01 Presentation I VS (8-55MB)- (3-28-08).pps

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Part I of Four part symposium: “Diagnostic Cytopathology of Serous Effusion” on April 19, 2007 at Neenah, WI, USA
(2008 Wisconsin Society of Cytology, 40th Anniversary)

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01 Presentation I VS (8-55MB)- (3-28-08).pps

  1. 1. 1 Diagnostic Cytopathology of Serous Effusions Vinod B. Shidham , MD, FRCPath, FIAC Professor Executive editor & coeditor-in-chief, CytoJournal ( www.cytojournal.com ) Department of Pathology Medical College of Wisconsin 9200 W Wisconsin Av, Milwaukee, WI 53226, USA [email_address] Sessions I ( 8.05-8.45 ) To view on web copy-paste the following URL into your browser: http://www.slideshare.net/vshidham/01-presentation-i-vs-32-10-26-07 2008 Wisconsin Society of Cytology SPRING MEETING, 40TH ANNIVERSARY Holiday Inn – Riverwalk, Neenah, WI Saturday, April 19, 2008 (7.30 to 3.30)
  2. 2. 2 Objectives 1. Cytopathology of benign and malignant conditions. 2. Approach to immunohistochemical evaluation. 3. Emphasize diagnostic pitfalls .
  3. 3. Shidham VB and Atkinson BF . Cytopathologic Diagnosis of Serous Fluids Elsevier ( W. B. Saunders Company) First edition, 2007. (ISBN-13: 9781416001454). Acknowledgment Most of the images and tables are based on chapters in: Elsevier link related to the book- http://www.us.elsevierhealth.com/product.jsp?isbn=9781416001454#description
  4. 4. 3 Outline Session I (40 minutes): Anatomy, histology, cytology, and effusions Collection, transportation, and processing of effusion fluids Factors leading to potential diagnostic pitfalls Approach to diagnostic cytopathology of effusions The panorama of different face of mesothelial cells Session II (45 minutes): Benign conditions with and without specific cellular patterns Mesothelioma Metastatic carcinoma Metastatic sarcoma and melanoma Hematolymphoid disorders (Lymphomas and leukemias) Session III (45 minutes): Evaluation of unknown primary sites of origin- Where do they come from? Immunocytochemistry of effusion fluids: SCIP (Subtractive Coordinate Immunoreactivity Pattern) approach Flow cytometry, molecular techniques, and other special techniques Session IV (45 minutes): Diagnostic cytopathology of peritoneal washings Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases
  5. 5. 3 Outline Session I (40 minutes): 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 face of mesothelial cells Session II (45 minutes): Benign conditions with and without specific cellular patterns Mesothelioma Metastatic carcinoma Metastatic sarcoma and melanoma Hematolymphoid disorders (Lymphomas and leukemias) Session III (45 minutes): Evaluation of unknown primary sites of origin- Where do they come from? Immunocytochemistry of effusion fluids: SCIP (Subtractive Coordinate Immunoreactivity Pattern) approach Flow cytometry, molecular techniques, and other special techniques Session IV (45 minutes): Diagnostic cytopathology of peritoneal washings Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases
  6. 6. 2 Anatomy, histology, cytology, and effusions a. Peritoneal cavity b. Left & right pleural cavities c. Pericardial cavity Four major serous cavities
  7. 7. 3 Session I Anatomy, histology, cytology, and effusions (continued) Histology of serous lining (inguinal hernia sac). The mesothelial cells lining the fibrous tissue are flat (1). Focal reactive changes are seen as hypertrophy of some cells which assume a cuboidal contour (2,3). [a-d, HE stain (a, 10X; b-d, 100X)]. 1 3 2 a d c b
  8. 8. 4 Session I Anatomy, histology, cytology, and effusions (continued) Mesothelial cells (peritoneal fluid): show outer faintly stained ectoplasm (1) with inner denser endoplasm (2) rich in intermediate filaments. The nucleus is usually central or near central (a), but may be eccentric (b). Nucleoli are readily observed. The vacuolation generally begins at the periphery in ectoplasm (1). [a,b, Papanicolaou stained SurePath® Preparation (a,b, 100XZoomed)] Peripheral light ectoplasm (1) Inner darker endoplasm (2) Slightly off-center nucleus Nucleolus 1 2 1 2 a b
  9. 9. 5 Session I Anatomy, histology, cytology, and effusions (continued) 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)] Peripheral ectoplasm Inner endoplasm Central to slightly eccentric nucleus Ruffled cell border with blebs
  10. 10. 6 Session I Anatomy, histology, cytology, and effusions (continued) Causes Transudate Exudate Chylous 1. Congestive heart failure 2. Pulmonary atelectasis 3. Nephrotic syndrome 4. Postpartum effusion 5. Peritoneal dialysis 6. Superior vena cava obstruction 7. Portal vein hypertension secondary to cirrhosis, schistosomiasis, and diffuse metastatic neoplasm in liver 8. Postoperative abdominal surgery 9. Meigs syndrome 10. Chronic renal diseases with impaired renal function 11. Inferior vena cava hypertension 12. Coagulation disorder and anticoagulant therapy 1. Malignant neoplasms 2. Infections, including bacterial pneumonia, lung abscess, tuberculosis, fungal infections, viral infections, and parasitic diseases 3. Collagen vascular diseases including systemic lupus erythematosus and rheumatoid pleuritis 4. Pulmonary embolism/infarction 5. Some abdominal diseases including pancreatitis, subphrenic abscess, esophageal rupture, and hepatic abscess 6. Radiotherapy 7. Bile peritonitis 8. Trauma 9. Myocardial infarction and post-myocardial infarction syndrome 10. Aortic dissection 11. Cardiac rupture 1. Metastatic cancer 2. Trauma, including blunt trauma and operative trauma 3. Retroperitoneal cancers and lymphoma 4. Tuberculosis 5. Congenital lymphatic anomalies
  11. 11. 7 Session I Anatomy, histology, cytology, and effusions (continued) Biochemical features Transudate Exudate Chylous Accumulation of fluid as an ultra-filtrate of plasma. a. Total protein 3.0 g/dL (30 g/L) or lower. b. Specific gravity 1.015 or less. c. Ratio of fluid lactic dehydrogenase to serum lactic dehydrogenase less than 0.6. d. Does not coagulate. Associated with increased permeability of the capillaries leading to exudation of protein rich fluid. a. Total protein 3.0 g/dL (30 g/L) or more. b. Specific gravity 1.015 or more. c. Ratio of fluid lactic dehydrogenase to serum lactic dehydrogenase more than 0.6. d. May coagulate on standing. Leakage of lymphatic fluid secondary to trauma or the obstructed thoracic duct or cisterna chyli, caused by malignant neoplasms including lymphomas and carcinomas. a. Milky white fluid. b. Wet preparation show usually small free fat droplets.
  12. 12. 8 Session I Anatomy, histology, cytology, and effusions (continued) Cellular features Transudate Exudate Chylous Hypocellular specimens. Mostly mesothelial cells . Hypercellular specimens. Predominantly inflammatory cells with reactive mesothelial cells with or without malignant cells . The specimens are rich in lymphocytes and some lipid-laden macrophages .
  13. 13. 9 Session I 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. Bloody effusions (Hemothorax, hemopericardium and hemoperitoneum) underlying malignancy Anatomy, histology, cytology, and effusions (continued) Para pneumonic effusions Post traumatic effusions -Post cardiothoracic procedures / surgeries -Thoracic cavity vascular damage Pulmonary embolism Asbestos exposure associated pleural effusion Pancreatitis Acute aortic dissection Endometriosis Sarcoidosis Intralobar pulmonary sequestration Some Infections –e.g B. Anthrax Reactive conditions associated with Bloody Effusions
  14. 14. Session I Collection, transportation, and processing 10 Biopsy vs effusion cytology The sampling benefit of effusion cytology generally provides a higher yield of diagnostic material than biopsy of the serous lining. In contrast to a focal biopsy from a small area of an extensive serosal surface, an effusion represents cells exfoliated from the entire serosal surface .
  15. 15. Session I Collection, transportation, and processing 11 Biopsy vs effusion cytology
  16. 16. Session I Collection, 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 µ mol/ml [e.g. di-sodium EDTA dihydrate (EDTA Na 2 , 2H 2 O) 1.4 mg per ml of effusion fluid] 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 4 o C (with precaution not to freeze the specimen). 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 Ficoll 12
  17. 17. Collection, transportation, and processing 13 Concentrate the effusion fluid specimen Concentrate the remaining fluid similar to specimen without clot Specimen with clot Cell-block preparation Histogel Plasma-Thrombin Celloidin bag Use homogenized specimen after mixing (For paucicelluar fluids: Use cell-rich sediment) Session I Concentrate by centrifugation (at 600g for 10 minutes) Pour off supernatant and vortex to resuspend cell pellet Collect fresh effusion fluid with or without anticoagulant Process for paraffin-embedding after formalin fixation. Direct smear of unconcentrated specimen for semi-quantitative evaluation Remove the clot and process for cell-block preparation <ul><li>Direct smears </li></ul><ul><li>Cytospin </li></ul><ul><li>SurePrep (Autocyte) </li></ul><ul><li>ThinPrep </li></ul><ul><li>Other methods </li></ul>Smear preparation
  18. 18. Session I Collection, transportation, and processing (continued) 14 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- 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 ( SurePath TM or ThinPrep TM or other non-proprietary methods) i. Wet fixed- Pap stain
  19. 19. Session I Collection, transportation, and processing (continued) 15 Preparation Purpose A Diff-Quik stained direct smear of the undiluted specimen Semiquantitative evaluation of cellularity. B Diff-Quik stained air-dried Shandon EZ Megafunnel ™ preparation Rapid initial detection of second population and cytomorphologic evaluation of hematopoietic cells . C Pap stained preparation Cytomorphologic evaluation especially nuclear details . D Cell-block preparation in HistoGel TM Evaluation of- a. Immunoprofile , b. Other special stains, c. Architecture,
  20. 20. Session I Approach to diagnostic cytopathology of effusions ► GENERAL FEATURES ► PROCESSING RELATED ► INTERPRETATION STRATEGY ► CYTOMORPHOLOGY ► IMMUNOCYTOCHEMISTRY 16
  21. 21. Approach to diagnostic cytopathology of effusions (continued) ► GENERAL FEATURES 17 Any general pathology laboratory may receive effusion- all general pathologists and 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 from those seen in exfoliative, brushing, and FNA cytology. ► 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. ►
  22. 22. Session I 1. Second population of foreign cells 2. Nuclear details of the ‘second population’. 3. Semiquantitative evaluation 4. Objective confirmation and differential diagnosis of primary neoplasm. ► PROCESSING RELATED 18 Approach to diagnostic cytopathology of effusions (continued)
  23. 23. 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’ g. Identical orientation of serial sections for immunocytochemistry h. Quantitative and qualitative clues for mesothelioma i. Apoptosis ► INTERPRETATION STRATEGY 19 Approach to diagnostic cytopathology of effusions (continued)
  24. 24. Session I 1. Cell groups and intercellular cohesion Non-cohesive, good intercellular cohesion, proliferation spheres 2. Arrangement of neoplastic cells P apillary configurations 3. Cytoplasm of neoplastic cells 4. Special structures and cytological features 5. Other features ► CYTOMORPHOLOGY 20 Approach to diagnostic cytopathology of effusions (continued)
  25. 25. 21 The panorama of different face of mesothelial cells REACTIVE MESOTHELIAL CELLS Binucleation and 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
  26. 26. The panorama of different face of mesothelial cells (continued) 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)]. 22 a b c
  27. 27. The panorama of different face of mesothelial cells (continued) Panorama of mesothelial cells (ascitic fluid). Central to near central nuclei. Rare mesothelial cells may show eccentric nuclei touching the cell membrane, but usually there is a narrow rim of cytoplasm separating the nucleus from the cell border (arrows). [a-x, Diff-Quik stained cytospin preparations (a-x, 100x Zoomed)]. 23 a c b h g f e d p o n m l k j i x w v u t s r q
  28. 28. The panorama of different face of mesothelial cells (continued) 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)]. 24 a b c 1
  29. 29. The panorama of different face of mesothelial cells (continued) Mesothelial cells with central to eccentric nuclei (ascitic fluid). Spectrum of cytomorphological features. [a-zc, Papanicolaou stained ThinPrep preparation (a-zc, 100x Zoomed)] 25 a c b h g f e d p o n m l k j i x w v u t s r q z y za zb zc
  30. 30. Mesothelial cells with eccentric nuclei (ascitic fluid). Careful scrutiny usually shows a narrow rim of cytoplasm separating the nucleus from the cell border (arrows). [Papanicolaou stained ThinPrep preparation, 100XZoomed]. The panorama of different face of mesothelial cells (continued) 26 l x
  31. 31. Algorithm for evaluation of a ‘second foreign population’ . 27 Cells in effusion fluid 1 Non-mesothelial cells 3 Mesothelial cells 2 Reactive- Usually single cells without large 3-D groups 4 Hematopoietic cells (Non-cohesive cells) 6 Neoplastic - Lymphoma 6b Reactive- Inflammatory cells 6a Neoplastic- Mesothelioma ♦ Quantity- Many cells ♦ Quality- Many large groups 5 Neoplastic - 8 ( 2nd foreign population) Carcinoma (Cohesive cells) Sarcoma (Spindle cells may be present. Known history of sarcoma is usually crucial for proper interpretation) Melanoma (Non-cohesive cells) 7 ¶ Metastatic cancer cells may be the predominant cells without being seen as a ‘second population’. They may be present as scattered solitary cells with cytomorphology overlapping with floridly reactive mesothelial cells. If indicated, immunocytochemistry would facilitate confirmation of these cells as non-mesothelial. 8
  32. 32. a b NC RM NC RM IC IC Metastatic adenocarcinoma (pleural fluid). An example with morphologically identifiable unequivocal ‘ second foreign population’ (red arrow NC) other than mesothelial cells (blue arrow RM) and inflammatory cells (brown arrow ‘IC) in DQ and PAP stained preparations. This ‘second population’ of cells ’ (red arrow 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, 100x Zoomed)]. DQ Pap The panorama of different face of mesothelial cells (continued) 28
  33. 33. Pulmonary adenocarcinoma (pleural fluid). The non-cohesive metastatic cancer cells is the predominant cell population without being seen as a ‘second population’ (a-d). Some apoptotic tumor cells (arrows in c & d) are present. Differential includes anaplastic large cell lymphoma [a-d: PAP stained Cytospin preparation (a, 10X; b, 40X; c,d, 100X)]. The panorama of different face of mesothelial cells (continued) 29 a b c d
  34. 34. Session I ► IMMUNOCYTOCHEMISTRY 30 Approach to diagnostic cytopathology of effusions (continued) If a ‘second foreign population’ is suggested in a Diff-Quik stained preparation but can not be confirmed with PAP stain, it may be analyzed further by immunostaining of cell-block sections - effusions secondary to the well differentiated adenocarcinomas especially that of ovary and breast. ► Confirmation of second population by evaluating ‘subtractive coordinate immunoreactivity pattern’ (SCIP) in cell-block sections ( session III ). ►
  35. 35. Session I Factors leading to potential diagnostic pitfalls a. Surface tension related alterations in cytomorphology 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 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 31
  36. 36. Session I Factors leading to potential diagnostic pitfalls (continued) a. Blockage of lymphatics without exfoliation of neoplastic cells b. Increased capillary permeability due to VEGF. c. Lack of exfoliation by low-grade sarcomas and spindle cell mesotheliomas. d. Organized thick fibrin serosal covering preventing exfoliation of neoplastic cells. e. Decrease or total disappearance of neoplastic cells over a time TRUE NEGATIVE RESULTS IN EFFUSIONS CAUSED BY CANCER 32
  37. 37. Thank you Milwaukee Art Museum [email_address] End Diagnostic Cytopathology of Serous Effusions 

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