Handout for Round table #19 at American Society of Cytopathology (Nov 9, 2013, Orlando, FL, USA)- VB...

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Vinod B. Shidham, MD, FRCPath, FIAC

Professor & Vice chair- AP

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

J Clin Pachol 1997;50:985-990

Vinod B. Shidham, MD, FRCPath, FIAC

Professor & Vice chair- AP

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

Shidham & EppleCh 14 ‘Cytopathologic Diagnosis of Serous Fluids’Elsevier (W. B. Saunders Company)

Vinod B. Shidham, MD, FRCPath, FIAC

Professor & Vice chair- AP

vshidham@med.wayne.edu

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.

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Vinod B. Shidham, MD, FRCPath, FIAC

Professor & Vice chair- AP

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

http://www.cellientsystem.com/

Automated cell block system

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Vinod B. Shidham, MD, FRCPath, FIAC

Professor & Vice chair- AP

vshidham@med.wayne.edu

Wagner DG et. Diagn Cytopathol 2011 Oct;39(10):730-6

Vinod B. Shidham, MD, FRCPath, FIAC

Professor & Vice chair- AP

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

Micro-fragments& cells inspecimen

Vinod B. Shidham, MD, FRCPath, FIAC

Professor & Vice chair- AP

vshidham@med.wayne.edu

Aligning the cells along the cutting surface

Depth of section cutting

Vinod B. Shidham, MD, FRCPath, FIAC

Professor & Vice chair- AP

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

Vinod B. Shidham, MD, FRCPath, FIAC

Professor & Vice chair- AP

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

23

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Immunocytochemistry of effusion fluids

(continued)

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Vinod B. Shidham, MD, FRCPath, FIAC

Professor & Vice chair- AP

vshidham@med.wayne.edu

Shidham & AtkinsonCh 5. ‘Cytopathologic Diagnosis of Serous Fluids’Elsevier (W. B. SaundersCompany)

Vinod B. Shidham, MD, FRCPath, FIAC

Professor & Vice chair- AP

vshidham@med.wayne.edu

Shidham & AtkinsonCh 5. ‘Cytopathologic Diagnosis of Serous Fluids’Elsevier (W. B. SaundersCompany)

Vinod B. Shidham, MD, FRCPath, FIAC

Professor & Vice chair- AP

vshidham@med.wayne.edu

Study cases

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Vinod B. Shidham, MD, FRCPath, FIAC

Professor & Vice chair- AP

vshidham@med.wayne.edu

Vinod B. Shidham, MD, FRCPath, FIAC

Professor & Vice chair- AP

vshidham@med.wayne.edu

pC

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Vinod B. Shidham, MD, FRCPath, FIAC

Professor & Vice chair- AP

vshidham@med.wayne.edu

H&E CD177 vimentin

SMAWS cytokeratin S-100 protein

GIST

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Vinod B. Shidham, MD, FRCPath, FIAC

Professor & Vice chair- AP

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

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40X

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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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu

Am J Clin Pathol 2011;136:564-571

Vinod B. Shidham, MD, FRCPath, FIAC

Professor & Vice chair- AP

vshidham@med.wayne.edu

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Vinod B. Shidham, MD, FRCPath, FIAC

Professor & Vice chair- AP

vshidham@med.wayne.edu

Vinod B. Shidham, MD, FRCPath, FIAC

Professor & Vice chair- AP

vshidham@med.wayne.edu

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

vshidham@med.wayne.edu