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Handout for Round table #19 at American Society of Cytopathology (Nov 9, 2013, Orlando, FL, USA)- VB Shidham, MD, FRCPath, FIAC
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Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
November 9, 2013 (ASC Round Table #11, 12:00 – 1:45 PM)
Vinod B. Shidham, MD, FRCPath, FIAC Vice-chair- AP, Professor, Director of Cytopathology, Cytotechnology School, Cytopathology fellowship, andGI PathologyExecutive editor & co-editor-in-chief, CytoJournal (www.cytojournal.com)
Dept of Pathology, Wayne State University Medical SchoolKarmanos Cancer Institute & Detroit Medical CenterDetroit, MI 48201, USA
This presentation is also available at:http://alturl.com/245zh
Cell Blocks for Molecular Tests & Immunocytochemistry Applications-
Frustrations & Triumphs!
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
Disclaimer
VS is co-editor of‘Cytopathologic Diagnosis ofSerous Fluids’Elsevier (W. B. Saunders Company)cited for various methods of cell block preparation (the sketches and tables used are from this reference).
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
DisclaimerVS has indirect financial interest (through spouse) in AV marker referenced.
http://www.bioinnovationllc.com/Home_Page.html
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Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
Cell block- microbiopsy-Role in cytopathologic evaluation/patient managementCritical issues to be consideredDifferent methods of cell block preparation
Aligning the cells along the cutting surfaceDepth of section cutting
Immunophenotyping and cell blocks-Immunoreactivity interferenceMarker for SCIP approach
A few study cases
Outline
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
Cell block of cytopathology specimen isEquivalent to microbiopsy evaluation
the interface between cytology and histopathology (bridge to histopathology/surgical pathology)
Routine example is Endocervical curretage (ECC), But without cytology preparation
Cell block with cytology preparation hasAdded benefit of excellent cytomorphologic details
in concert with architectural insight
Cyto-histo-pathology
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
Cell blocksConglomeration of
minute tissue fragments and
isolated cells / small cell groups
in specimens collected or submitted
as suspensions
To processed as
Paraffin embedded tissue sections and
long term archival for future elective evaluations
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Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
Samples that may be ‘Cell blocked’
Clinical samplesFluids
Effusion fluids (ascitic, pleural, pericardial, etc)
Other fluids (drainage, cysts, cavities, etc)
Washings (peritoneal, pelvic, bronchial washing)
Exfoliated cells- Cervical cytology specimensBrushings- Endocervical, bronchial, bile duct etcCurretages- EndocervicalFNA passes-rinsesScrapings of cytology smears (stained or unstained)Any cytology specimen with micro-fragments
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
Samples that may be ‘Cell blocked’(contd)
Research samples Cell cultures- scrapings of surface grown and suspensions
Other animal experiments related (comparable to clinical samples)
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
Role of cell blocks in cytopathologic evaluation/patient management
1. Immunophenotyping2. Special stains- Mucicarmine, Congo red, organism stains3. Architectural evaluation-
Trabecular-sinusoidal pattern in HCC,Hollow or solid proliferation spheres without cores
in carcinoma versus mesothelioma in effusionsEvaluate for invasionComparative evaluation with surgical pathology material
E.g- Peritoneal/pelvic washingQuantification of some features such as mitotic figures
4. Enhanced sampling of FNAB rinses5. Molecular test
e.g. FISH, CISH, In-situ PCR6. Archival for future studies
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Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
Critical issues to be considered
Depending on the primary indication,the method of cell block preparation vary
Multiple variants should be considered for selecting the method and modifying it as needed for
individual specimens
Specimen type- Fresh versus fixed cells Cellularity of the specimenNature of cell distribution-
predominantly solitary cells versus microfragments/aggregatesAncillary tests anticipatedAvailable resources/infrastructure in the labInstitutional and regional biases
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
J Clin Pachol 1997;50:985-990
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
Different methods of cell block preparation
A. Cell block from specimen with clot or significant sediment- FNAB
B. HistoGel methodC. Gelatin embeddingD. Agar embedding methodE. Plasma-thrombin methodF. Collodion (Celloidin) bag method
G. From scraped material from cytology smearsH. From Millipore filtersI. From cells lifted selectively from the cytology preparation
(Kaneko C et al. Diagn Cytopathol 2000;22:117–119)
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Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
Shidham & EppleCh 14 ‘Cytopathologic Diagnosis of Serous Fluids’Elsevier (W. B. Saunders Company)
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
Processing of FNA aspirate to be submitted to laboratory for Cell block
Let the remainingaspirate clot in thesyringe for 5 to 7minutes (slightlylonger than theclotting time).
1
Transfer the aspiratedformalin withdislodged cot in to thespecimen containerwith 10% formalinfixative
4
Gently and firmlyremove the plunger ofthe syringe .
3
Aspirate 10% formalinfrom the container inwhich the specimen isto be submitted forcell block processing.This dislodges the clotfrom syringe wall.
2
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
Protocol for plasma-thrombin method for cell block preparation
Shidham & EppleCh 14 ‘Cytopathologic Diagnosis of Serous Fluids’Elsevier (W. B. Saunders Company)
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Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
Protocol for cell block preparation with collodion bag
Shidham & EppleCh 14 ‘Cytopathologic Diagnosis of Serous Fluids’Elsevier (W. B. Saunders Company)
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
Protocol for preparing cell blocks with HistoGel
Shidham & EppleCh 14 ‘Cytopathologic Diagnosis of Serous Fluids’Elsevier (W. B. Saunders Company)
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
http://www.cellientsystem.com/
Automated cell block system
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Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
Wagner DG et. Diagn Cytopathol 2011 Oct;39(10):730-6
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
Shidham et al. Diag Cytopathol 2003 Oct;29(4):217-21.
Immunocytochemistry of imprint smears(continued)
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
However, all these methods face
the critical challenge due
unpredictable nature of the section cutting
of paraffin blocks with scant scattered cells
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Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
Micro-fragments& cells inspecimen
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
Aligning the cells along the cutting surface
Depth of section cutting
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
Varsegi GM, Shidham V (2009)Cell Block Preparation from Cytology Specimen with Predominance of IndividuallyScattered Cells.Journal of Visualized Experiments (JoVE) 2009 Jul 21;(29). pii: 1316.doi: 10.3791/1316. PMID: 19623160
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Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
Video of JoVE article (8 minutes 15 sec)
Video article is available FREE on web in open access at-
http://www.jove.com/index/Details.stp?ID=1316Video of JoVE article (8 minutes 15 sec)
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
From:Varsegi GM, Shidham V (2009)Journal of Visualized Experiments(JoVE) 2009 Jul 21;(29). pii: 1316.doi: 10.3791/1316. PMID: 19623160
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Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
From:Varsegi GM, Shidham V (2009)Journal of Visualized Experiments(JoVE) 2009 Jul 21;(29). pii: 1316.doi: 10.3791/1316. PMID: 19623160
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
Modified from:Varsegi GM, Shidham V (2009)Journal of Visualized Experiments(JoVE) 2009 Jul 21;(29). pii: 1316.doi: 10.3791/1316. PMID: 19623160
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
From:Varsegi GM, Shidham V (2009)Journal of Visualized Experiments(JoVE) 2009 Jul 21;(29). pii: 1316.doi: 10.3791/1316. PMID: 19623160
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Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
Immunophenotyping and cell blocks-
Factors affecting immunoreactivity-
Loss, reduction, or
enhancement of antigen immunoreactivity
Exposure to different reagents and fixative(s)
Temperature
Storage of specimen with or without fixative
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
Subtractive Coordinate Immunoreactivity Pattern (SCIP) approach
Shidham & AtkinsonCh 5. Immunocytochemistry of effusion fl uids: introduction to SCIP approach. ‘Cytopathologic Diagnosis of Serous Fluids’Elsevier (W. B. Saunders Company)
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
23
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SCIP approach
Immunocytochemistry of effusion fluids
(continued)
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Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
Shidham & AtkinsonCh 5. ‘Cytopathologic Diagnosis of Serous Fluids’Elsevier (W. B. SaundersCompany)
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
Shidham & AtkinsonCh 5. ‘Cytopathologic Diagnosis of Serous Fluids’Elsevier (W. B. SaundersCompany)
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
Study cases
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Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
pC
EA
CD
10
pC
EA
(+co
ntr
ol)
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
H&E CD177 vimentin
SMAWS cytokeratin S-100 protein
GIST
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Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
a b
c dCongo red stained 10 micron thick sections: Orange yellow birefringence under polarized light.The color changes to apple green when the axis of polarizer (blue arrows) is changed by 90 degree
Cell block- Fat pad aspiration
Positive control
Shidham VB et al. J Vis Exp. (2010) 44.http://www.jove.com/index/Details.stp?ID=1747
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
SCIP approach
F. CDX2Immunoreactivenuclear
HEstained cell blocksection
40X
B. Pan-cytokeratinImmunoreactive
C. LCA (CD45)Non-immunoreactive
A. VimentinNon-immunoreactive
D. CalretininNon-immunoreactive(Inset {2}-Mesothelial cell immunoreactivenuclear-cytoplasmic)
E. WT-1Non-immunoreactive(Arrow 2 with inset: Mesothelial cell-immunoreactivenuclear-cytoplasmic)
RM
RM
NC
10X
10X
10X
10X
10X
10X
40X
40X
40X
40X
40X
100X40X
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
RM
RM
NC
Metastatic colonic adenocarcinoma, (peritoneal fluid).
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Shidham & Atkinson.Cytopathologic Diagnosis ofSerous FluidsElsevier.
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
SCIP approachHEstained cell blocksection
Metastatic colonic adenocarcinoma, (peritoneal fluid).
Shidham & Atkinson.Cytopathologic Diagnosis ofSerous FluidsElsevier.
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Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
12
a b CalretininCalretinin
Calretinin immunoreactivity pattern (epithelioid mesothelioma, pleural fluid).Mesothelioma cells (arrow in a) show nuclear (arrowhead 1) immunoreactivity usually withcytoplasmic immunostaining (arrowhead 2) imparting the so called ‘fried-egg’ appearance.
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
C. CalretininNon-immunoreactive(Rare mesothelial cell [blue arrow] is immunoreactivenuclear-cytoplasmic)
D. BerEP4Immunoreactive
E. Estrogen receptorsImmunoreactive
B. CD68 (PGM1)Non-immunoreactive(inflammatory cells are immunoreactive)
A. VimentinNon-immunoreactive(Mesothelial & inflammatory cells are immunoreactive)
‘Subtractive coordinate immunoreactivity pattern’ (SCIP) in cell block sections
20X
20X
20X
20X
20X
40X
40X
40X
40X
40X
NC
RM
NC
NC
Metastatic mammary adenocarcinoma, (pleural effusion).
SCIP approach
(continued)
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
A. Pap smear dx – LSIL, B. H&E cell block sections, C. p16 stained cell block sections, D. biopsy showing CIN II-III
A DCB
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Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
A. Pap smear dx – HSIL , B. H&E cell block section containing “microbiopsies”, C. p16 stained cell block section showing true nuclear positivity, D. biopsy showing invasive squamous cell carcinoma .
A DCB
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
Am J Clin Pathol 2011;136:564-571
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
17
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
open access, teaching material with many pictures.
Hard copy and online availability.Opportunity for frequent updates
Vinod B. Shidham, MD, FRCPath, FIAC
Professor & Vice chair- AP
