Ser fl cytol ico pune jan 7, 2012 (handout)

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This is the handout for the short course to be presented at International CME on Oncopathology, Hotel O, Pune, India January 7, 2012

This is the handout for the short course to be presented at International CME on Oncopathology, Hotel O, Pune, India January 7, 2012

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  • 1. Diagnostic Cytopathology International CME on Hotel O, Pune, India of Effusion Fluids Oncopathology January 7, 2012 Short course Diagnostic Cytopathology of Effusion Fluids Short course Vinod B. Shidham, MD, FRCPath, FIAC Vice-chair & Professor Director of Cytopathology, Cytotechnology School, Cytopathology fellowship Dept of Pathology, Wayne State University Medical School Detroit, MI 48201, USA Co-editor-in-chief & Executive editor, CytoJournal (www.cytojournal.com) vshidham@med.wayne.eduThis presentation is available on web for all conference attendees at- http://alturl.com/3ucwx Diagnostic Cytopathology International CME on of Effusion Fluids Oncopathology Short course Disclosure I do not have conflict of any financial interest in the product if cited any in the course. I am the co-editor of the book * and Co-author of th chapter** C th f the h t on the same topic. *Shidham VB and Atkinson BF. Editors and contributors ‘Cytopathologic Diagnosis of Serous Fluids’ Multi-author book with 15 chapters, Elsevier (W. B. Saunders Company), 2007 (ISBN-13: 9781416001454). ** Shidham VB, Falzon M. Serous effusions: reactive, benign and malignant. In Gray & Kocjan, ed. Diagnostic Cytopathology, Elsevier, 3rd edition, Chapter 3. 1
  • 2. Diagnostic CytopathologyInternational CME on of Effusion Fluids Oncopathology Acknowledgment Short course Many images and tables are based on chapters in:Shidham VB and Atkinson BF.Cytopathologic Diagnosis of SerousFluidsElsevier (W. B. Saunders Company) Firstedition, 2007. Diagnostic Cytopathology OutlineInternational CME on of Effusion Fluids Oncopathology Short course Part I : Anatomy, histology, cytology, and effusions Collection, transportation, and processing Factors leading to potential diagnostic pitfalls Approach to diagnostic cytopathology of effusions The panorama of different faces of mesothelial cells Part II : Immunocytochemistry of effusion fluids: SCIP (Subtractive Coordinate Immunoreactivity Pattern) approach Evaluation of unknown primary sites of origin- Where do they come from? 2
  • 3. Diagnostic Cytopathology International CME on of Effusion Fluids Oncopathology Short course Anatomy, histology, cytology, and effusions a. Peritoneal cavity y b. Left & right pleural cavities c. Pericardial cavity Four major serous cavities Diagnostic Cytopathology International CME on of Effusion Fluids Oncopathology Short course Anatomy, histology, cytology, and effusions (continued) 1 Histology of serous li i Hi t l f lining (inguinal hernia sac). The mesothelial cells lining the fibrous tissue are flat (1). Focal reactive changes are seen as hypertrophy ofa b some cells which assume a 2 3 cuboidal contour (2,3). ( ,3) [a-d, HE stain (a, 10X; b-d, 100X)].c d 3
  • 4. Diagnostic Cytopathology International CME on of Effusion Fluids Oncopathology Short course Anatomy, histology, cytology, and effusions (continued) Peripheral light ectoplasm (1) Mesothelial cells (peritoneal fluid): show outer faintly stained Inner darker endoplasm (2) ectoplasm (1) with inner denser endoplasm (2) rich in Slightly off-center nucleus intermediate filaments. The nucleus is usually central or near Nucleolus central (a), but may be eccentric (b). Nucleoli are readily 1 1 observed. observed The vacuolation generally begins at the periphery in ectoplasm (1). 2 [a,b, Papanicolaou stained 2 SurePath® Preparation (a,b, 100XZoomed)] a b Diagnostic Cytopathology International CME on of Effusion Fluids Oncopathology Short courseAnatomy, histology, cytology, and effusions (continued) Peripheral ectoplasm Inner endoplasm Central to slightly eccentric nucleus Ruffled cell border with blebs Mesothelial cell (pleural fluid): shows outer ectoplasm which is denser than the inner endoplasm. The nucleus is central to slightly eccentric but not touching the cell periphery. The cell margin shows blebs and is ruffled. [Diff-Quik stained Cytospin preparation (100XZoomed)] 4
  • 5. Diagnostic Cytopathology International CME on of Effusion Fluids Oncopathology Short courseAnatomy, histology, cytology, and effusions (continued) Bloody effusions (Hemothorax, (Hemothorax hemopericardium and hemoperitoneum) Homogenously red or dark brown with hemosiderin pigment and the hematocrit of the effusion is 10% or more than that of the plasma hematocrit. (Occasional blood tinged fluids may be associated with a procedure trauma). Bloody effusions are more likely to be associated with an underlying malignancy. Reactive conditions associated with Bloody Effusions Para pneumonic effusions Pancreatitis Post traumatic effusions Acute aortic dissection underlying malignancy -Post cardiothoracic procedures / surgeries -Thoracic cavity vascular damage Pulmonary embolism Endometriosis Sarcoidosis Intralobar pulmonary sequestration Asbestos exposure associated pleural effusion Some Infections –e.g B. Anthrax Diagnostic Cytopathology International CME on of Effusion Fluids Oncopathology Short course Collection, transportation, and processing Biopsy vs effusion cytology 5
  • 6. Diagnostic CytopathologyInternational CME on of Effusion Fluids Oncopathology Short courseCollection, transportation, and processing (continued) To prevent clotting: Collected in anticoagulant such as- EDTA (sodium or potasium salts) in final concentration of 4.55+0.85 µmol/ml [e.g. di-sodium [e g di sodium EDTA dihydrate (EDTA Na2, 2H2O) 1 4 mg per ml of effusion fluid] 1.4 Amount of fluid sample: Variable (less than 1 ml to 100 ml or more). For optimum cellularity of cytology preparations and cell blocks, it is recommend to submit as much specimen as possible (up to 1000 ml). Each laboratory follows its own protocol for determining the amount of sample to be used. Submit as a fresh, unfixed sample: If delay is expected in transporting to the laboratory- refrigerate at 4oC (with precaution not to freeze the specimen). g ( p p ) Alternatively, although not recommended, it may be preserved in a weak fixative Fluids collected in fixative- require a wash prior to instrument processing. For blood rich specimens- start with smaller aliquots (such as 10 ml) of fresh fluid. Concentration of cells and removal of excess erythrocytes in the blood rich specimen may be achieved by density gradient centrifugation with FicollInternational CME on Collection, transportation, Diagnostic Cytopathology of Effusion Fluids Oncopathology and processing (continued) Short course Collect fresh effusion fluid Concentrate the with or without anticoagulant effusion fluid specimen Concentrate the remaining fluid similar to Direct smear of Specimen specimen without clot unconcentrated specimen for with clot semi-quantitative evaluation Use homogenized specimen after mixing (For paucicelluar fluids: Use cell-rich sediment) Concentrate by centrifugation Remove the clot (at 600g for 10 minutes) and process for cell-block preparation Pour off supernatant and vortex to resuspend cell pelletProcess for paraffin-embedding after formalin Cell-block preparation Smear preparationfixation. Histogel • Direct smears • SurePrep (Autocyte) Plasma-Thrombin • Cytospin • ThinPrep Celloidin bag • Other methods 6
  • 7. Diagnostic CytopathologyInternational CME on of Effusion Fluids Oncopathology Short courseCollection, transportation, and processing (continued) Different alternatives available for preparing permanent cytology preparations. a. Direct smears (Spreading a drop of specimen directly on slide. The specimen may be before concentration OR after concentration as sediment) i. Wet fixed- Pap stain ii. Air-dried- Diff-Quik stain* Pap stain: After rehydration in saline and post-fixation in 95%ethanol with 5% acetic acid (28) b. Cytospin preparations (Shandon EZ Megafunnel™ with Shandon Cytospin®) (31) i. Wet fixed- Pap stain ii. Air-dried- ii Ai d i d Diff-Quik stain Pap stain: After rehydration in saline and post-fixation in 95%ethanol with 5% acetic acid (28) c. Filters i. Wet fixed- Pap stain d. Liquid based cytology (SurePathTM or ThinPrepTM or other non-proprietary methods) i. Wet fixed- Pap stain Diagnostic CytopathologyInternational CME on of Effusion Fluids Oncopathology Short courseCollection, transportation, and processing (continued) Preparation Purpose Semiquantitative evaluation ofA Diff-Quik stained direct smear of the undiluted specimen cellularity. Diff-Quik stained air-dried Rapid initial detection of secondB Shandon EZ Megafunnel™ population and cytomorphologic preparation evaluation of hematopoietic cells. Pap stained preparation Cytomorphologic evaluationC especially nuclear details. details Cell-block preparation in Evaluation of-D HistoGelTM a. Immunoprofile, b. Other special stains, c. Architecture, 7
  • 8. Diagnostic Cytopathology International CME on of Effusion Fluids Oncopathology Short courseApproach to diagnostic cytopathology of effusions ► GENERAL FEATURES ► PROCESSING RELATED ► INTERPRETATION STRATEGY ► CYTOMORPHOLOGY ► IMMUNOCYTOCHEMISTRY Diagnostic Cytopathology International CME on of Effusion Fluids Oncopathology Short courseApproach to diagnostic cytopathology of effusions (continued) ► GENERAL FEATURES►A general pathology l b Any l h l laboratory may receive effusion- all general pathologists and i ff i ll l h l i d cytopathologists should be conversant with the diagnostic challenges and pitfalls of effusion fluid cytology.►Finding neoplastic cells in effusion specimens not only reveals that a patient has cancer, but it also denotes the advanced nature of the disease which at this stage is almost always incurable.►The morphologic features of most of the cancer cells in effusion smears are different f diff t from th those seen i exfoliative, b hi in f li ti brushing, and FNA cytology. d t l►The standard cytologic criteria of malignancy based on evaluation of single cell morphology are not applicable for most of the effusion cytology specimens (except in cases with high-grade neoplasms with pleomorphic cells).►Since cells in a fluid medium ‘round up’ because of surface tension, the native shapes of cancer cells cannot be a guiding feature. 8
  • 9. Diagnostic Cytopathology International CME on of Effusion Fluids Oncopathology Short courseApproach to diagnostic cytopathology of effusions (continued) ► PROCESSING RELATED1. Second population of foreign cells2. Nuclear details of the ‘second population’.3. Semiquantitative evaluation4. Objective confirmation and differential diagnosis of primary neoplasm. Diagnostic Cytopathology International CME on of Effusion Fluids Oncopathology Short courseApproach to diagnostic cytopathology of effusions (continued) ► INTERPRETATION STRATEGY a. Reactive mesothelial cells- morphologic overlap with neoplastic cells b. A single population- favor mesothelial cells c. Second foreign population consistent with metastatic neoplasm d. Sarcomas- previous history known e. Romanowsky stains highlight the ‘second population’ f. Immunocytochemistry- objective confirmation of ‘second population’ y y g. Identical orientation of serial sections for immunocytochemistry h. Quantitative and qualitative clues for mesothelioma i. Apoptosis 9
  • 10. Diagnostic Cytopathology International CME on of Effusion Fluids Oncopathology Short courseApproach to diagnostic cytopathology of effusions (continued) ► CYTOMORPHOLOGY 1. Cell groups and intercellular cohesion Non-cohesive, good intercellular cohesion, proliferation spheres 2. Arrangement of neoplastic cells Papillary configurations 3. Cytoplasm of neoplastic cells 4. Special structures and cytological features 5. Other features International CME on Algorithm for evaluation of Diagnostic Cytopathology of Effusion Fluids Oncopathology ‘second foreign population’ Short course Reactive- 4 Usually single cells without large 3-D groups Mesothelial cells 2 Neoplastic- Mesothelioma 5 ♦Quantity- Many cells ♦Quality- Many large groups Cells in effusion fluid 1 Reactive- 6a Hematopoietic cells 6 Inflammatory cells (Non-cohesive cells) Neoplastic- 6b Non mesothelial cells 3 Non-mesothelial Lymphoma L h 7 Neoplastic-8 (2nd foreign population) 8 ¶Metastatic cancer cells may be the predominant Carcinoma (Cohesive cells) cells without being seen as a ‘second population’. Sarcoma They may be present as scattered solitary cells with (Spindle cells may be present. Known cytomorphology overlapping with floridly reactive history of sarcoma is usually crucial for mesothelial cells. If indicated, immunocytochemistry proper interpretation) would facilitate confirmation of these cells as non- Melanoma (Non-cohesive cells) mesothelial. 10
  • 11. Diagnostic Cytopathology International CME on of Effusion Fluids Oncopathology Short course The panorama of different faces of mesothelial cellsREACTIVE MESOTHELIAL CELLS Binucleation and multinucleation- multinucleation Gigantic nuclei Phagocytic activity Cohesive Clusters and/or Papillary Structures ‘The two cell population approach’ Cell-in-cell configuration Cells in sheets Groups of reactive mesothelial cells I) Hepatomegaly, II) Ischemic conditions such as pulmonary infarction, ischemic colitis, and occlusion of mesenteric blood vessels, III) Trauma to organs covered with mesothelium such as spleen, liver, and lung etc, IV) Large retroperitoneal masses- Slowly growing retroperitoneal masses, V) Postoperative- following laparotomy and thoracotomy.‘ATYPICAL’ MESOTHELIAL CELLS Diagnostic Cytopathology International CME on of Effusion Fluids Oncopathology Short courseThe panorama of different face of mesothelial cells (continued) a b c Mesothelial cells with central to slightly eccentric nuclei (ascitic fluid). The cytoplasm shows a two-zone staining pattern. [a-c, Diff-Quik stained cytospin preparation (a-c, 100x Zoomed)]. 11
  • 12. Diagnostic Cytopathology International CME on of Effusion Fluids Oncopathology Short courseThe panorama of different face of mesothelial cells (continued) Panorama of mesothelial cells (asc t c u d) Ce t a (ascitic fluid). Central to near ea central nuclei. Rare mesothelial a b c d e f cells may show eccentric nuclei touching the cell membrane, but usually there is a narrow rim of cytoplasm separating the g h i j k l nucleus from the cell border (arrows). [a-x, Diff Q ik stained cytospin [ Diff-Quik t i d t i preparations (a-x, 100x m n o p q r Zoomed)]. s t u v w x Diagnostic Cytopathology International CME on of Effusion Fluids Oncopathology Short courseThe panorama of different face of mesothelial cells (continued) 1 a b c Mesothelial cells with central to slightly eccentric nuclei (ascitic fluid). The cytoplasm shows a two-zone staining pattern with peripheral vacuolation (red arrow 1). [a-c, Papanicolaou stained ThinPrep preparation (a-c, 100x Zoomed)]. 12
  • 13. Diagnostic Cytopathology International CME on of Effusion Fluids Oncopathology Short courseThe panorama of different face of mesothelial cells (continued) Mesothelial cells with central to eccentric nuclei ( ti l i (ascitic fl id) iti fluid). a b c d e f Spectrum of cytomorphological features. [a-zc, Papanicolaou stained g h i j k ThinPrep preparation (a-zc, 100x Zoomed)] l m n o p q r s t u v w x y z za zb zc Diagnostic Cytopathology International CME on of Effusion Fluids Oncopathology Short courseThe panorama of different face of mesothelial cells (continued) Mesothelial cells with eccentric nuclei (ascitic fluid). Careful scrutiny usuallyy shows a narrow rim of cytoplasm separating the nucleus from the cell border (arrows). [Papanicolaou stained ThinPrep preparation,l x 100XZoomed]. 13
  • 14. Diagnostic Cytopathology International CME on of Effusion Fluids Oncopathology Short courseThe panorama of different face of mesothelial cells (continued) NC NC RM IC RM ICDQ Pap a bMetastatic adenocarcinoma (pleural fluid). An example with morphologically identifiable unequivocal‘second foreign population’ (red arrow NC) other than mesothelial cells (blue arrow RM) and inflammatorycells (brown arrow ‘IC) in DQ and PAP stained preparations. This ‘second population’ of cells ’ (redarrow NC) is easy to be identified in DQ stain (a) as compared to that in PAP stain (b).[a, Diff-Quik stained cytospin preparation; b, Papanicolaou stained ThinPrepTM preparation (a,b, 100xZoomed)] Diagnostic Cytopathology International CME on of Effusion Fluids Oncopathology Short courseThe panorama of different face of mesothelial cells (continued)a b c dPulmonary adenocarcinoma (pleural fluid). The non-cohesive metastatic cancer cellsis the predominant cell population without being seen as a ‘second population’ (a-d).Some apoptotic tumor cells (arrows in c & d) are present. Differential includesanaplastic large cell lymphoma[a-d: PAP stained Cytospin preparation (a, 10X; b, 40X; c,d, 100X)]. 14
  • 15. Diagnostic Cytopathology International CME on of Effusion Fluids Oncopathology Short course Factors leading to potential diagnostic pitfalls a. Surface tension related alterations in cytomorphology b. b Improper specimen processing c. Many faces of reactive mesothelial cells d. Proliferation related features i) Proliferation spheres ii) Increased number of mitotic figures iii) Prominent nucleoli e. Degenerative changes Nuclear hyperchromasia Cytoplasmic vacuolation C l i l i f. Presence of some unexpected patterns and unusual structures i) Reactive lymphoid population ii) Polymorphic lymphocytes iii) Single population of cells due to predominance of tumor cells iv) Psammoma bodies v) Three dimensional benign cell groups Benign papillary inclusions, Gland-like epithelial structures, Mullerian inclusions vi) Megakaryocytes Diagnostic Cytopathology International CME on of Effusion Fluids Oncopathology Short course Factors leading to potential diagnostic pitfalls (continued)TRUE NEGATIVE RESULTS IN EFFUSIONS CAUSED BY CANCERa. Blockage of lymphatics without exfoliation of neoplastic cellsb. Increased capillary permeability due to VEGF.c. Lackc L k off exfoliation b llow-grade sarcomas and spindle cell mesotheliomas. f li ti by d d i dl ll th lid. Organized thick fibrin serosal covering preventing exfoliation of neoplastic cells.e. Decrease or total disappearance of neoplastic cells over a time 15
  • 16. Diagnostic Cytopathology International CME on of Effusion Fluids Oncopathology Short course Part II Diagnostic Cytopathology International CME on of Effusion Fluids Oncopathology Short course Immunocytochemistry of effusion fluidsThe most important issue to be considered when applyingimmunocytochemistry to effusion fluids is the significantvariation in results due to the many variables incurredfrom the time of collection of the specimen to its finalimmunostaining. 16
  • 17. Diagnostic Cytopathology International CME on Immunocytochemistry of of Effusion Fluids Oncopathology effusion fluids Short course UNIQUENESS OF EFFUSION IMMUNOCYTOCHEMISTRY Confirmation of a ‘ C fi ti f ‘second-foreign’ non-inflammatory population of cells other th df i ’ i fl t l ti f ll th than mesothelial cells in effusions correlate with metastatic cancer with objectivity. Intricacies of finding and locating the cells of interest in cell-block sections may adversely affect the final results. It is crucial to: ►Orient the serial sections identically on all slides (to identify more precisely the same cell (or small group of cells) in different sections). ►Know the sequence of these serial sections (to evaluate their co-ordinate immunoreactivity pattern). ►Immunocytochemistry does not have significant role in evaluation of peritoneal washings. Diagnostic Cytopathology International CME on Immunocytochemistry of of Effusion Fluids Oncopathology effusion fluids Short courseCell-blocks are the preferred choice.Formalin-fixed cell-block sections are recommended- Other protocols such as the evaluation of various cytology preparations(direct smears- wet fixed in alcohol or acetone, air-dried fixed with alcohol, air-driedsmears rehydrated and post-fixed in formol alcohol, liquid based cytologypreparations- SurePath or ThinPrep, cytospin preparations, etc) should beavoided.For reproducible results a standardized protocol with stepscomparable to the processing of formalin-fixed paraffin-embeddedtissue sections is essential. 17
  • 18. Diagnostic CytopathologyInternational CME on of Effusion Fluids Oncopathology Short course Critical issues leading to the best result with cell blocks of cytology specimens with singly scattered cells Aligning the cells along the cutting surface Monitor the depth of section cutting Diagnostic CytopathologyInternational CME on of Effusion Fluids Oncopathology Short course Varsegi GM, Shidham V (2009) Journal of Visualized Experiments (JoVE) 2009 Jul 21;(29). pii: 1316. doi: 10.3791/1316. PMID: 19623160 18
  • 19. Diagnostic CytopathologyInternational CME on of Effusion Fluids Oncopathology Short course From: Varsegi GM, Shidham V (2009) Journal of Visualized Experiments (JoVE) 2009 Jul 21;(29). pii: 1316. doi: 10.3791/1316. PMID: 19623160 Diagnostic CytopathologyInternational CME on of Effusion Fluids Oncopathology Short course From: Varsegi GM, Shidham V (2009) Journal of Visualized Experiments (JoVE) 2009 Jul 21;(29). pii: 1316. doi: 10.3791/1316. PMID: 19623160 19
  • 20. Diagnostic CytopathologyInternational CME on of Effusion Fluids Oncopathology Short course Modified from: Varsegi GM, Shidham V (2009) Journal of Visualized Experiments (JoVE) 2009 Jul 21;(29). pii: 1316. doi: 10.3791/1316. PMID: 19623160 Diagnostic CytopathologyInternational CME on of Effusion Fluids Oncopathology Short course From: Varsegi GM, Shidham V (2009) Journal of Visualized Experiments (JoVE) 2009 Jul 21;(29). pii: 1316. doi: 10.3791/1316. PMID: 19623160 20
  • 21. Diagnostic CytopathologyInternational CME on of Effusion Fluids Oncopathology Short courseThe video article is available FREE on the web under open access at:http://www.jove.com/index/Details.stp?ID=1316 Video f J VE ti l Vid of JoVE article (8 minutes 15 sec) i t ) Video of JoVE article (8 minutes 15 sec) Diagnostic CytopathologyInternational CME on of Effusion Fluids Oncopathology Short course 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 Imm noc tochemistr of effusion fl uids: introd ction to SCIP approach 5. Immunocytochemistry eff sion ids introduction approach. ‘Cytopathologic Diagnosis of Serous Fluids’ Elsevier (W. B. Saunders Company) Shidham VB. Effusion Fluid Evaluation Made Simple: A brief review of cytomorphologic and SCIP approach CytoJournal (In press). 21
  • 22. Diagnostic CytopathologyInternational CME on Immunocytochemistry of of Effusion Fluids Oncopathology effusion fluids Short courseDiscrepant results between formalin-fixed paraffin-embedded tissue sections ofsurgical pathology material and effusion fluid cell block sections are not uncommon cell-block uncommon.Reasons for variable reports: The variables responsible for such discrepancies include: sample size (tiny cell groups or single cells), selection of fixatives, antigen retrieval methods (i.e., heat-induced (i e heat induced epitope retrieval enzyme digestion etc ) retrieval, digestion, etc.), antibody clones used, antibody titer, and other variations in immunostaining protocols.The proteinaceous effusion fluid around suspended cells may contribute tounexpected nonspecific immunoreactivity. Diagnostic CytopathologyInternational CME on Immunocytochemistry of of Effusion Fluids Oncopathology effusion fluids Short courseIf findings are equivocal: It is prudent to be conservative and recommend to repeat. Malignant effusions usually re-accumulate quickly. Acquiring a new sample is generally not a problem. However, it is not uncommon to submit only a small fraction of a large volume of effusion fluid collected. To avoid inadequate resubmission, it may be specifically communicated in the recommendation as comment : “Recommend submission of most of the drained effusion fluid (up to 1000 mL). Larger volume of specimen facilitates retrieval of adequate cellular material in cell-block sections for immunocytochemical evaluation”. 22
  • 23. Diagnostic CytopathologyInternational CME on Immunocytochemistry of of Effusion Fluids Oncopathology effusion fluids Short courseInterpretation:I t t ti All aspects of individual and complimentary immunomarkers should be considered collectively rather than applying a reflexive positive-negative approach. Evaluation of co-ordinate immunoreactivity in different cell y population. Diagnostic CytopathologyInternational CME on Immunocytochemistry of of Effusion Fluids Oncopathology effusion fluids Short course Immunostaining None Nuclear Nuclear & Cytoplasmic Membranous Microvillous pattern cytoplasmicImmunomarkerCalretinin X XWT-1 X XD2-40 (Podoplanin) XCytokeratin* XVimentin XLCA (CD45) XPGM1 (CD68) XEMA X AdCa X mesoHBME-1 X AdCa X meso 23
  • 24. Diagnostic CytopathologyInternational CME on Immunocytochemistry of of Effusion Fluids Oncopathology effusion fluids Short course Immunostaining None Nuclear Nuclear & Cytoplasmic Membranous Microvillous pattern cytoplasmicImmunomarkerB72.3 XBerEP4 XCadherins XMOC-31 XCD44S XMesothelin XmCEA XCK 5/6 XCD15 (Lue-M1) XCA19.9 XTtf-1 X Diagnostic CytopathologyInternational CME on Immunocytochemistry of of Effusion Fluids Oncopathology effusion fluids Short course Immunoreactivity pattern with EMA (epithelioid mesothelioma, pleural fluid). EMA Mesothelioma cells with membranous (arrow) and cytoplasmic immunostaining. Note the microvilli (arrowhead). [Immunostained cell-block section (100XZoomed)]. 24
  • 25. Diagnostic Cytopathology International CME on Immunocytochemistry of of Effusion Fluids Oncopathology effusion fluids Short course a HBME-1 b HBME-1HBME-1 immunoreactivity pattern (epithelioid mesothelioma, pleural fluid).Mesothelioma cells with membranous (arrow in a) and cytoplasmic immunostaining.Note the microvilli (arrowhead in b). Diagnostic Cytopathology International CME on Immunocytochemistry of of Effusion Fluids Oncopathology effusion fluids Short course Pan-cytokeratin immunoreactivity pattern (pleural fluid). Reactive mesothelial cells with cytoplasmic immunostaining (arrow in inset). Some reactive mesothelial cells may show a concentric immunostaining i t i i pattern tt around the nucleus better appreciated by adjusting fine focus. Pan-cytokeratin 25
  • 26. Diagnostic Cytopathology International CME on Immunocytochemistry of of Effusion Fluids Oncopathology effusion fluids Short course Cytokeratin 7 immunoreactivity pattern (epithelioid mesothelioma, pleural fluid). Neoplastic mesothelial cells with cytoplasmic immunostaining. Note the bushy microvilli (arrowhead). Cytokeratin 7 Diagnostic Cytopathology International CME on Immunocytochemistry of of Effusion Fluids Oncopathology effusion fluids Short course 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. 26
  • 27. Diagnostic CytopathologyInternational CME on Immunocytochemistry of of Effusion Fluids Oncopathology effusion fluids Short course Calretinin immunoreactivity pattern (pleural fluid). Reactive mesothelial cells (blue arrows). The effusion also contains metastatic RM mammary y carcinoma cells (red arrow NC). NC Calretnin Diagnostic CytopathologyInternational CME on Immunocytochemistry of of Effusion Fluids Oncopathology effusion fluids Short course WT-1 immunoreactivity p pattern (Metastatic colonic adenocarcinoma, peritoneal fluid). Reactive mesothelial cells (arrow RM) show nuclear immunoreactivity (arrowhead in inset) with some cytoplasmic immunostaining. y p g Rare adenocarcinoma cells demonstrating nuclear immunoreactivity for CDX2 RM were also seen in other section. WT-1 27
  • 28. Diagnostic CytopathologyInternational CME on Immunocytochemistry of of Effusion Fluids Oncopathology effusion fluids Short course B72.3 immunoreactivity pattern (Metastatic mammary adenocarcinoma, pleural fluid). Metastatic adenocarcinoma cells (red arrow NC) show a cytoplasmic immunoreactivity pattern. NC B72.3 Diagnostic CytopathologyInternational CME on Immunocytochemistry of of Effusion Fluids Oncopathology effusion fluids Short course Vimentin immunoreactivity pattern (peritoneal wash). Reactive mesothelial cells (arrow RM) show cytoplasmic immunoreactivity pattern (arrowhead in inset) inset). RM vimentin 28
  • 29. Diagnostic Cytopathology International CME on Immunocytochemistry of of Effusion Fluids Oncopathology effusion fluids Short course LCA (CD45 ) immunoreactivity pattern (pleural fluid). Reactive mesothelial cells (blue arrow RM) with chronic RM inflammatory cells (red arrows). The inflammatory cells show a strong cytoplasmic immunoreactivity pattern obscuring the nucleus (arrowhead in inset). LCA Diagnostic Cytopathology International CME on Immunocytochemistry of of Effusion Fluids Oncopathology effusion fluids Short course CD 68 (PGM1 ) immunoreactivity pattern (M tt (Metastatic i mammary adenocarcinoma H with proliferation spheres H (red arrow NC), pleural fluid). Histiocytes show CD68H immunoreactivity (blue arrows H). In our experience, PGM1 does not show non non- specific immunostaining usually associated with KP1. NC Inset- Histiocytes (blue arrow H) with cytoplasmic H immunoreactivity pattern CD68 around the nucleus. 29
  • 30. Diagnostic Cytopathology International CME on Immunocytochemistry of of Effusion Fluids Oncopathology effusion fluids Short course NC NC NCa BerEP4 b BerEP4BerEP4 immunoreactivity pattern (Metastatic mammary adenocarcinoma, pleural fluid).a. The neoplastic cells in proliferation spheres (red arrow NC)- membranous immunostainingwith a honey comb-like pattern. b. Solitary adenocarcinoma cells (red arrow NC)- membranousimmunostaining pattern along the cell membrane (arrowhead in inset). Diagnostic Cytopathology International CME on Immunocytochemistry of of Effusion Fluids Oncopathology effusion fluids Short course NC NC Comparison of immunoreactivity with NC BerEP4 and B72.3 (Metastatic mammary adenocarcinoma, pleural fluid). As Compared to B72.3,a BerEP4 b NC BerEP4 most of the adenocarcinoma cells (red NC arrows NC) show strong ) g NC membranous BerEP4 immunoreactivity.c B72.3 d B72.3 30
  • 31. Diagnostic CytopathologyInternational CME on Immunocytochemistry of of Effusion Fluids Oncopathology effusion fluids Short course Monoclonal CEA (mCEA) m immunoreactivity pattern (Metastatic ovarian carcinoma, peritoneal fluid). Metastatic adenocarcinoma cells show cytoplasmic (c) and membranous (m) c immunostaining. mCEA Diagnostic CytopathologyInternational CME on Immunocytochemistry of of Effusion Fluids Oncopathology effusion fluids Short course CDX2 y immunoreactivity pattern NC (Metastatic colonic adenocarcinoma, peritoneal fluid). The adenocarcinoma cells show nuclear immunoreactivity (arrow NC). Compare with non- immunoreactive nuclei with blue hematoxylin counterstain (arrowhead). CDX2 31
  • 32. Diagnostic CytopathologyInternational CME on Immunocytochemistry of of Effusion Fluids Oncopathology effusion fluids Short course TTF-1 immunoreactivity y pattern (Metastatic pulmonary carcinoma, pleural fluid). The solitary adenocarcinoma cells as the predominant population (arrows NC) show NC nuclear immunoreactivity (arrowheads in inset). NC TTF-1 Diagnostic CytopathologyInternational CME on Immunocytochemistry of of Effusion Fluids Oncopathology effusion fluids Short course MOC-31 immunoreactivity m pattern (Metastatic mammary carcinoma, pleural fluid). The adenocarcinoma cells show predominantly membranous (m) with cytoplasmic (c) immunoreactivity. c MOC-31 32
  • 33. Diagnostic Cytopathology International CME on Immunocytochemistry of of Effusion Fluids Oncopathology effusion fluids Short course Diffuse malignant mesothelioma of epithelial type, EMA (pleural fluid). Neoplastic cells are immunoreactive for EMA (a & b) and HBME-1 (c, d, & e) a b with a membranous immunostaining pattern (arrows) highlighting long, slender, microvilli HBME-1 (arrowheads). c d e Diagnostic Cytopathology International CME on Immunocytochemistry of of Effusion Fluids Oncopathology effusion fluids Short course a EMA b HBME-1Adenocarcinoma, peritoneal fluid.Cytoplasmic immunostaining pattern (arrows) with focal blotchy immunostaining forEMA (a) and HBME-1 (b) along the cell membrane . 33
  • 34. Diagnostic Cytopathology International CME on Immunocytochemistry of of Effusion Fluids Oncopathology effusion fluids Short course For reproducible results, it is important to select any immunopanel which will fundamentally identify most of the mesothelial and inflammatory cells to create the basic map for confirmation of a ‘second-foreign’ population by ‘Subtractive Coordinate Immunoreactivity Pattern’ (SCIP) approach* Shidham VB, Atkinson BF. Immunocytochemistry of effusion fluids: Introduction to the SCIPapproach. In: Shidham VB and Atkinson BF. Editors ‘Cytopathologic Diagnosis of Serous Fluids’First edition, Elsevier (W. B. Saunders Company); 2007. Ch 5, pp. 55-78.*Shidham VB. Diagnostic Cytopathology of Serous Fluids- A brief review of cytomorphologic andSCIP approach. CytoJournal (In press). Diagnostic Cytopathology International CME on Immunocytochemistry of of Effusion Fluids Oncopathology effusion fluids Short course Evaluation of ‘Subtractive Coordinate Immunoreactivity Pattern’ (SCIP) 34
  • 35. Mesothelial & X Metastasis Y Metastasis Z ry inflammator cells (carcinoma) (non-carcinoma) vimentin 1 2 1 2 1 2 A 3 3 4 3 4 6 5 6 5 4 7 6 8 7 5 7 SCIP 1 2 1 2 2 B Pan CK 1 approach (Mixture of AE1/AE3 3 3 & CAM5.2) 4 3 6 5 4 6 4 Oncopathology Oncopathology 5 7 6 International CME on International CME on 8 7 5 7 1 2 1 2 2 C LCA(CD45) 1 [or PGM1(CD68) 3 3 or 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 effusion fluids effusion fluids 7 6 8 7 5 7 Immunocytochemistry of Immunocytochemistry of 1 2 1 2 E 1 WT-1 3 3 4 3 6 5 4 6 5 4 7 6 8 7 5 7 SCIP Short course Short course of Effusion Fluids of Effusion Fluids approach Diagnostic Cytopathology Diagnostic Cytopathology35
  • 36. Diagnostic Cytopathology International CME on Immunocytochemistry of of Effusion Fluids Oncopathology effusion fluids Short course SCIPapproach a b Diagnostic Cytopathology International CME on Immunocytochemistry of of Effusion Fluids Oncopathology effusion fluids Short course Immunoreactive for ‘negative’ lretinin, D2-40) mesothelial markers such as- BerEP4, B72.3, MOC-31, mCEA, . Proceed with: CK+,vim –/+ Carcinoma Immunopanel for unknown primary CIP ORBasic panel for evaluation by SC (vimentin, PanCK,CK CK20, Ber/EP4, B72.3, MOC31Cal Restricted panel for known primary With ‘second-foreign’ population LCA+ Lymphoma panel Lymphoma Cytogenetics Gene rearrangement CK-,vim+ Melanoma/ S-100 protein & LCA– Sarcoma Melanoma markers Without Qualitative & quantitative – + K7, ‘second foreign’ population features of mesothelioma p second-foreign Melanoma Sarcoma Absent Present Negative for Malignant malignancy mesothelioma EMA/HBME-1: Immunopanel for sarcoma Microvillous pattern OR B72.3–, BerEP4– Restricted panel for known primary 36
  • 37. ‘Subtractive coordinate immunoreactivity pattern’ (SCIP) in cell block sections Diagnostic CytopathologyInternational CME on of Effusion Fluids Oncopathology A. Vimentin Short course Non-immunoreactive NC NC 10X 40X SCIP B. Pan-cytokeratin Immunoreactive approach NC 10X NC 40X C. LCA (CD45) Non-immunoreactive NC Metastatic colonic NC 10X 40X adenocarcinoma, D. Calretinin Non-immunoreactive (peritoneal fluid). (Inset {2}- RM Mesothelial cell NC immunoreactive nuclear-cytoplasmic) RM y p ) NC 10X 40X E. WT-1 Non-immunoreactive RM HE (Arrow 2 with inset: stained Mesothelial cell- NC cell block immunoreactive RM NC section nuclear-cytoplasmic) 10X 40X NC F. CDX2 Immunoreactive nuclear NC NC 40X 10X 40X 100X ‘Subtractive coordinate immunoreactivity pattern’ (SCIP) in cell block sections Diagnostic CytopathologyInternational CME on RM of Effusion Fluids Oncopathology A. Vimentin Short course Non-immunoreactive NC 10X Zoomed SCIP B. Pan-cytokeratin Immunoreactive approach 10X RM C. Calretinin NC Non-immunoreactive [Mesothelial cells (RM) immunoreactive nuclear-cytoplasmic] 10X Zoomed RM HE D. BerEP4 stained Immunoreactive NC cell block sect o section 10X Zoomed E. Cytokeratin 7 Immunoreactive Metastatic ovarian carcinoma, (peritoneal fluid). 10X 10X NC RM F. Cytokeratin 20 Non-immunoreactive Zoomed 10X 37
  • 38. ‘Subtractive coordinate immunoreactivity pattern’ (SCIP) in cell block sections Diagnostic Cytopathology International CME on of Effusion Fluids Oncopathology Short course A. Vimentin Non-immunoreactive 40X SCIP approach B. CD68 (PGM1) Non-immunoreactive 40X RM C. Calretinin Non-immunoreactive Mesothelial cell (RM) immunoreactive Metastatic mammary nuclear-cytoplasmic) 40X adenocarcinoma, (pleural effusion). RM D. Cytokeratin 7 HE Immunoreactive stained cell block NC section 40X RM NC E. BerEP4 Immunoreactive NC 40X 40X ‘Subtractive coordinate immunoreactivity pattern’ (SCIP) in cell block sections Diagnostic Cytopathology International CME on of Effusion Fluids Oncopathology A. Vimentin Short course Non-immunoreactive (Mesothelial & inflammatory cells are immunoreactive) 20X 40X SCIP Metastatic B. CD68 (PGM1)approach Non-immunoreactive (inflammatory cells are mammary adenocarcinoma, immunoreactive) (pleural effusion). 20X 40X RM C. Calretinin Non-immunoreactive (Rare mesothelial cell [blue arrow] is immunoreactive nuclear-cytoplasmic) 20X 40X D. BerEP4 Immunoreactive NC 20X 40X NC E. Estrogen receptors NC Immunoreactive 20X 40X 38
  • 39. ‘Subtractive coordinate immunoreactivity pattern’ (SCIP) in cell block sections Diagnostic Cytopathology International CME on NC of Effusion Fluids A. Cytokeratin 7 Oncopathology Immunoreactive Short course cytoplasmic 40X 100X SCIP B. Cytokeratin 20 Non-immunoreactive approach 40X 100X C. TTF-1 Immunoreactive NC Metastatic small cell Nuclear carcinoma, 40X 100X (pleural fluid). D. Chromogranin NC Immunoreactive cytoplasmic 40X 100X E. Synaptophysin Weak immunoreactive NC cytoplasmic 40X 100X F. CD56 Immunoreactive cytoplasmic 40X 100X ‘Subtractive coordinate immunoreactivity Pattern’ (SCIP) in cell block sections Diagnostic Cytopathology International CME on of Effusion Fluids Oncopathology 7 A. Cytokeratin Short course Non-immunoreactive RMPAP stained [Mesothelial cellCytospin preparation (a-c) Immunoreactive (red arrow) Cytoplasmic] 40X B. Calretinin SCIP Non-immunoreactive [Mesothelial cell [M th li l ll Immunoreactive RM approacha 10X (red arrow) (continued) nuclear-cytoplasmic] 40X C. CD 20 Large B-cell lymphoma, Immunoreactiveb 40X Cytoplasmic (peritoneal fluid). (red arrow) NC 40X NC D. Bcl2NC Immunoreactive Cytoplasmicc (red arrow)HE stained cell blockSection (d) 40X NC NC E. CD3 Non-immunoreactived 40X 40X 39
  • 40. Diagnostic CytopathologyInternational CME on Immunocytochemistry of of Effusion Fluids Oncopathology effusion fluids Short courseVimentin (Brown)-Cytokeratin 7 (Red) Vimentin (Brown)-Cytokeratin 7 (Red) SCIP with dual staining methoda b Mammary carcinoma,Vimentin (Brown)-Cytokeratin 7 (Red) Vimentin (Brown)-Cytokeratin 7 (Red) (effusion fluid). Shidham V.B., Varsegi G, D’Amore K. Two-color immunocytochemistry for evaluation of effusion fluids for metastatic adenocarcinoma.c d CytoJournal 2010 Feb 10;7:1 Diagnostic CytopathologyInternational CME on Immunocytochemistry of of Effusion Fluids Oncopathology effusion fluids Short course SCIP with dual staining method Metastatic mammary adenocarcinoma, pleural fluid. Vimentin (Brown)-Cytokeratin 7 (R d) Vi ti (B )C t k ti (Red) Calretinin (B C l ti i (Brown)-BerEP4 (R d) ) B EP4 (Red)a b c 40
  • 41. Diagnostic CytopathologyInternational CME on Immunocytochemistry of of Effusion Fluids Oncopathology effusion fluids Short course SCIP with dual staining method Metastatic gastric adenocarcinoma, peritoneal fluid. Vimentin (Brown)-Cytokeratin 7 (Rred) Calretinin (Brown)-BerEP4 (Red)a b c Diagnostic CytopathologyInternational CME on Immunocytochemistry of of Effusion Fluids Oncopathology effusion fluids Short course Evaluation of unknown primary sites of origin- Where do they come from? Equivocal 2 1 for malignant cells Effusio cytology Negative 3 for on malignant cells Unequivocal 4 for malignant cells 41
  • 42. Diagnostic Cytopathology International CME on Evaluation of of Effusion Fluids Oncopathology unknown primary sites of origin Short courseEvaluation of unknown primary sites of origin- Where do they come from? (continued) Suspicious for malignant cells with recommendation to N t available 9 Not il bl 13 submit additional specimen for Report Or insufficient confirmation with additional 2 cytopathological evaluation with cell-block preparation if effusion malignant cells reaccumilatesb. Equivocal 5 17 for Cell-block Report Negative Immunocytochemical for malignant cells characterization on cell- 18 block sections to confirm Report 8 presence of second population by SCIP with Positive Available for malignant cells, characterization of this second population for 12 depending on results of possible primary site immunocytochemistry, comment about the primary site Immunocytochemistry of Diagnostic Cytopathology International CME on effusion fluids of Effusion Fluids Oncopathology Evaluation of Primary Short courseEvaluation of unknown primary sites of origin- Where do they come from? (continued) 6 Negative 3 Report for Negative malignant cells for malignant cells 42
  • 43. Immunocytochemistry of Diagnostic Cytopathology International CME on effusion fluids of Effusion Fluids Oncopathology Evaluation of Primary Short course Evaluation of unknown primary sites of origin- Where do they come from? (continued) 10 Comparative 144 Possible review of primary lesion malignant cells nequivocal Clinical 7 for correlationcUn 11 Not possible Diagnostic Cytopathology International CME on Evaluation of of Effusion Fluids Oncopathology unknown primary sites of origin Short course Cytomorphological features suggestive of a primary site Cytomorphological patterns y p g p Possible primary carcinoma p y 1 Three dimensional round cell groups- Breast adenocarcinoma proliferation spheres or ‘cannonballs’ Ovarian adenocarcinoma Mesothelioma of epithelioid type Reactive mesothelial proliferations 2 Acini / glands Adenocarcinomas of Breast, Lung, Colorectum, Stomach, Ovary, Endometrium, etc. Mesothelioma of epithelioid type 3 Predominantly scattered isolated Gastric adenocarcinoma malignant cells Non-cohesive variant of lung adenocarcinoma Breast lobular carcinoma Adrenocortical carcinoma (Also Lymphoma, Melanoma, & Sarcoma) 43
  • 44. Diagnostic Cytopathology International CME on Evaluation of of Effusion Fluids Oncopathology unknown primary sites of origin Short courseCytomorphological features suggestive of a primary site (continued) Cytomorphological patterns Possible primary carcinoma4 Carcinoma cells in chains and rows Breast- Lobular and ductal carcinoma (‘Indian file’ pattern) Poorly differentiated small cell carcinoma Gastric adenocarcinoma Ovarian adenocarcinoma5 Extensive cytoplasmic vacuolization Renal cell adenocarcinoma (glycogen, fat) Adrenocortical carcinoma (fat) Benign mesothelial cells Pancreatic adenocarcinoma (mucin) ( ) Ovarian adenocarcinoma (mucin) Lung adenocarcinoma Clear cell carcinoma endometrium6 Signet ring cells Gastric adenocarcinoma Colorectal adenocarcinoma7 Intracytoptasmic lumina Breast adenocarcinoma Diagnostic Cytopathology International CME on Evaluation of of Effusion Fluids Oncopathology unknown primary sites of origin Short courseCytomorphological features suggestive of a primary site (continued) Cytomorphological patterns y p g p Possible primary carcinoma p y8 Giant tumor cells Lung large cell carcinoma- giant cell type Pancreatic adenocarcinoma Thyroid anaplastic carcinoma Squamous cell carcinoma (Also Melanoma & pleomorphic sarcoma)9 Targetoid intracytoplasmic vacuole Breast adenocarcinoma (especially containing secretion lobular) Thyroid carcinoma (colloid) Ovarian carcinoma Pancreatic carcinoma10 Three dimensional groups in papillary Bronchioloalveolar carcinoma configurations Colonic adenocarcinoma Endometrial adenocarcinoma Mammary adenocarcinoma 44
  • 45. Diagnostic Cytopathology International CME on Evaluation of of Effusion Fluids Oncopathology unknown primary sites of origin Short course Cytomorphological features suggestive of a primary site (continued) Cytomorphological patterns Possible primary carcinoma 11 Three dimensional papillary groups Ovarian carcinoma- serous papillary containing psammoma bodies Thyroid papillary carcinoma Pancreatic papillary carcinoma 12 Cell groups of tall columnar cells with a Colonic adenocarcinoma picket fence pattern Pancreato-biliary carcinoma 13 Cellular pleomorphism Poorly differentiated carcinomas of lung, pancreas, ovary, thyroid, urothelium 14 Large polyhedral cells g p y Hepatocellular carcinoma p Transitional cell carcinoma Large c ell type squamous cell carcinoma 15 Cytoplasmic pigment Hepatocellular carcinoma- Bile, (Melanoma- melanin) 16 Prominent nucleoli Hepatocellular carcinoma Renal cell carcinoma Prostatic adenocarcinoma Immunocytochemistry of Diagnostic Cytopathology International CME on effusion fluids of Effusion Fluids Oncopathology Evaluation of Primary Short course Evaluation of unknown primary sites of origin- 19 Where do they come from? (continued) Report Positive Cytomorphology consistent with for f malignant cells, li t ll primary site 15 consistent with 10 Comparative 14 metastatic cancer from4 Possible review of previous neoplasm. primary lesion Cytomorphology malignant cells not classical for nequivocal the known 26 7 primary Report Clinical 16 for neoplasm correlationc Positive for malignant cells with broadUn Not available 22 cytomorphological Or insufficient characterization (such as 11 20 Not possible Cell-block non-small cell carcinoma vs small cell carcinoma vs Available 21 lymphoma). Recommend additional specimen for cell-block 23 for immunocharacterization of Immunocytochemical neoplastic cells if effusion characterization reaccumilates. 45
  • 46. Immunocytochemistry of Diagnostic Cytopathology International CME on effusion fluids of Effusion Fluids Oncopathology Evaluation of Primary Short courseEvaluation of unknown primary sites of origin- Where do they come from? (continued) 27 Report Positive Immunoprofile of the 24 23 for malignant cells, second population is nocytochemical consistent with XYZ consistent with primary aracterization primary. neoplasm. 28 Immunoprofile of the 25 Immun cha Report second population is not Positive distinct for primary for malignant cells, neoplasm. And suggest a differential diagnosis for the primary sites. Diagnostic Cytopathology International CME on Oncopathology End of Effusion Fluids Short course CytoJournal www.cytojournal.com www cytojournal com vshidham@med.wayne.eduThis presentation is available on web for all conference attendees at- http://alturl.com/3ucwx 46