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Round table #19 at American Society of Cytopathology (Nov 5, 2012, Las Vegas, NV, USA)- VB Shidham, MD, FRCPath, FIAC
 

Round table #19 at American Society of Cytopathology (Nov 5, 2012, Las Vegas, NV, USA)- VB Shidham, MD, FRCPath, FIAC

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Round table #19 at American Society of Cytopathology (Nov 5, 2012, Las Vegas, NV, USA)- VB Shidham, MD, FRCPath, FIAC

Round table #19 at American Society of Cytopathology (Nov 5, 2012, Las Vegas, NV, USA)- VB Shidham, MD, FRCPath, FIAC

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    Round table #19 at American Society of Cytopathology (Nov 5, 2012, Las Vegas, NV, USA)- VB Shidham, MD, FRCPath, FIAC Round table #19 at American Society of Cytopathology (Nov 5, 2012, Las Vegas, NV, USA)- VB Shidham, MD, FRCPath, FIAC Presentation Transcript

    • Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.edu Cell Blocksfor Molecular Tests & Immunocytochemistry Applications- Frustrations & Triumphs! Vinod B. Shidham, MD, FRCPath, FIAC Vice-chair- AP, Professor, Director of Cytopathology, Cytotechnology School, Cytopathology fellowship, and GI Pathology Executive editor & co-editor-in-chief, CytoJournal (www.cytojournal.com) Dept of Pathology, Wayne State University Medical School Karmanos Cancer Institute & Detroit Medical Center Detroit, MI 48201, USA November 5, 2012 (ASC Round Table #19, 12:00 – 1:45 PM)
    • Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.edu DisclaimerVS is co-editor of‘Cytopathologic Diagnosis ofSerous Fluids’Elsevier (W. B. Saunders Company)cited for various methods ofcell block preparation (thesketches and tables used arefrom 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
    • Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.edu OutlineCell block- microbiopsy-Role in cytopathologic evaluation/patient managementCritical issues to be consideredDifferent methods of cell block preparation Aligning the cells along the cutting surface Depth of section cuttingImmunophenotyping and cell blocks- Immunoreactivity interference Marker for SCIP approachA few study cases
    • Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.edu Cell block of cytopathology specimen is Equivalent to microbiopsy evaluationthe 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 blocks Conglomeration of minute tissue fragments and isolated cells / small cell groups in specimens collected or submitted as suspensions To processed as Paraffin embedded tissue sections andlong term archival for future elective evaluations
    • 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 samplesCell cultures- scrapings of surface grown and suspensionsOther animal experiments related (comparable to clinical samples)
    • Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.eduRole of cell blocks in cytopathologic evaluation/patient management 1. Immunophenotyping 2. Special stains- Mucicarmine, Congo red, organism stains 3. Architectural evaluation- Trabecular-sinusoidal pattern in HCC, Hollow or solid proliferation spheres without cores in carcinoma versus mesothelioma in effusions Evaluate for invasion Comparative evaluation with surgical pathology material E.g- Peritoneal/pelvic washing Quantification of some features such as mitotic figures 4. Enhanced sampling of FNAB rinses 5. Molecular test e.g. FISH, CISH, In-situ PCR 6. Archival for future studies
    • 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 specimensSpecimen type- Fresh versus fixed cellsCellularity 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.eduJ 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 preparationA. Cell block from specimen with clot or significant sediment- FNABB. HistoGel methodC. Gelatin embeddingD. Agar embedding methodE. Plasma-thrombin methodF. Collodion (Celloidin) bag methodG. 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)
    • Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.eduShidham & 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.eduLet the remaining Aspirate 10% formalin Gently and firmly Transfer the aspiratedaspirate clot in the from the container in remove the plunger of formalin withsyringe for 5 to 7 which the specimen is the syringe . dislodged cot in to theminutes (slightly to be submitted for specimen containerlonger than the cell block processing. with 10% formalinclotting time). This dislodges the clot fixative from syringe wall. 1 2 3 4Processing of FNA aspirate to be submitted to laboratory for Cell block
    • Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.eduProtocol for plasma-thrombin method for cell block preparation Shidham & Epple Ch 14 ‘Cytopathologic Diagnosis of Serous Fluids’ Elsevier (W. B. Saunders Company)
    • Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.eduProtocol for cell block preparation with collodion bag Shidham & Epple Ch 14 ‘Cytopathologic Diagnosis of Serous Fluids’ Elsevier (W. B. Saunders Company)
    • Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.eduProtocol for preparing cell blocks with HistoGel Shidham & Epple Ch 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 However, all these methods face the critical challenge dueunpredictable nature of the section cuttingof paraffin blocks with scant scattered cells
    • 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.eduAligning the cells along the cutting surface Depth of section cutting
    • Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.eduVarsegi 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
    • Vinod B. Shidham, MD, FRCPath, FIACProfessor & Vice chair- APvshidham@med.wayne.edu
    • Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.edu Video article is available FREE on web in open access at-http://www.jove.com/index/Details.stp?ID=1316 Video of JoVE article (8 minutes 15 sec) Video of JoVE article (8 minutes 15 sec)
    • Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.eduFrom: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.eduFrom: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.eduModified 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.eduFrom: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.eduImmunophenotyping 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.eduSubtractive Coordinate Immunoreactivity Pattern (SCIP) approach Shidham & Atkinson Ch 5. Immunocytochemistry of effusion fl uids: introduction to SCIP approach. ‘Cytopathologic Diagnosis of Serous Fluids’ Elsevier (W. B. Saunders Company)
    • Mesothelial & X Metastasis Y Metastasis Z inflammatory cells (carcinoma) (non-carcinoma) vimentin 1 2 1 2 1 2A 3 3 4 3 4 6 5 6 5 4 7 6 8 7 5 7 1 2 1 2 2B Pan CK 1(Mixture of AE1/AE3 3 3 & CAM5.2) 4 3 6 5 4 6 5 4 7 6 8 7 5 7 1 2 1 2 2C LCA(CD45) 1[or PGM1(CD68) 3 3or mixture of LCA 4 3 6 5 4 6& PGM1] 4 5 7 6 8 7 5 7 1 2 1 2 2 D Calretinin 1 3 3 4 3 6 5 4 6 5 4 7 6 8 7 5 7 1 2 1 2E 1 WT-1 3 3 4 3 6 5 4 6 5 4 7 6 8 7 5 7 Professor & Vice chair- AP vshidham@med.wayne.edu (continued) SCIP of effusion fluids approach Vinod B. Shidham, MD, FRCPath, FIAC Immunocytochemistry
    • Vinod B. Shidham, MD, FRCPath, FIACProfessor & Vice chair- APvshidham@med.wayne.edu Shidham & Atkinson Ch 5. ‘Cytopathologic Diagnosis of Serous Fluids’ Elsevier (W. B. Saunders Company)
    • Vinod B. Shidham, MD, FRCPath, FIACProfessor & Vice chair- APvshidham@med.wayne.edu Shidham & Atkinson Ch 5. ‘Cytopathologic Diagnosis of Serous Fluids’ Elsevier (W. B. Saunders Company)
    • Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.eduStudy cases
    • Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.eduCell block section of FNA of From: Çoban S et al. BMC Cancer 2004, 4:89 Open access article from:Hepatocellular Carcinoma http://www.biomedcentral.com/1471-2407/4/89
    • Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.edu H&E CD177 vimentin GISTWS cytokeratin S-100 protein SMA
    • Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.edu Cell block- Fat pad aspiration a b Positive control 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
    • Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP 35 A. Vimentin vshidham@med.wayne.edu Non-immunoreactive NC NC 10X 40X B. Pan-cytokeratin Immunoreactive NC NC 10X 40X SCIP C. LCA (CD45) Non-immunoreactive approach NC NC 10X 40X D. Calretinin Non-immunoreactive (Inset {2}- RM Mesothelial cell NC immunoreactive nuclear-cytoplasmic) RM NC Metastatic colonic 10X 40X adenocarcinoma, E. WT-1 Non-immunoreactive RM (peritoneal fluid).HE (Arrow 2 with inset:stained Mesothelial cell- NCcell block immunoreactive RM NCsection nuclear-cytoplasmic) 10X 40X NC Shidham & Atkinson. F. CDX2 Immunoreactive Cytopathologic Diagnosis of nuclear NC Serous Fluids NC Elsevier. 40X 10X 40X 100X
    • Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.eduHEstained SCIP approachcell blocksection Shidham & Atkinson. Metastatic colonic Cytopathologic Diagnosis of adenocarcinoma, Serous Fluids (peritoneal fluid). Elsevier.
    • Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.edu 2 1a Calretinin b CalretininCalretinin 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.
    • ‘Subtractive coordinate immunoreactivity pattern’ (SCIP) in cell block sections Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- APA. Vimentin vshidham@med.wayne.eduNon-immunoreactive(Mesothelial &inflammatory cells areimmunoreactive) 20X 40XB. CD68 (PGM1) MetastaticNon-immunoreactive(inflammatory cells are mammary adenocarcinoma,immunoreactive) (pleural effusion). 20X 40X RMC. CalretininNon-immunoreactive(Rare mesothelialcell [blue arrow] isimmunoreactivenuclear-cytoplasmic) 20X 40XD. BerEP4Immunoreactive NC SCIP 20X 40X approach NC (continued)E. Estrogenreceptors NCImmunoreactive 20X 40X
    • Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.eduA B C D A. Pap smear dx – LSIL, B. H&E cell block sections, C. p16 stained cell block sections, D. biopsy showing CIN II-III
    • Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.eduA B C D 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 .
    • Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.eduAm J Clin Pathol 2011;136:564-571
    • Vinod B. Shidham, MD, FRCPath, FIACProfessor & Vice chair- APvshidham@med.wayne.edu
    • Vinod B. Shidham, MD, FRCPath, FIACProfessor & Vice chair- APvshidham@med.wayne.edu
    • Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.edu Peer-reviewed, open access,teaching material with many pictures. Hard copy and online availability. Opportunity for frequent updates
    • Vinod B. Shidham, MD, FRCPath, FIACProfessor & Vice chair- APvshidham@med.wayne.edu
    • Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.eduAdd-Bile canalicular patternTwo color immunoSmall biopsies and scattered cells