Role of cell blocks in cytopathology

1. May 17 & 19, 2010 Vinod B. Shidham , MD, FRCPath, FIAC Professor Co-editor-in-chief & Executive editor, CytoJournal (www.cytojournal.com) Vice-chair Director of Cytopathology, Cytotechnology School, Cytopathology fellowship, & GI fellowship Dept of Pathology, Wayne State University Medical School Detroit, MI 48201, USA [email_address] (Workshop# 5 & 43 ) Cell Blocks In Cytopathology

2. 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 ).

3. Disclaimer VS has indirect financial interest (through spouse) in AV marker mentioned in the workshop. http://www.bioinnovationllc.com/Page_2.html

4. Cell block- microbiopsy - Role 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 blocks- Immunoreactivity interference Marker for SCIP approach A few study cases Q/A session Outline

5. Cell block of cytopathology specimen is Equivalent to microbiopsy evaluation the interface between cytology and histopathology ( bridge to histopatholog y/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

6. Role 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

7. 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 distribution- predominantly solitary cells versus microfragments/aggregates Ancillary tests anticipated Available resources/infrastructure in the lab Institutional and regional biases

8. 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)

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

10. Processing of FNA aspirate to be submitted to laboratory for Cell block Let the remaining aspirate clot in the syringe for 5 to 7 minutes (slightly longer than the clotting time). 1 Transfer the aspirated formalin with dislodged cot in to the specimen container with 10% formalin fixative 4 Gently and firmly remove the plunger of the syringe . 3 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

11. Protocol for plasma-thrombin method for cell block preparation Shidham & Epple Ch 14 ‘Cytopathologic Diagnosis of Serous Fluids’ Elsevier ( W. B. Saunders Company)

12. Protocol for cell block preparation with collodion bag Shidham & Epple Ch 14 ‘Cytopathologic Diagnosis of Serous Fluids’ Elsevier ( W. B. Saunders Company)

13. Protocol for preparing cell blocks with HistoGel Shidham & Epple Ch 14 ‘Cytopathologic Diagnosis of Serous Fluids’ Elsevier ( W. B. Saunders Company)

14. Aligning the cells along the cutting surface Depth of section cutting

15. 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

17. Video of JoVE article (8 minutes 15 sec) At this stage, please note the URL and observe the full FREE video in the new internet window Video of JoVE article (8 minutes 15 sec) Video article FREE on web as open access at- http://www.jove.com/index/Details.stp?ID=1316 Or at http://alturl.com/zkns

18. From: Varsegi GM, Shidham V (2009) Journal of Visualized Experiments (JoVE) 2009 Jul 21;(29). pii: 1316. doi: 10.3791/1316. PMID: 19623160

19. From: Varsegi GM, Shidham V (2009) Journal of Visualized Experiments (JoVE) 2009 Jul 21;(29). pii: 1316. doi: 10.3791/1316. PMID: 19623160

20. Modified from: Varsegi GM, Shidham V (2009) Journal of Visualized Experiments (JoVE) 2009 Jul 21;(29). pii: 1316. doi: 10.3791/1316. PMID: 19623160

21. From: Varsegi GM, Shidham V (2009) Journal of Visualized Experiments (JoVE) 2009 Jul 21;(29). pii: 1316. doi: 10.3791/1316. PMID: 19623160

22. 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 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)

23. SCIP approach Immunocytochemistry of effusion fluids (continued) 2 3 1 6 5 4 8 7 Mesothelial & inflammatory cells 2 3 1 6 5 4 8 7 2 3 1 6 5 4 8 7 2 3 1 6 5 4 8 7 2 3 1 6 5 4 8 7 X Metastasis (non-carcinoma) 2 3 1 6 5 4 7 2 3 1 6 5 4 7 2 3 1 6 5 4 7 3 1 6 5 4 7 2 3 1 6 5 4 7 Z Metastasis (carcinoma) 2 1 5 4 3 7 6 2 1 5 4 3 7 6 2 1 5 4 3 7 6 2 1 5 4 3 7 6 2 1 5 4 3 7 6 Y vimentin Pan CK (Mixture of AE1/AE3 & CAM5.2) Calretinin WT-1 LCA (CD45) [or PGM1(CD68) or mixture of LCA & PGM1] A B C D E

24. Shidham & Atkinson Ch 5. ‘Cytopathologic Diagnosis of Serous Fluids’ Elsevier (W. B. Saunders Company)

25. Shidham & Atkinson Ch 5. ‘Cytopathologic Diagnosis of Serous Fluids’ Elsevier (W. B. Saunders Company)

26. SCIP approach Metastatic colonic adenocarcinoma, (peritoneal fluid). 35 F. CDX2 Immunoreactive nuclear HE stained cell block section 40X B. Pan-cytokeratin Immunoreactive C. LCA (CD45) Non-immunoreactive A. Vimentin Non-immunoreactive D. Calretinin Non-immunoreactive (Inset {2}- Mesothelial cell immunoreactive nuclear-cytoplasmic) E. WT-1 Non-immunoreactive (Arrow 2 with inset: Mesothelial cell- immunoreactive nuclear-cytoplasmic) RM RM NC 10X 10X 10X 10X 10X 10X 40X 40X 40X 40X 40X 100X 40X NC NC NC NC NC NC NC NC NC NC NC RM RM NC

27. Study cases

28. A. Pap smear dx – LSIL, B. H&E cell block sections, C. p16 stained cell block sections, D. biopsy showing CIN II-III A D C B

29. 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 carc inoma . A D C B

30. 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. 14 1 2 a b Calretinin Calretinin

31. 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 Cell block- Fat pad aspiration Positive control a b c d

32. Metastatic mammary adenocarcinoma, (pleural effusion). SCIP approach (continued) 38 C. Calretinin Non-immunoreactive (Rare mesothelial cell [blue arrow] is immunoreactive nuclear-cytoplasmic) D. BerEP4 Immunoreactive E. Estrogen receptors Immunoreactive B. CD68 (PGM1) Non-immunoreactive (inflammatory cells are immunoreactive) A. Vimentin Non-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

33. GIST H&E CD177 vimentin SMA WS cytokeratin S-100 protein

34. From: Ç oban S et al. BMC Cancer 2004, 4:89 Open access article from: http://www.biomedcentral.com/1471-2407/4/89 Cell block section of FNA of Hepatocellular Carcinoma

35. Q/A

36. Peer-reviewed, open access, teaching material with many pictures. Hard copy and online availability. Opportunity for frequent updates

37. Thank you Milwaukee Art Museum [email_address] Detroit After June 1, 2010 [email_address]