Shidham's protocol for cell block preparation

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Emerging technologies in cytopathology with special reference to cell block preparation
TATA Memorial Cancer Center, Mumbai, India, Dec 21, 2010

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Shidham's protocol for cell block preparation

  1. 1. Vinod B. Shidham , MD, FRCPath, FIAC Professor Co-editor-in-chief & Executive editor, CytoJournal (www.cytojournal.com) Vice-chair - AP Director of Cytopathology, Residency training program, Cytotechnology School, Cytopathology fellowship, & GI fellowship Dept of Pathology, Wayne State University Medical School Karmanos Cancer Institute & Detroit Medical Center Detroit, MI 48201, USA [email_address] Emerging technologies in cytopathology  with special reference to cell block preparation TATA Memorial Cancer Center Mumbai, India Dec 21, 2010
  2. 2. Acknowledgement Shidham & Atkinson Cytopathologic Diagnosis of Serous Fluids Elsevier (W. B. Saunders Company) Some of the sketches and tables used are from the following reference.
  3. 3. 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 Acknowledgement Some of the sketches and tables used are from the following reference.
  4. 4. Disclaimer VS has indirect financial interest (through spouse) in AV marker mentioned in the workshop. http://www.bioinnovationllc.com/Page_2.html
  5. 5. Liquid Based Cytology (SurePath & ThinPrep) Molecular pathology techniques on Cytology specimens Intraoperative immunocytochemistry 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 Introduction
  6. 6. Pap stained Conventional, SurePath, & ThinPrep Conventional SurePath ThinPrep
  7. 7. Intraoperative evaluations
  8. 8. Intraoperative evaluation of SLN Authors Intraoperative evaluation Sensitivity (%) Specificity (%) Gibbs et al . Ann Surg Oncol 1999; 6: 699-704. FS 43 100 Clary et al . Eur J Nucl Med 1999; 26: S68.  FS 56 100 Tanis et al . Ann Surg Oncol 2001; 8: 222-26. FS 47 100 Stojadinovic A et al. Ann Surg 2002;235:92-8. FS 59 100 Creager et al. Cancer. 2002;94:3016-22. IS 38 100 Eudy et al. Hum Pathol 2003;34:797-802. FS with IHC 86 97 Our study ICIS 90 100
  9. 9. The discriminatory immunostaining pattern with ‘ MCW melanoma cocktail’ facilitated the possibility of immunocytochemical evaluation of imprint smears of Sentinel Lymph Nodes Immunocytochemistry of imprint smears (continued)
  10. 10. SLN Positive for melanoma micrometastases- IS immunostained with MCW melanoma cocktail IHC section 40X ICC 40X ICC 100X HE section 40X
  11. 11. Imprint smear 1 2 OR 3
  12. 12. a1 b2 b3 c4 Imprint smears for intra-operative immunocytochemistry MCW Melanoma Cocktail Immunocytochemistry of imprint smears (continued) a c b a b c 1 4 2 3 2-3 mm
  13. 13. Shidham et al. Diag Cytopathol 2003 Oct;29(4):217-21. Immunocytochemistry of imprint smears (continued)
  14. 14. ADS WFS WFS WFS
  15. 15. Rapid intra-operative immunostaining with MCW melanoma cocktail with 17 minute protocol. Immunocytochemistry of imprint smears (continued)
  16. 16. Shidham et al. CytoJournal 2004 Aug 06;1(1):2.
  17. 17. HE section 40X ICC 10X ICC 40X ICC 100X SLN Positive for melanoma micrometastases- IS immunostained with MCW melanoma cocktail
  18. 18. Melanoma cells Mast cells
  19. 19. Melanoma cells
  20. 20. Mast cells
  21. 21. Melanoma cell Mast cell Immunocytochemistry of imprint smears (continued)
  22. 22. 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 of cytopathology specimens
  23. 23. 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 Cell block of cytopathology specimens
  24. 24. 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
  25. 25. Critical issues to be considered Depending on the primary indication, the method of cell block preparation vary All potential variables 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
  26. 26. 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)
  27. 27. Shidham & Epple Ch 14 ‘Cytopathologic Diagnosis of Serous Fluids’ Elsevier ( W. B. Saunders Company) Different methods of cell block preparation (contd)
  28. 28. 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
  29. 29. 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 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
  30. 30. 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 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
  31. 31. 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
  32. 32. Shidham V.B., Hunt B., Jardeh S.S., Barboi A.C., Devata S., Hari P. (2010). Performing and Processing FNA of Anterior Fat Pad for Amyloid. JoVE. 44 . doi: 10.3791/1747 Video article is available FREE on web as open access at- http://www.jove.com/index/Details.stp?ID=1747
  33. 33. Shidham V.B., Hunt B., Jardeh S.S., Barboi A.C., Devata S., Hari P. (2010). Performing and Processing FNA of Anterior Fat Pad for Amyloid. JoVE. 44 . doi: 10.3791/1747
  34. 34. 9.41 minutes Video Article (Step by Step demo) Performing FNAB predominantly for cell block FREE on web at- http://www.jove.com/index/Details.stp?ID=1747
  35. 35. Protocol for plasma-thrombin method for cell block preparation Shidham & Epple Ch 14 ‘Cytopathologic Diagnosis of Serous Fluids’ Elsevier ( W. B. Saunders Company)
  36. 36. Protocol for cell block preparation with collodion bag Shidham & Epple Ch 14 ‘Cytopathologic Diagnosis of Serous Fluids’ Elsevier ( W. B. Saunders Company)
  37. 37. Protocol for preparing cell blocks with HistoGel Shidham & Epple Ch 14 ‘Cytopathologic Diagnosis of Serous Fluids’ Elsevier ( W. B. Saunders Company)
  38. 38. Aligning the cells along the cutting surface Depth of section cutting
  39. 39. Varsegi G.M., Shidham V. (2009). Cell Block Preparation from Cytology Specimen with Predominance of Individually Scattered Cells. J Vis Exp. ( JoVE - Journal of Visualized Experiments) 2009 Jul 21;(29). pii: 1316. doi: 10.3791/1316. PMID: 19623160 Video article is available FREE on web as open access at- http://www.jove.com/index/Details.stp?ID=1316
  40. 40. Varsegi GM, Shidham V (2009) Journal of Visualized Experiments (JoVE) 2009 Jul 21;(29). pii: 1316. doi: 10.3791/1316. PMID: 19623160
  41. 41. Cell Block Preparation 8.19 minutes Video Article (Step by Step demo) FREE on web at- http://www.jove.com/index/Details.stp?ID=1316
  42. 42. From: Varsegi GM, Shidham V (2009) Journal of Visualized Experiments (JoVE) 2009 Jul 21;(29). pii: 1316. doi: 10.3791/1316. PMID: 19623160
  43. 43. From: Varsegi GM, Shidham V (2009) Journal of Visualized Experiments (JoVE) 2009 Jul 21;(29). pii: 1316. doi: 10.3791/1316. PMID: 19623160
  44. 44. Modified from: Varsegi GM, Shidham V (2009) Journal of Visualized Experiments (JoVE) 2009 Jul 21;(29). pii: 1316. doi: 10.3791/1316. PMID: 19623160
  45. 45. From: Varsegi GM, Shidham V (2009) Journal of Visualized Experiments (JoVE) 2009 Jul 21;(29). pii: 1316. doi: 10.3791/1316. PMID: 19623160
  46. 46. 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
  47. 47. 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)
  48. 48. 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
  49. 49. Q/A
  50. 50. Peer-reviewed, open access, teaching material with many pictures. Hard copy and online availability. Opportunity for frequent updates
  51. 51. Detroit [email_address] Thank you

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