eur Respir J 1999; 14: 245-248
6. Meyer KC et al. An official ATS clinical practice guideline: The clinical utility of BAL
cellular analysis in interstitial lung disease. Am J Respir Crit Care Med 2012; 185: 10041014
7. Costabel U, Guzman J. Bronchoalveolar lavage in interstitial lung disease. Curr Opin
Pulm Med 2001; 7: 255 – 261
8. Reynolds HY. Use of bronchoalveolar lavage in humans – past necessity and future
imperative. Lung 2000; 178: 271 – 293
9. Costabel U. CD4/CD8 ratios in bronchoalveolar lavage fluid: of value for diagnosing
sarcoidosis? Eur Respir J 1997; 10: 2699-2700
10. Costabel U. Atlas of bronchoalveolar lavage. London, Chapman and Hall, 1998
11. Hunninghake GW, Costabel U, Ando M, et al. ATS/ERS/WASOG Statement on
Sarcoidosis. Sarc Vasc Diffuse Lung Dis 1999; 16:149-173
12. ATS/ERS Statement. Idiopathic pulmonary fibrosis: Diagnosis and treatment. Am J
Respir Crit Care Med 2000; 161: 646-664
13. Wells AU, Hirani N et al. Interstitial lung disease guideline: the British Thoracic Society
in collaboration with the Thoracic Society of Australia and New Zealand and the Irish
Thoracic Society. Thorax 2008; 63(Suppl V): v1-v58
14. Bonella F, Ohshimo S, Bauer P, Guzman J, Costabel U: Bronchoalveolar lavage. Eur
Respir Mon 2010; 48: 59-72
131
15. Costabel U, Uzaslan E, Guzman J. Bronchoalveolar lavage in drug-induced lung disease.
Clin Chest Med 2004; 25: 25-35
16. Kinder BW, Brown KK, Schwarz MI, et al. Baseline BAL neutrophilia predicts early
mortality in idiopathic pulmonary fibrosis. Chest 2008; 133: 226-232
17. Ryu YJ, Chung MP, Han J, et al. Bronchoalveolar lavage in fibrotic idiopathic interstitial
pneumonias. Respir Med 2007; 101: 665-660.
132
Table 1. Diagnostic BAL findings
BAL finding
Pneumocystis carinii, fungi, CMV
transformed cells
Diagnosis
Opportunistic infections
Milky effluent, PAS-positive
noncellular corpuscles, amorphous
debris, foamy macrophages
Alveolar proteinosis
Haemosiderin-laden macrophages,
intracytosplasmic fragments of red
blood cells in macrophages, free red
blood cells
Alveolar haemorrhage syndrome
Malignant cells of solid tumours,
lymphoma, leukaemia
Malignant infiltrates
Dust particles in macrophages,
quantifying asbestos bodies
Dust exposure
Eosinophils >25%
Eosinophilic lung disease
Positive lymphocyte transformation
test to beryllium
Chronic beryllium disease
CD1-positive Langerhans cells
increased
Langerhans cell histiocytosis
Atypical hyperplastic type II
pneumocytes
Diffuse alveolar damage, drug
Toxicity
133
Table 2. BAL cellular patterns as an adjunct to diagnosis
Lymphocytic
Extrinsic allergic alveolitis
Berylliosis
Sarcoidosis
Tuberculosis
NSIP (mainly cellular type)
LIP
Connective-tissue disorders
Drug-induced pneumonitis
Malignant infiltrates
Silicosis
Early asbestosis
Crohn’s disease
Primary biliary cirrhosis
HIV infection
Viral pneumonia
Netrophilic (+/- eosinophilic)
Idiopathic pulmonary fibrosis
Desquamative interstitial pneumonia
Fibrotic NSIP
Acute interstitial pneumonia
Acute respiratory distress syndrome
Bacterial pneumonia
Connective tissue disorders
Asbestosis
Wegener’s granulomatosis
Diffuse panbronchiolitis
Transplant bronchiolitis obliterans
Idiopathic bronchiolitis obliterans
Drug-induced reaction
Eosinophilic
Eosinophilic pneumonia
Churg-Strauss syndrome
Hypereosinophilic syndrome
Allergic bronchopulmonary aspergillosis
Idiopathic pulmonary fibrosis
Mixed cellularity
BOOP
NSIP
Inorganic dust disease
134
Table 3. CD4/CD8 ratio in diseases with lymphocytic alveolitis
CD4/CD8 increased
CD4/CD8 normal
CD4/CD8 decreased
Sarcoidosis
Tuberculosis
Beryllium disease
Lymphangitic
carcinomatosis
Drug-induced pneumonitis
Asbestos-induced alveolitis
BOOP
Silicosis
Crohn’s disease
HIV infection
135
Table 4. Diagnostic yield of BAL in ILD
BAL without biopsy usually sufficient (high sensitivity and high specificity)
Pneumocystis pneumonia
Bronchoalveolar carcinoma
Alveolar hemorrhage
BAL in combination with clinical and HRCT features frequently sufficient (high
sesitivity, low specificity)
IPF (neutrophils  eosinophils)
Extrinsic allergic alveolitis (lymphocytes, plasma cells, foamy macrophages)
RB-ILD (smokers´macrophages)
BOOP (mixed cellularity, CD4/CD8 )
Lymphangioleiomyomatosis (alveolar hemorrhage)
BAL typical in only 50% of patients, biopsy often needed (if CT atypical) (moderate
sensitivity, high specificity)
Sarcoidosis (CD4/CD8 )
Langerhans cell histiocytosis (CD1a>4%)
BAL mostly not diagnostic, biopsy required (low sensitivity  low specificity)
Hodgkin´s disease
Invasive aspergillosis
BAL = bronchoalveolar lavage; HRCT = high-resolution computed tomography; IPF=
idiopathic pulmonary fibrosis; RB-ILD = respiratory bronchiolitis-associated
interstitial lung disease; BOOP = bronchiolitis obliterans organizing pneumonia.
136
Analysis of bronchoalveolar
lavage (BAL) fluid
Prof. Ulrich Costabel
University of Duisburg-Essen
Ruhrlandklinik
Essen, Germany
137
 I have no real or perceived conflicts of interest that relate to this presentation:
This event is accredited for CME credits by EBAP and EACCME and speakers are required to disclose their potential conflict of interest.
The intent of this disclosure is not to prevent a speaker with a conflict of interest (any significant financial relationship a speaker has with
manufacturers or providers of any commercial products or services relevant to the talk) from making a presentation, but rather to provide listeners
with information on which they can make their own judgments. It remains for audience members to determine whether the speaker’s interests, or
relationships may influence the presentation. The ERS does not view the existence of these interests or commitments as necessarily implying bias or
decreasing the value of the speaker’s presentation.
Drug or device advertisement is forbidden.
138
Bronchoalveolar Lavage
• Purpose: Differential diagnosis of interstitial lung
disease or unclear parenchymal infiltrates
• Important: BAL is different from
- bronchial washings used for
bacteriology / tumor cytology
(small volumes, 10 - 30 ml)
- whole lung lavage used for alveolar proteinosis
(large volumes, 10 - 40 liters)
139
Comparison of Biopsy/Cytology
procedures
BAL
TBB
Invasiveness
+
++
+++
Days in
hospital
Mortality
0
0-1
3-5
0%
0.1-0.2%
Open Biopsy
0.4-10%
140
BAL
 How to do it
141
Bronchoalveolar Lavage
Good local anesthesia
No coughing
No putride secretions
Wedge position
142
Technical recommendations and guidelines
for bronchoalveolar lavage (BAL)
• Report of the European Society of Pneumology
Task Group on BAL
Klech H, Pohl W (eds)
Eur Respir J 1989; 2: 561-585
• Haslam P, Baughman R. Report of ERS Task
Force. Eur Respir J 1999; 14: 245-248
• BAL Cooperative Steering Committee. Am Rev
Respir Dis 1990; 141: S169-S202
143
Site of BAL
• diffuse disease: ML or lingula
• Patchy disease: involved segment
144
Methods to instil and recover the fluid
Instillation
• Syringe, 20-50 ml bolus
Recovery
• Manual aspiration back into the syringe
• Suction trap with mechanical suction
Total instillation volume: 100 - 300 ml
Recovery rate > 30%
145
146
147
148
149
150
151
152
BAL Fluid Analysis
• Cell differentials (profile of inflammatory cells)
• Immunocytology (CD4/CD8, CD1)
• Cellular morphology:
tumor cells, alveolar proteinosis, haemorrhage,
foamy macrophages, plasma cells
• Dust particles and fibres (e.g. asbestos bodies)
• Infectious organisms
153
BAL
 How
to read it
154
Normals:
Non-smokers and smokers
155
NORMAL NONSMOKER SUBJECT:
YIELD APPROXIMATELY 10 MIO CELLS, WITH
> 85% Alveolar macrophages
< 15% Lymphocytes
< 3%
Neutrophils
< 0.5% Eosinophils
< 0.5% Basophils, Mast cells.
156
Nonsmoker
Smoker
157
BAL
 How
to use it
158
BAL as a clinical tool
• Specific findings may replace biopsy
• BAL cell differentials are nonspecific, but a useful
adjunct to diagnosis in the appropriate clinical
setting
• Value of BAL in assessment of prognosis and
treatment efficacy: still an open question
159
Diseases that can be diagnosed by BAL alone
(high sensitivity and specificity)
• Alveolar filling diseases
(CT: ground glass, alveolar consolidation)
- alveolar proteinosis
- pneumocystis jiroveci
- eosinophilic lung disease
- alveolar haemorrhage
- bronchoalveolar carcinoma
160
Alveolar Proteinosis
161
162
BAL may reveal infection: Pneumocystis
163
Toluidine Blue Stain
164
CMV Cell
165
BAL may reveal eosinophilic lung disease
166
167
BAL may reveal alveolar haemorrhage
168
BAL may reveal significant dust exposure
169
170
BAL may reveal asbestos exposure
171
172
173
174
BAL may reveal malignancy
175
Hyperplastic Type II Pneumocytes
176
Diagnostic yield of BAL in diffuse malignancy
(Semenzato, Poletti, Respiration 1992)
• Bronchoalveolar carcinoma
93%
• Adenocarcinoma
77%
• Squamous cell carcinoma
50%
• Non-Hodgkin lymphoma
67%
• Hodgkin lymphoma
33%
• Total (n=162)
76%
177
BAL as a clinical tool
• BAL cell differentials are nonspecific, but a useful
setting
178
BAL pattern
• Neutrophilic
IPF etc.
• Lymphocytic
Sarcoidosis, extrinsic allergic alveolitis, etc.
• Eosinophilic
CEP, Churg-Strauss-Syndrome, etc.
• Mixed cellularity
BOOP, etc.
179
Increase of neutrophils (+ eosinophils)
in BAL
•
•
•
•
•
•
•
•
•
IPF
DIP
AIP
ARDS
Collagen vascular disease
Wegener´s granulomatosis
Asbestosis
Bronchopulmonary infections
etc...
180
Role of BAL in Diagnosis of IPF
• BAL may reveal alternative specific
diagnoses: malignancy, infections,
eosinophilic pneumonia,
alveolar proteinosis
• Increase in neutrophils +/eosinophils (in 90%) suggests
a fibrosing process:
IPF, collagen/vascular disease,
asbestosis
• An increase in lymphocytes
>30% is uncommon, exclude:
EAA, BOOP, NSIP, LIP,
sarcoidosis
181
Increase of lymphocytes in BAL
•
•
•
•
•
•
•
•
Sarcoidosis
Beryllium disease
Tuberculosis
BOOP
Drug induced diseases
Lymphangitic carcinomatosis
etc...
182
BAL Profile in Sarcoidosis
• Lymphocytes  in 90% of patients
• Clinically active disease:
Lymphocytes range 20~80%, mean ~40%
• Clinically inactive disease:
Lymphocytes lower, mean ~30%, but
broad overlap
• Neutrophils may be increased in late or
advanced disease
183
BAL in Sarcoidosis
184
BAL in EAA
185
Lymphocytes (% of total cells)
186
Diagnostic value of BAL CD4/CD8
ratio for sarcoidosis
CD4/CD8 Sensitivity Specificity Author
> 3.5
59%
92%
> 4.0
59%
96%
> 4.0
55%
94%
Costabel
Milan 1987
Winterbauer
Chest 1993
Thomeer
WASOG1997
187
Value of various cut-offs of CD4/CD8
ratios for the diagnosis of sarcoidosis
CD4/CD8 ratio
Sensitivity (%)
Specificity (%)
2.0
80
73
3.5
54
93
5.0
38
98
10.0
12
100
Sarc. n=129; Non-sarc. n=353
Costabel et al 1987
188
Value of various cut-offs of CD4/CD8
ratios for the diagnosis of sarcoidosis
CD4/CD8 ratio
Sensitivity (%)
Specificity (%)
2.0
80
73
3.5
54
93
5.0
38
98
10.0
12
100
Sarc. n=129; Non-sarc. n=353
Costabel et al 1987
189
BAL advantages in sarcoidosis
• Low risk
• narrows differential diagnosis
• supports the diagnosis (lymphocytes ,
CD4/CD8 ) in up to 70% of patients
• Characteristic BAL plus characteristic
clinical presentation may obviate biopsy
190
BAL profile in EAA
•
•
•
•
•
•
Very high total cell count (> 4 fold)
Very high lymphocyte % (> 50%)
Mild increase in neutrophil % and mast cells
Presence of plasma cells
Presence of foamy macrophages
CD4/CD8 usually < 1.0 (but may be normal or
increased)
• Profiles may change with chronic disease
191
EAA and BAL CD4/CD8 ratio
Barrera L et al. AJRCCM 2008 177: 44-55
192
Histological pattern in chronic pigeon breeder’s disease:
correlation with clinical data
Typical pattern
n = 58
NSIP pattern
n = 22
UIP-like pattern
n = 10
p
Finger clubbing
30/56 (53)
10/21 (47.6)
8/10 (80)
0.26
BAL lymphocytes %
65 ± 21
52 ± 23
36 ± 23
0.0011
BAL macrophages %
34 ± 20
45 ± 23
59 ± 18
0.0028
BAL eosinophils %
1 (0–9)
0 (0–13)
2 (0–13)
0.11
BAL neutrophils %
0 (0–10)
1 (0–10)
1 (0–4)
0.61
Inflammation (%)
30/40 (75)
11/16 (69)
1/7 (14)
<0.007
Fibrosis (%)
10/40 (25)
5/16 (31)
6/7 (86)
<0.007
HRCT
Gaxiola M et al, Respir Med 2011
193
Clinical significance of BAL in EAA
• A normal BAL widely excludes EAA
• BAL is the most sensitive tool in detecting signs of
alveolitis
• BAL cannot differentiate between symptomatic ILD
and subclinical alveolitis in a given patient
• In the follow-up, persistent BAL abnormalities
indicate that complete allergen avoidance has not
been achieved
194
Diseases which can largely be excluded
by a normal BAL
• Extrinsic allergic alveolitis
• Alveolar haemorrhage
• Alveolar proteinosis
• Any siginificant „active“ ILD
195
How to interpret BAL findings
• Not in isolation but in context with
clinical presentation and HRCT
findings!
196
197
Ground Glass Pattern on HR-CT
BAL findings
Diagnosis
• Lymphocytes > 50%
Total cell count high
• Eosinophils > 25%
• Pneumocystis
Infection
• Iron laden macrophages
Alveolar haemorrhage
• Smoker´s macrophages
+ mild increase in
neutros/eos
DIP/RBILD
198
IPF
199
Reticular/linear Pattern on HR-CT
BAL findings
Diagnosis
• Neutros + eos,
+ lymphocytes < 30%
IPF (UIP)
Collagen disease
Asbestosis
• Lymphocytosis
+ neutros, eos, mast cells
Chronic EAA
• Malignant cells
+ lymphocytosis
Lymphangitic
carcinomatosis
200
201
Nodular or reticulonodular pattern
on HR-CT
BAL findings
• lymphocytosis, normal
appearance of macrophages,
CD4/CD8 n or
Diagnosis
Sarcoidosis
• lymphocytosis, dust particles
in macrophages,
CD4/CD8 n or
Silicosis
• malignant cells,
± lymphocytosis
carcinomatosis
Lymphangitic
202
Langerhans cell histiocytosis (LCH)
203
Cystic Pattern on HR-CT
BAL findings
Diagnosis
• Smoker´s macrophages,
mild increase in neutros/eos,
CD1a+ cells > 4%
LCH
• Moderate to severe
alveolar haemorrage
Lymphangioleiomyomatosis
204
How to integrate BAL
into the management of ILD patients
• Cell differentials are nonspecific but a useful
setting
205
Role of BAL in ILD
• In idiopathic interstitial pneumonia :
BAL differential may guide choice of
treatment and determine prognosis?
206
BAL cells in NSIP vs IPF
Author
Nagai
Lymphocytes
NSIP IPF
37%
7%
Neutrophils
NSIP IPF
8%
6%
1998
Daniil
9%
8%
8%
10%
46%
8%
23%
13%
21%
6%
7%
7%
1999
Park
2000
Suga
2000
207
Overall survival (%)
Baseline BAL Neutrophilia Predicts Early
Mortality in Idiopathic Pulmonary Fibrosis
N= 156
Follow-up Time (years)
Kinder BW et al. Chest 2008
208
Lymphocytosis on BAL in
Fibrotic NSIP and IPF and Prognosis
ALL IIP (N=122)
NSIP (N=35) and IPF (N=87)
Ryu YJ, et al. Respir. Med. 2007 209
What the guidelines tell us
•
ATS/ERS IIP Statement 2002
•
New IPF Guidelines 2011
•
Sarcoidosis Statement 1999
•
BTS ILD Guideline 2008
210
ATS/ERS Statement 2002:
HRCT
Atypical clinical Features diagnostic Suspected
Confident CT
other DPLD
of another
diagnosis of IPF with or CT features
for IPF
DPLD e.g. HX
consistent clinical
features
TBBx or If non-diagnostic TBBx, BAL or
BAL?
other relevant
test
Surgical lung biopsy
UIP
NSIP
RB
DIP
DAD
OP
LIP
non-IIP
confirmed
211
Diagnostic algorithm for IPF
Am J Respir Crit Care Med 2011
Suspected IPF
Yes
Identifiable causes
for ILD?
No
HRCT
UIP *
Possible
*
PossibleUIP
UIP
Inconsistent
UIPUIP
*
Inconsistentw/
with
Surgical Lung
Biopsy
UIP
†
Not UIP †
Probable
UIP † // Possible UIP
UIP / Probable
UIP†
Non-classifiable
Non-classifiable fibrosis
fibrosis †
MDD
IPF
IPF/Not IPF per Table 4
Not IPF
212
Diagnostic algorithm for IPF
Am J Respir Crit Care Med 2011
Suspected IPF
Yes
Identifiable causes
for ILD?
No
HRCT
UIP *
Where is the
Surgical Lung
BAL/TBB
??
Possible
*
PossibleUIP
UIP
Inconsistent
UIPUIP
*
Inconsistentw/
with
Not UIP †
Biopsy
UIP
†
Probable
UIP † // Possible UIP
UIP / Probable
UIP†
Non-classifiable
Non-classifiable fibrosis
fibrosis †
MDD
IPF
IPF/Not IPF per Table 4
Not IPF
213
Diagnosis of IPF
Should BAL cellular analysis be performed in the
diagnostic evaluation of suspected IPF?
Recommendation: weak against (low)
- should not be done in the majority, but may be
appropriate in a minority
(vote: 4 for use, 18 against use, 1 abstention)
Should transbronchial lung biopsy be used in the
evaluation of suspected IPF?
Recommendation: weak against (low)
(vote: none for use, 23 against use, no abstentions)
Am J Respir Crit Care Med, 2011
214
Should BAL be performed in IPF ?
• BAL cellular analysis should be considered in the
evaluation of patients with IPF at the discretion of
the treating physician based on availability and
experience at their institution/regional laboratory.
• In the evaluation of patients with suspected IPF, the
most important application of BAL is in the
exclusion of chronic hypersensitivity pneumonitis;
prominent lymphocytosis (>40%) should suggest
the diagnosis.
Am J Respir Crit Care Med, 2011215
Significance of Bronchoalveolar
Lavage for the Diagnosis of
Idiopathic Pulmonary Fibrosis
Shinichiro Ohshimo, Francesco Bonella, Ai Cui, Martin Beume, Nobuoki Kohno,
Josune Guzman, and Ulrich Costabel
Am J Respir Crit Care Med 2009; 179: 1043-1047
216
Study Design
and Results
Suspicious IPF based
on confident HRCT
findings
n = 101
Application of
ATS/ERS criteria
2002
Eligible Patients
n = 74
Addition of BAL
cell differential
analysis
No
Lymphocytosis in
BALF
(cut off 30%)
No
Yes
Yes
n = 68
n=6
Granulocytosis
in BALF
(cut off 3%)
Granulocytosis
in BALF
(cut off 3%)
No
Yes
No
n = 63
n=5
n=4
n=2
IPF
IPF
NSIP 3; 1 EAA
EAA
217
Wells AU, Thorax 2008
218
Recommendations for BAL
in ILD
•
BAL or TBLB, when required, should be performed before the
initiation of treatment. [D]
•
BAL should be considered in all patients with suspected
infection, malignancy and some rare ILDs. In such cases, BAL
may be diagnostic. [C]
•
BAL is not required as a diagnostic tool in patients
with clinical features and HRCT appearances typical of IPF. [C]
219
Recommendations for BAL
in ILD
•
In patients for whom the diagnosis is uncertain after clinical
assessment and HRCT scanning, typical BAL cellular profiles
may allow a diagnosis of HP or sarcoidosis to be made with
greater confidence. [C]
•
In cases in which the diagnosis is uncertain and BAL is
considered, the procedure should be performed in a regional
centre with technical expertise in the procedure and the analysis
of the BAL samples. [D]
•
BAL should be performed in all patients undergoing TBLB. [D]
220
Am J Respir Crit
Care
2009;
1043-1047
Meyer
KCMed
et al.,
BAL 179:
Cinical
Practice Guideline,
AJRCCM 2012
221
Summary: BAL contribution to ILD
• Specific diagnosis is possible.
• Level of confidence in a certain diagnosis can be
enhanced or weakened.
• BAL and HR-CT combined may obviate the need for
biopsy.
• BAL is included as a diagnostic tool in major guidelines
on ILD.
222
Thank you for your attention
Interstitial and Rare Lung Disease Unit
Prof. U. Costabel
Dr. F. Bonella
Dr. T.E. Wessendorf
Dr. E. Börner
Dr. X. Long
Prof. J. Guzman
Dr. S. Ohshimo
Prof. D. Theegarten
223
Recommended reading list and E-learning resources
WORKSTATION 1 - Clinical cytology
WORKSTATION 2 - Histology
WORKSTATION 3 - Sputum collection
1. Brightling CE. Clinical applications of induced sputum. Chest 2006; 129; 1344–1348.
2. European Respiratory Society Task Force Standardised methodology of sputum induction and
processing. Eur Respir J 2002; 20: Suppl. 37, 1s-55s.
3. Pizzichini E, et al. Indices of airway inflammation in induced sputum: reproducibility and validity
of cell and fluid-phase measurements. Am J Respir Crit Care Med 1996; 154: 308-317
4. Spanevello A, et al. Induced sputum cellularity. Reference values and distribution in normal
volunteers. Am J Respir Crit Care Med 2000; 62: 1172-1174.
5. Spanevello A, et al. Induced sputum to assess airway inflammation: a study of reproducibility.
Clin Exp Allergy 1997; 27: 1138-1144.
6. Spanevello A, Beghé B, Bianchi A, et al. Comparison of two methods of processing induced
sputum: selected versus entire sputum. Am J Respir Crit Care Med. 1998; 157:665-8
7. Pizzichini MM, Popov TA, Efthimiadis A, et al. Spontaneous and induced sputum to measure
indices of airway inflammation in asthma. Am J Respir Crit Care Med. 1996; 154:866-9
8. Bhakta NR, Woodruff PG. Human asthma phenotypes: from the clinic, to cytokines, and back
again. Immunol Rev. 2011; 242:220-32.
9. Louis R, Lau LC, Bron AO, et al. The relationship between airways inflammation and asthma
severity. Am J Respir Crit Care Med. 2000; 161:9-16
10. Romagnoli M, Vachier I, Tarodo de la Fuente Pet al. Eosinophilic inflammation in sputum of
poorly controlled asthmatics. Eur Respir J. 2002;20:1370-7
11. Louis R, Sele J, Henket M, et al. Sputum eosinophil count in a large population of patients with
mild to moderate steroid-naive asthma: distribution and relationship with methacholine bronchial
hyperresponsiveness. Allergy. 2002;57:907-12.
12. Pignatti P, Zanini A, Della Patrona S, Gumiero F, Cherubino F, Spanevello A. Sputum and
exhaled breath analysis. ERS Handbook: Respiratory Medicine ERH-0025-2012.3d 2014
13. Green RH, Brightling CE, McKenna S, et al. Asthma exacerbations and sputum eosinophil
counts: a randomised controlled trial. Lancet. 2002; 360:1715-21.
14. Grootendorst DC, Gauw SA, Verhoosel RM, et al. Reduction in sputum neutrophil and eosinophil
numbers by the PDE4 inhibitor roflumilast in patients with COPD. Thorax. 2007; 62:1081-7.
15. Bafadhel M, McKenna S, Terry S, et al. Acute exacerbations of chronic obstructive pulmonary
disease: identification of biologic clusters and their biomarkers. Am J Respir Crit Care Med.
2011;184:662-71
16. Schleich FN, Chevremont A, Paulus V, Henket M, Manise M, Seidel L, Louis R. Importance of
concomitant local and systemic eosinophilia in uncontrolled asthma. Eur Respir J. 2014; 44:97108.
17. Korevaar DA, Westerhof GA, Wang J, Cohen JF, Spijker R, Sterk PJ, Bel EH, Bossuyt PM.
Diagnostic accuracy of minimally invasive markers for detection of airway eosinophilia in
asthma: a systematic review and meta-analysis.Lancet Respir Med. 2015; 3:290-300.
WORKSTATION 4 - Analysis of BAL fluids
1. U. Costabel, Atlas of bronchoalveolar lavage, Thieme publisher, 1994
224
Faculty disclosures
Prof. Helmut H. Popper has received unrestricted research grants from Eli Lilly (IALT-BIO), Astra
Zeneca, Pfizer, Hofman La Roche, Boehringer-Ingelheim, as well as advisory board honoraria from
Hofman La Roche, Eli Lilly, Boehringer-Ingelheim, Novartis and Pfizer.
Dr Erik Thunnissen has received honoraria or consultation fees from Eli - Lilly that supported
meeting European experts 2010.
225
Faculty contact information
Prof. Dr Ulrich Costabel
Ruhrlandklinik
Dept Pneumology/Allergy
Tueschener Weg 40
45239 Essen
GERMANY
[email protected]
Prof. Christian Kaehler
Department of Internal Medicine
Innsbruck Medical University
Anichstrasse 35
6020 Innsbruck
AUSTRIA
[email protected]
27100 Pavia
ITALY
[email protected]
Prof. Helmut H. Popper
Pathology
Medical University Graz
Auenbruggerplatz 25
8036 Graz
AUSTRIA
[email protected]
Prof. Dr Philipp A. Schnabel
Instit. Alllgemeine&Spezielle Pathologie
University of Saarlandes
66421 Homburg/Saar
GERMANY
[email protected]
Via Roncaccio 16
21049 Tradate
ITALY
Department of Clinical and Experimental
Medicine
Via Guicciardini 9
21100 Varese
ITALY
[email protected]
Dr Eva Schmutz
Amalienstrasse 5
69126 Heidelberg
GERMANY
[email protected]
Dr Erik Thunnissen
Pathologie
VU University Medical Center
De Boelelaan 1117
1081 HV Amsterdam
NETHERLANDS
[email protected]
Dr Matthias Wiebel
Amalienstrasse 5
69126 Heidelberg
GERMANY
[email protected]
226
Answers to evaluation questions
Please find all correct answers in bold below
WS2. Histology – Prof. Helmut H. Popper, Dr Erik Thunnissen
1. Rapid on site evaluation of during cytology sampling for lung cancer is performed with EBUS/
EUS /TTBA. What is appropriate for predictive analysis?
a. When a node is negative continue sampling.
b. When node is positive continue sampling same node.
c. When node is negative go to next enlarged node.
d. When node is positive go to next enlarged node.
2. If a transbronchial biopsy did not yield in diagnostic tissue in lung cancer or an interstitial
disease, what would you do next?
a. repeat TBA
b. proceed to VATS.
c. perform EBUS TBNA
d. perform BAL
3. Can an UIP diagnosis we made on cryobiopsies?
a. no
b. yes
c. yes, but a differential diagnosis of underlying diseases is most often not possible
d. no, the material is not sufficient
4. On cytologic specimen the diagnosis of mucin negative NSCLC NOS was made. What should be
done next?
a. Immunohistochemistry for TTF1 and p40
b. Immunohistochemistry for p63 and cytokeratin 5/6
c. Immunohistochemsirty for Surfactant Apoprotein A and cytokeratin 7
d. Immunohistochemsirty for cytokeratin 7 and napsinA
5. Does it matter, if a lung tissue specimen is placed in a large container with formalin, or is any
container suitable?
a. does not matter
b. does matter, but I don’t know why
c. does matter, because of fixation artifacts induced by squeezing the tissue
d. does matter, because the tissue can stay therein for many hours
6. Can molecular testing be done on bone biopsies?
a. yes, bone metastasis is equal to any other metastasis specimen
b. no, bone metastasis shows less genetic changes than other metastasis
c. no, DNA is degraded by acidic solutions used for decalcification
d. yes, by decalcification normal tissue is destroyed and cancer tissue remains
WS3. Sputum collection - Dr Patrizia Pignatti, Prof. Antonio Spanevello
1. A pre-treatment with short acting 2 agonist
a. Reduces the bronchoconstriction caused by the inhalation of hypertonic saline solution
b. Increases the amount of sputum collection
c. Avoids contamination of the collected sample
d. It is useless
2. You can always perform the induction independently of patient’s FEV1
a. Yes
b. No
c. It depends on the smoking history of the patient
3. The amount of total sputum cells should be expressed as
a. Cells/ml
b. Cells/mg
c. Both the previous response
4. Sputum sample of a healthy subject is mainly composed by:
a. macrophages
b. neutrophils
c. eosinophils
d. epithelial cells
5. Sputum analysis in COPD patients is mainly useful to:
a. Predict reduction in FEV1
b. Evaluate severity of the disease
c. Evaluate the effects of smoking
d. Evaluate eosinophilic inflammation and predict response to corticosteroid therapy

BAL

Transcription

Similar documents

Le Bal des Débutantes is synonymous with high fashion, good

Electrical Contact Solutions for Medical Active Implantables (TMB-10)

Powder choice with ease

1) Varing distance `Y`will increase or decrease the

Folder IPF English

Pulmonary Grand Rounds - American Lung Association

Bal de Maison Secondary - Bayou Vermilion District