1. Vinod B. Shidham, MD, FRCPath, FIACProfessor & Vice chair- APvshidham@med.wayne.edu Cell Blocksfor Molecular Tests & Immunocytochemistry Applications- Frustrations & Triumphs! Vinod B. Shidham, MD, FRCPath, FIACVice-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 School Karmanos Cancer Institute & Detroit Medical Center Detroit, MI 48201, USA November 5, 2012(ASC Round Table #19, 12:00 1:45 PM)

2. Vinod B. Shidham, MD, FRCPath, FIACProfessor & Vice chair- APvshidham@med.wayne.eduDisclaimerVS is co-editor ofCytopathologic Diagnosis ofSerous FluidsElsevier (W. B. Saunders Company)cited for various methods ofcell block preparation (thesketches and tables used arefrom this reference). 3. Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.eduDisclaimerVS has indirect financial interest (through spouse) in AV marker referenced. http://www.bioinnovationllc.com/Home_Page.html 4. 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 preparationAligning the cells along the cutting surfaceDepth of section cuttingImmunophenotyping and cell blocks-Immunoreactivity interferenceMarker for SCIP approachA few study cases 5. Vinod B. Shidham, MD, FRCPath, FIACProfessor & Vice chair- APvshidham@med.wayne.eduCell 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 preparationCell block with cytology preparation hasAdded benefit of excellent cytomorphologic details in concert with architectural insight Cyto-histo-pathology 6. Vinod B. Shidham, MD, FRCPath, FIACProfessor & Vice chair- APvshidham@med.wayne.edu Cell blocks Conglomeration ofminute tissue fragments andisolated cells / small cell groupsin specimens collected or submittedas suspensionsTo processed asParaffin embedded tissue sections andlong term archival for future elective evaluations 7. Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.edu Samples that may be Cell blocked Clinical samplesFluidsEffusion 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 8. 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) 9. 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 coresin carcinoma versus mesothelioma in effusionsEvaluate for invasionComparative evaluation with surgical pathology materialE.g- Peritoneal/pelvic washingQuantification of some features such as mitotic figures 4. Enhanced sampling of FNAB rinses 5. Molecular teste.g. FISH, CISH, In-situ PCR 6. Archival for future studies 10. Vinod B. Shidham, MD, FRCPath, FIACProfessor & Vice chair- APvshidham@med.wayne.eduCritical issues to be consideredDepending on the primary indication,the method of cell block preparation varyMultiple 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 11. Vinod B. Shidham, MD, FRCPath, FIACProfessor & Vice chair- APvshidham@med.wayne.eduJ Clin Pachol 1997;50:985-990 12. Vinod B. Shidham, MD, FRCPath, FIACProfessor & Vice chair- APvshidham@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;22:117119) 13. Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.eduShidham & EppleCh 14 Cytopathologic Diagnosis of Serous FluidsElsevier (W. B. Saunders Company) 14. Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.eduLettheremaining Aspirate 10% formalin Gentlyandfirmly Transfer the aspiratedaspirate clot in thefrom the container in remove the plunger of formalin withsyringe for5 to 7 which the specimen is the syringe . dislodged cot in to theminutes (slightly to be submitted for specimen containerlongerthan thecell block processing.with 10% formalinclotting time). This dislodges the clot fixativefrom syringe wall. 12 3 4Processing of FNA aspirate to be submitted to laboratory for Cell block 15. Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.eduProtocol for plasma-thrombin method for cell block preparationShidham & EppleCh 14 Cytopathologic Diagnosis of Serous FluidsElsevier (W. B. Saunders Company) 16. Vinod B. Shidham, MD, FRCPath, FIACProfessor & Vice chair- APvshidham@med.wayne.eduProtocol for cell block preparation with collodion bag Shidham & Epple Ch 14 Cytopathologic Diagnosis of Serous Fluids Elsevier (W. B. Saunders Company) 17. Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.eduProtocol for preparing cell blocks with HistoGelShidham & EppleCh 14 Cytopathologic Diagnosis of Serous FluidsElsevier (W. B. Saunders Company) 18. 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 19. Vinod B. Shidham, MD, FRCPath, FIACProfessor & Vice chair- APvshidham@med.wayne.eduMicro-fragments & cells inspecimen 20. Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.eduAligning the cells along the cutting surface Depth of section cutting 21. 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 22. Vinod B. Shidham, MD, FRCPath, FIACProfessor & Vice chair- APvshidham@med.wayne.edu 23. Vinod B. Shidham, MD, FRCPath, FIACProfessor & Vice chair- APvshidham@med.wayne.edu Video article is available FREE on web inopen access at-http://www.jove.com/index/Details.stp?ID=1316Video of JoVE article (8 minutes 15 sec) Video of JoVE article (8 minutes 15 sec) 24. 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 25. 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 26. 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 27. 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 28. Vinod B. Shidham, MD, FRCPath, FIACProfessor & Vice chair- APvshidham@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 29. Vinod B. Shidham, MD, FRCPath, FIACProfessor & Vice chair- APvshidham@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) 30. Mesothelial & XMetastasis YMetastasisZinflammatory cells(carcinoma) (non-carcinoma) vimentin 1 2 1 21 2A334 3 46 565 47 68 7 5 71 2 1 22BPan CK 1(Mixture of AE1/AE3 33 & CAM5.2)4 36 5 465 47 68 75 71 2 1 22CLCA(CD45) 1[or PGM1(CD68)33or mixture of LCA 4 36 5 46& PGM1] 457 68 75 71 2 1 22 D Calretinin1334 36 5 465 47 68 75 71 2 1 2E1 WT-1 3 34 36 5 465 47 68 75 7Professor & Vice chair- AP vshidham@med.wayne.edu(continued)SCIPof effusion fluidsapproach Vinod B. Shidham, MD, FRCPath, FIACImmunocytochemistry 31. Vinod B. Shidham, MD, FRCPath, FIACProfessor & Vice chair- APvshidham@med.wayne.eduShidham & AtkinsonCh 5. CytopathologicDiagnosis of Serous FluidsElsevier (W. B. SaundersCompany) 32. Vinod B. Shidham, MD, FRCPath, FIACProfessor & Vice chair- APvshidham@med.wayne.eduShidham & AtkinsonCh 5. CytopathologicDiagnosis of Serous FluidsElsevier (W. B. SaundersCompany) 33. Vinod B. Shidham, MD, FRCPath, FIACProfessor & Vice chair- APvshidham@med.wayne.eduStudy cases 34. Vinod B. Shidham, MD, FRCPath, FIACProfessor & Vice chair- APvshidham@med.wayne.eduCell block section of FNA of From: oban S et al. BMC Cancer 2004, 4:89 Open access article from:Hepatocellular Carcinoma http://www.biomedcentral.com/1471-2407/4/89 35. Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.edu H&ECD177 vimentin GISTWS cytokeratin S-100 protein SMA 36. Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.edu Cell block- Fat pad aspiration a bPositive 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 37. Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP 35 A. Vimentin vshidham@med.wayne.edu Non-immunoreactive NC NC 10X40X B. Pan-cytokeratin ImmunoreactiveNC NC 10X40X SCIP C. LCA (CD45) Non-immunoreactive approachNCNC 10X40X D. Calretinin Non-immunoreactive (Inset {2}-RM Mesothelial cell NC immunoreactive nuclear-cytoplasmic) RMNC Metastatic colonic 10X40X adenocarcinoma, E. WT-1 Non-immunoreactiveRM(peritoneal fluid).HE (Arrow 2 with inset:stainedMesothelial cell-NCcell block immunoreactive RM NCsectionnuclear-cytoplasmic)10X40XNC Shidham & Atkinson. F. CDX2 ImmunoreactiveCytopathologic Diagnosis of nuclearNC Serous FluidsNC Elsevier. 40X 10X 40X100X 38. 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. 39. Vinod B. Shidham, MD, FRCPath, FIACProfessor & Vice chair- APvshidham@med.wayne.edu 2 1a Calretininb 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. 40. Subtractive coordinate immunoreactivity pattern (SCIP) in cell block sectionsVinod B. Shidham, MD, FRCPath, FIACProfessor & Vice chair- APA. Vimentin vshidham@med.wayne.eduNon-immunoreactive(Mesothelial &inflammatory cells areimmunoreactive)20X 40XB. CD68 (PGM1)MetastaticNon-immunoreactive(inflammatory cells aremammary adenocarcinoma,immunoreactive) (pleural effusion).20X 40X RMC. CalretininNon-immunoreactive(Rare mesothelialcell [blue arrow] isimmunoreactivenuclear-cytoplasmic)20X 40XD. BerEP4ImmunoreactiveNC SCIP20X 40Xapproach NC(continued)E. Estrogenreceptors NCImmunoreactive20X 40X 41. Vinod B. Shidham, MD, FRCPath, FIACProfessor & Vice chair- APvshidham@med.wayne.eduA B C DA. Pap smear dx LSIL,B. H&E cell block sections,C. p16 stained cell block sections,D. biopsy showing CIN II-III 42. Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.eduABCDA. 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 . 43. Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.eduAm J Clin Pathol 2011;136:564-571 44. Vinod B. Shidham, MD, FRCPath, FIACProfessor & Vice chair- APvshidham@med.wayne.edu 45. Vinod B. Shidham, MD, FRCPath, FIACProfessor & Vice chair- APvshidham@med.wayne.edu 46. Vinod B. Shidham, MD, FRCPath, FIACProfessor & Vice chair- APvshidham@med.wayne.eduPeer-reviewed, open access,teaching material with many pictures.Hard copy and online availability.Opportunity for frequent updates 47. Vinod B. Shidham, MD, FRCPath, FIACProfessor & Vice chair- APvshidham@med.wayne.edu 48. Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.eduAdd-Bile canalicular patternTwo color immunoSmall biopsies and scattered cells