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

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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  • 1. Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.edu 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 Cell Blocks for Molecular Tests & Immunocytochemistry ApplicationsFrustrations & Triumphs! November 9, 2013 (ASC Round Table #11, 12:00 – 1:45 PM) This presentation is also available at: http://alturl.com/245zh Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.edu Disclaimer VS is co-editor of ‘Cytopathologic Diagnosis of Serous 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 Disclaimer VS has indirect financial interest (through spouse) in AV marker referenced. http://www.bioinnovationllc.com/Home_Page.html 1
  • 2. Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.edu Outline Cell block- microbiopsyRole in cytopathologic evaluation/patient management Critical issues to be considered Different methods of cell block preparation Aligning the cells along the cutting surface Depth of section cutting Immunophenotyping and cell blocksImmunoreactivity interference Marker for SCIP approach A 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 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 has Added 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 and long term archival for future elective evaluations 2
  • 3. Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.edu Samples that may be ‘Cell blocked’ Clinical samples Fluids Effusion fluids (ascitic, pleural, pericardial, etc) Other fluids (drainage, cysts, cavities, etc) Washings (peritoneal, pelvic, bronchial washing) Exfoliated cells- Cervical cytology specimens Brushings- Endocervical, bronchial, bile duct etc Curretages- Endocervical FNA passes-rinses Scrapings 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. Immunophenotyping 2. Special stains- Mucicarmine, Congo red, organism stains 3. Architectural evaluationTrabecular-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 3
  • 4. 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 specimen Nature of cell distributionpredominantly solitary cells versus microfragments/aggregates Ancillary tests anticipated Available resources/infrastructure in the lab Institutional 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 method C. Gelatin embedding D. Agar embedding method E. Plasma-thrombin method F. Collodion (Celloidin) bag method G. From scraped material from cytology smears H. From Millipore filters I. From cells lifted selectively from the cytology preparation (Kaneko C et al. Diagn Cytopathol 2000;22:117–119) 4
  • 5. Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.edu 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 Let the remaining aspirate clot in the syringe for 5 to 7 minutes (slightly longer than the clotting time). 1 Aspirate 10% formalin from the container in which the specimen is to be submitted for cell block processing. This dislodges the clot from syringe wall. 2 Gently and firmly remove the plunger of the syringe . 3 Transfer the aspirated formalin with dislodged cot in to the specimen container with 10% formalin fixative 4 Processing of FNA aspirate to be submitted to laboratory for Cell block Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.edu Protocol for plasma-thrombin method for cell block preparation Shidham & Epple Ch 14 ‘Cytopathologic Diagnosis of Serous Fluids’ Elsevier (W. B. Saunders Company) 5
  • 6. Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.edu Protocol 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.edu Protocol 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 Automated cell block system http://www.cellientsystem.com/ 6
  • 7. 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 Immunocytochemistry & Vice chair- AP Professor of imprint smears (continued) vshidham@med.wayne.edu Shidham et al. Diag Cytopathol 2003 Oct;29(4):217-21. 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 7
  • 8. Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.edu Microfragments & cells in specimen 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 Individually Scattered Cells. Journal of Visualized Experiments (JoVE) 2009 Jul 21;(29). pii: 1316. doi: 10.3791/1316. PMID: 19623160 8
  • 9. 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 article is available FREE on web in open access athttp://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.edu From: Varsegi GM, Shidham V (2009) Journal of Visualized Experiments (JoVE) 2009 Jul 21;(29). pii: 1316. doi: 10.3791/1316. PMID: 19623160 9
  • 10. 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 10
  • 11. Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.edu Immunophenotyping and cell blocksFactors affecting immunoreactivityLoss, 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 & Atkinson Ch 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 7 6 7 6 2 5 3 7 4 5 1 4 5 5 7 1 4 5 3 3 2 1 5 7 3 4 5 1 5 7 6 7 6 7 2 6 2 3 4 1 Immunocytochemistry of effusion fluids (continued) 7 5 2 3 6 4 SCIP approach 8 2 8 6 6 3 6 8 3 2 7 5 4 E WT-1 1 5 7 4 1 D Calretinin 4 [or PGM1(CD68) or mixture of LCA & PGM1] 7 5 3 3 C LCA(CD45) 1 5 7 4 1 4 2 1 6 3 6 2 8 2 3 1 B Pan CK (Mixture of AE1/AE3 & CAM5.2) 7 4 5 3 1 6 6 3 1 4 4 2 2 4 6 8 6 3 1 2 1 A vimentin Mesothelial & X inflammatory cells 7 5 Metastasis (carcinoma) 2 Y Z Metastasis (non-carcinoma) vshidham@med.wayne.edu 11
  • 12. Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@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.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.edu Study cases 12
  • 13. Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.edu Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP CD10 pCEA (+control) pCEA vshidham@med.wayne.edu Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.edu H&E CD177 vimentin GIST WS cytokeratin S-100 protein SMA 13
  • 14. Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.edu Shidham VB et al. J Vis Exp. (2010) 44. http://www.jove.com/index/Details.stp?ID=1747 Cell block- Fat pad aspiration a b c d Positive control Congo 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 vshidham@med.wayne.edu A. Vimentin Non-immunoreactive NC NC 10X 40X B. Pan-cytokeratin Immunoreactive NC NC 10X 40X C. LCA (CD45) Non-immunoreactive SCIP approach NC NC 10X 40X D. Calretinin Non-immunoreactive (Inset {2}RM Mesothelial cell immunoreactive nuclear-cytoplasmic) RM NC 10X NC 40X E. WT-1 Non-immunoreactive (Arrow 2 with inset: Mesothelial cellimmunoreactive RM nuclear-cytoplasmic) HE stained cell block section RM NC NC 10X 40X Shidham & Atkinson. Cytopathologic Diagnosis of Serous Fluids NC F. CDX2 Immunoreactive nuclear NC NC 40X Metastatic colonic adenocarcinoma, (peritoneal fluid). 10X 40X Elsevier. 100X Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.edu SCIP approach HE stained cell block section Metastatic colonic adenocarcinoma, (peritoneal fluid). Shidham & Atkinson. Cytopathologic Diagnosis of Serous Fluids Elsevier. 14
  • 15. Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.edu 2 a Calretinin b 1 Calretinin Calretinin immunoreactivity pattern (epithelioid mesothelioma, pleural fluid). Mesothelioma cells (arrow in a) show nuclear (arrowhead 1) immunoreactivity usually with cytoplasmic 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- AP A. Vimentin Non-immunoreactive (Mesothelial & inflammatory cells are immunoreactive) vshidham@med.wayne.edu 20X 40X Metastatic mammary adenocarcinoma, (pleural effusion). B. CD68 (PGM1) Non-immunoreactive (inflammatory cells are immunoreactive) 20X 40X RM C. Calretinin Non-immunoreactive (Rare mesothelial cell [blue arrow] is immunoreactive nuclear-cytoplasmic) 20X 40X 20X 40X D. BerEP4 Immunoreactive SCIP approach NC (continued) NC E. Estrogen receptors NC Immunoreactive 40X 20X Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.edu A 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 15
  • 16. Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.edu A 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.edu Am J Clin Pathol 2011;136:564-571 Vinod B. Shidham, MD, FRCPath, FIAC Professor & Vice chair- AP vshidham@med.wayne.edu 16
  • 17. 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 17

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