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

1. 1 Diagnostic Cytopathology of Serous Effusions Session IV ( 10.50-11.35 ) Q&A- 11.35-12.00 To view this session on web copy-paste the following URL into your browser: http://www.slideshare.net/vshidham/04-presentations-iv-vs-8mb-1042708b-pps 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] 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 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/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): Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

3. 2 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/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): Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

4. 3 Washings, lavages, brushings, scrapings, and touch imprints Involvement of serosa by cancer cells, even without effusion correlates with poor prognosis . Upstaging of such cases has been recommended. Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases Washings or lavages - Retrieval of irrigated physiologic saline Peritoneal washing (peritoneal lavage, pelvic washing)- routine for staging of gynecologic cancers. Pleural lavages- for staging of lung cancer and esophageal cancer Other methods include- Cul-de-sac (pouch of Douglas) aspirate or as intraoperative collections Pleural brushings I ntraoperative touch imprint cytology of visceral serosa Scrapings and brushings such as diaphragmatic scrapings Peritoneal dialysis fluid- Cytologic atypia a potential pitfall The methods of evaluation in this subset involves-

5. 4 1. INTRODUCTION 1a. Specimen collection procedure 1b. Interpretation approach 2. BENIGN CONDITIONS 2a. Mesothelial cell polymorphism 2b . Mesothelial surface reactions 2bi. Endosalpingiosis 2bii. Endometriosis 2c. Benign Ovarian Tumors 2d. Miscellaneous Benign Changes in Peritoneal Washings 3. MALIGNANT DISEASE 3a. Ovarian neoplasms 3b. Endometrial carcinoma 3c. Cervical carcinoma Peritoneal washings Washings, lavages, brushings, scrapings, and touch imprints (continued) Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

6. For staging purposes , the peritoneal cytologic sample must be obtained as soon as the peritoneal cavity is entered. Preexisting spontaneous fluid in the pelvis- collected and labeled as “ peritoneal fluid ” or, if excessive, as “ ascites ”. Peritoneal washings (continued) 5 The significance of tumor cells identified in washings performed after iatrogenic contamination is not known. While artificial contamination of the peritoneum with tumor is disquieting, the prognostic value of the peritoneal washings is predicated on the assumption that the results reflect the natural biology of the patient’s tumor. This dilemma can be avoided when the cytologic samples are collected as an initial step in the exploration . Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

7. 100 ml of saline, preferably a balanced salt solution such as peritoneal dialysis fluid, is instilled into the peritoneal cavity, agitated, aspirated and sent for cytologic evaluation. Collection Peritoneal washings (continued) 6 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

8. Although washing the peritoneal cavity with saline or balanced salt solution may be expected to yield a cell sample similar to ascites fluid , there are differences that must be appreciated in order to accurately evaluate these samples. Peritoneal washings (continued) Interpretation approach (continued) 7 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases Rather than the freely desquamated cells seen in spontaneous ascitic fluid, many of the cells present in peritoneal washings have been mechanically stripped from the underlying connective tissue. The cells that are uncommon in ascites (probably because they are normally shed in very low numbers) may be present.

9. The differences between ascites and peritoneal washings An awareness of these differences aid optimum interpretation. Peritoneal washings (continued) Interpretation approach (continued) 8 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases FEATURE ASCITES WASHINGS Collection Spontaneous exfoliation Cells abraded Aggregation 3-dimensional groups 2-dimensional sheets Cell shape Rounded shapes Flat mesothelial cells

10. BENIGN CONDITIONS It is important that benign conditions that can be confused with malignancy be understood and excluded. The benign changes found in peritoneal washings are the key differences between intra-operative washings and spontaneous ascites. Peritoneal washings (continued) Interpretation approach (continued) 9 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

11. Peritoneal washings (continued) Interpretation approach (continued) 10 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases BENIGN CONDITION CYTOLOGIC FINDING PITFALL Inflammatory conditions Mixture of mesothelial patterns in the same sample Can be confused with cancer Bulky benign tumors Leiomyomas Ovarian fibrothecomas Mixture of mesothelial patterns in the same sample Can be confused with cancer Adhesions Capillary tangles Detached ciliary tufts Anucleated cytoplasmic fragments with cilia Ruptured benign epithelial cyst or cystic tumor Sheets and groups of epithelial cells Can be confused with carcinoma

12. Peritoneal washings (continued) Interpretation approach (continued) Surface reactions 11 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases BENIGN CONDITION CYTOLOGIC FINDING PITFALL Endosalpingiosis Epithelial cell clusters with psammoma bodies Pattern overlaps with serous tumors of low malignant potential and carcinoma Endometriosis Endometrial epithelial cells and stroma; hemosiderin-laden macrophages Can be confused with cancer Collagen balls Small round bodies of collagenized stroma and flat mesothelial cells Surface component of benign tumors Epithelial cells forming cast like-group of ovarian surface clefts Overlaps with low grade carcinomas

13. SPECIALIZED CRITERIA FOR PERITONEAL WASHING CYTOLOGY Peritoneal washings (continued) Interpretation approach (continued) Comparative review of cell-block sections of washing & surgical pathology of primary neoplasm is simple and crucial . 12 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases Classical criteria for malignancy including Abnormal single cells and groups Dyspolaric nuclei Nuclear enlargement Irregular nuclear outline Chromatin abnormalities Nucleolar abnormalities Abnormal cells outside the spectrum of reactive mesothelial cells and benign surface reactions- Second population Abnormal cells compare well with those of the primary tumor .

14. Peritoneal washings (continued) Interpretation approach (continued) Comparison of cytomorphologic features of serous lesions in peritoneal washings 13 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases Feature Endosalpingiosis Benign tumor Low malignant potential Micropapillary (Low grade serous carcinoma) Carcinoma Aggregation Small 3-D papillary groups Cast-like Large 3-D groups Large 3-D groups Finger-like 3-D groups and single cells Periphery Cohesive Cohesive Cohesive Cohesive Frayed Single cells Absent Absent Rare Variable Characteristic N/C ratio Increased Variable Variable Variable Variable Nuclear size Normal Normal Normal-Variable Increased Increased; pleomorphic

15. Peritoneal washings (continued) Interpretation approach (continued) Comparison of cytomorphologic features of serous lesions in peritoneal washings (continued) 14 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases Feature Endosalpingiosis Benign tumor Low malignant potential Micropapillary (Low grade serous carcinoma) Carcinoma Nuclear shape Round-oval Round-oval Variable Variable Pleomorphic Chromatin Uniform, finely granular Uniform, finely granular Variable Variable Clumped; variable Nucleoli Small, inconspicuous Small Small Variable Prominent Psammoma bodies Frequently present Infrequent Variable Variable Variable

16. Peritoneal washings (continued) Nuclear size in mesothelial cells can vary considerably in peritoneal washings and it is important not to over-call in such cases. All of the cells in this field are benign . 15 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

17. Peritoneal washings (continued) Two structures composed of tangled fibrillary material appear to represent capillaries and associated connective tissue material that reflect lysed adhesions. This pattern can be seen associated with healed inflammation or endometriosis . 16 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

18. Peritoneal washings (continued) Mesothelial sheets associated with large ovarian fibromas and spontaneous peritoneal fluid can have a rolled-up, pseudopapillary appearance. 17 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

19. Peritoneal washings (continued) Psammoma bodies are surrounded by cuboidal epithelium. This pattern is typical of endosalpingiosis , although it can be seen with low grade serous tumors . Comparative review of the histology with primary tumor is often helpful. 18 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

20. Peritoneal washings (continued) Cast-like groups that appear to have been removed as a unit from an irregular ovarian surface have been found in association with benign tumors, particularly adenofibromas . Although they can occasionally be found in the peritoneal washings of women with malignant tumors, the diagnosis of malignancy should only be made in cases that have other cells or groups with the typical features of malignancy. 19 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

21. Peritoneal washings (continued) Collagen balls are microscopic nodules that appear to represent small, mesothelial lined surface protrusions . 20 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

22. Peritoneal washings (continued) Detached ciliary tufts are seen as anucleated cell fragments composed of cilia and a small fragment of residual cytoplasm. 21 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

23. Peritoneal washings (continued) A large fragment of tumor cells can be dislodged from intraperitoneal malignancy in the course of taking the washings. This pattern is rare in spontaneous effusions. 22 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

24. Peritoneal washings (continued) Groups of abnormal cells surrounding psammoma bodies in a washing from a woman with an ovarian serous borderline tumor involving the peritoneal cavity. 23 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

25. Peritoneal washings (continued) Groups of atypical cells interpreted as “low grade serous tumor” are present in this washing from a woman with intraperitoneal micropapillary serous tumor . While the cytologic pattern is that of a neoplasm, the precise categorization is difficult by cytology alone . Indeed, it is unclear whether this lesion is a well differentiated carcinoma or a tumor of low malignant potential (borderline). 24 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

26. Peritoneal washings (continued) Endometrioid carcinoma. The columnar cells in the three-dimensional group, the are haphazardly arranged with dyspolaric nuclei. This distinguishes endometrioid carcinoma from benign tubal epithelium. 25 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

27. Peritoneal washings (continued) Serous carcinoma of endometrium in peritoneal washings are typically seen as three-dimensional papillary groups and single cells with large nuclei and prominent nucleoli. Unlike endometrioid adenocarcinomas, peritoneal involvement is common and the abnormal cells are readily recognized as malignant . 26 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

28. Peritoneal washings (continued) Cervical squamous carcinoma three-dimensional groups or single cells in peritoneal washings. This group of non-keratinizing squamous carcinoma cells shows enlarged nuclei and increased nuclear cytoplasmic ratio. Unless the histologic type is known, it is possible to overlook non-keratinizing squamous carcinoma as reactive mesothelial cells . 27 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

29. Peritoneal washings (continued) Three-dimensional cell groups are present associated with a sheet of mesothelial cells. Some of the unusual cells are ciliated . 28 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

30. Peritoneal washings (continued) Atypical cell aggregate associated with microcalcification. The cells have a hob-nail shape with enlarged nuclei and prominent large nucleoli. Despite the sparse number of cells in the sample, these cells were interpreted as neoplastic. 29 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

31. Peritoneal washings (continued) Higher power view of isolated atypical cells with rounded shape, enlarged irregular nuclei and prominent nucleoli. A similar cell group is present in the cell block. 30 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

32. Peritoneal washings (continued) Histologic section of non-invasive implant of low grade serous tumor . These cells compare well with the cytology. (Peritoneal biopsy at re-exploration) 31 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases Cell block section of washing

33. Diagnostic pitfalls in cytopathology of serous cavity fluids False-positivity in up to 0.5 % and false negativity in up to 30% of cases, have been reported in effusion fluid cytology. Most of these discrepant diagnoses, especially false positive results , should be prevented. The interpreter should be conversant various factors responsible for potential diagnostic pitfalls in this area of cytopathology. 32 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

34. 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 FACTORS LEADING TO DIAGNOSTIC POTENTIAL PITFALLS Diagnostic pitfalls in cytopathology of serous cavity fluids (continued) 33 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

35. Diagnostic pitfalls in cytopathology of serous cavity fluids (continued) Classical example in this category would be predominance of polyhedral cells in metastatic sarcoma , in contrast to spindle cells in other cytology specimens such as fine-needle aspiration biopsy (FNAB) smears. Surface tension related alterations in cytomorphology 34 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

36. Diagnostic pitfalls in cytopathology of serous cavity fluids (continued) If staining, cell-block preparation, immunostaining, and other specimen processing steps are not organized properly to address various objectives associated with interpretation of effusion cytology, it may lead to suboptimal results . Improper specimen processing Different steps associated with possible failure include: Improper collection or storage to Failure of making preparations for proper staining such as Diff-Quik staining and Lack of cell-block preparation Improper orientation of immunostained cell-block sections for evaluation of ‘ Subtractive Coordinate Immunoreactivity Pattern ’ (SCIP) may further compromise the final interpretation of effusion fluid immunocytochemistry. 35 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

37. Diagnostic pitfalls in cytopathology of serous cavity fluids (continued) Clinical conditions associated with the pitfall of malignant misinterpretation Many faces of reactive mesothelial cells 36 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases Effusion fluid Clinical association Peritoneal fluid Cirrhosis Renal failure with uremia Pancreatitis Bile peritonitis Huge intra-abdominal benign masses e.g. ovarian fibroma (with florid reactive mesothelial hyperplasia of the overlying serosal surface) Pleural fluid Pulmonary infarction Infection/inflammation Pancreatitis Pericardial fluid Pericarditis- viral Congestive heart failure All fluids Collagen vascular diseases Chemotherapy Radiation therapy

38. Diagnostic pitfalls in cytopathology of serous cavity fluids (continued) Some proliferation spheres may join together , especially during specimen processing, to form groups that may resemble papillary configuration Proliferation related features Metastatic ovarian endometrioid carcinoma (ascitic fluid). Cohesive clusters of neoplastic cells appear to be conglomerations of ‘proliferation spheres’ (arrows) leading to papillary-like configurations . The patient had endometrioid carcinoma of ovary without papillary component. 37 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases a b c d e

39. Diagnostic pitfalls in cytopathology of serous cavity fluids (continued) Degenerative and other changes secondary to improper storage and handling of specimens introduce various atypical morphological features including nuclear hyperchromasia and cytoplasmic vacuolations . They are not uncommon in free floating cells in a fluid medium. Degenerative hyperchromasia seen in Pap stained preparations and other changes in Pap and Diff-Quik stained preparations may lead to the pitfall of misinterpreting such cells in effusions as malignant cells. Degenerative changes 38 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

40. Diagnostic pitfalls in cytopathology of serous cavity fluids (continued) Degenerative changes 39 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases May lead to false positive interpretation of these floridly reactive atypical mesothelial cells as cancer cells. RM Adenocarcinoma, colon Compare

41. Diagnostic pitfalls in cytopathology of serous cavity fluids (continued) Degenerative changes Reactive mesothelial cells, with degenerative intracytoplasmic vacuoles may be misinterpreted as adenocarcinoma cells with mucin vacuoles Secretion in the lumen ( targetoid vacuole) Well-defined borders Ancillary tests , including histochemistry, such as PAS stain with diastase digestion and mucicarmine stain may help 40 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

42. Diagnostic pitfalls in cytopathology of serous cavity fluids (continued) Secretory cytoplasmic vacuoles. Metastatic papillary carcinoma of thyroid (pleural fluid). Targetoid secretory vacuole with colloid in neoplastic cell 41 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

43. Metastatic mucinous adenocarcinoma (pleural fluid). Intracytoplasmic mucicarmine positive mucin [Cell block section, Mucicarmine stain] Diagnostic pitfalls in cytopathology of serous cavity fluids (continued) 42 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

44. Presence of some unexpected patterns and unusual structures i) Reactive lymphoid population ii) Polymorphic lymphocytes iii) Single population 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 43 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

45. Presence of some unexpected patterns and unusual structures (continued) Reactive lymphoid population May be misinterpreted as- Lymphoma, Neuroblastoma, Ewing’s / Primitive neuroectodermal tumors (PNETs), Wilms tumor, or Desmoplastic small round cell tumor (DSRCT) leading to false positive interpretation, especially in children and in patients with a history of any of the aforementioned neoplasms. 44 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

46. Polymorphic lymphocytes Presence of some unexpected patterns and unusual structures (continued) 45 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases Polymorphic lymphocytes of low grade lymphoproliferative neoplasms such as some follicular lymphomas may be misinterpreted as reactive chronic inflammatory cells especially in Papanicolaou stained preparations . Immunophenotyping with flow cytometry and immunostaining of cell-block sections or cytospin preparations are very useful ancillary to cytomorphology for objective interpretation. Additional tests include cytogenetics , in-situ hybridization , and other tests as indicated. ► ► ►

47. Presence of some unexpected patterns and unusual structures (continued) Polymorphic lymphocytes Chronic inflammatory cells with a few reactive mesothelial cells (pleural fluid). a. With Diff-Quik stain, the typical nuclear morphology helps to interpret them as polymorphic lymphocytes which is consistent with chronic inflammatory cells. b. With PAP stain, these chronic inflammatory cells may resemble cells of lymphoma (compare with 4.12 d) and round blue cell tumors, especially in children. 46 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases a b RM RM

48. Presence of some unexpected patterns and unusual structures (continued) Follicular lymphoma (peritoneal fluid). The lymphoid population with rare reactive mesothelial cells (arrowheads RM in a,d) resemble chronic inflammatory cells and cells of round blue cell tumors, especially in PAP stained preparations (d). Polymorphic lymphocytes 47 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases PAP 100X a b RM c d e f g c b e f RM DQ 100X HE 100X

49. Presence of some unexpected patterns and unusual structures (continued) Single population of cells due to predominance of tumor cells A predominant population of scattered isolated cells of low grade carcinoma from some primary sites, such as ovary and breast , may resemble mesothelial cells with marked reactive changes. As DQ stain highlights second population more distinctly , the failure of including DQ stained preparation in the evaluation protocol may compromise proper interpretation in such clinical situation and lead to the pitfall of misinterpreting such specimens as negative for neoplastic cells because of failure to detect the second population in PAP stained preparation alone. Further immunocytochemical evaluation with properly tailored immunopanel , after initial suggestion of two cell population in DQ stained preparation, is helpful to demonstrate their non-mesothelial nature and confirm the second population . 48 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

50. Presence of some unexpected patterns and unusual structures (continued) Large cell carcinoma of lung (pleural fluid). Numerous isolated carcinoma cells (arrow in c) seen as the predominant population. Occasional mitotic figures (arrowhead in c) are present. Single population of cells due to predominance of tumor cells 49 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases a b c

51. Presence of some unexpected patterns and unusual structures (continued) Psammoma bodies Psammoma body : Round accellular bodies with concentric lamination ( peritoneal fluid ). 50 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

52. Presence of some unexpected patterns and unusual structures (continued) Psammoma bodies Encountered in 3.7% of effusions Pleural and pericardial effusions - Usually associated with various neoplasms : Such as metastatic papillary carcinoma of thyroid, bronchioloalveolar carcinoma of lung, and serous papillary cystadenocarcinoma of ovary. However, in up to 30% peritoneal effusions, washings , and cul-de-sac aspirates May be associated with benign processes: Such as papillary mesothelial hyperplasia, endometriosis, endosalpingiosis, and ovarian cystadenoma / cystadenofibroma 51 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

53. Presence of some unexpected patterns and unusual structures (continued) Psammoma bodies A significant pitfall and may lead to a false positive interpretation as malignant . An experience graciously shared by Dr. Naylor emphasizes this point: A benign case with psammoma bodies in culdocentesis fluid presented at diagnostic seminar in 1968 was misinterpreted by all four cytopathologists, including him, as adenocarcinoma . 52 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

54. Presence of some unexpected patterns and unusual structures (continued) Three dimensional benign cell groups Especially in peritoneal washings, may be misinterpreted as malignant. They may represent Mullerian inclusions Tubular or papillary structures. Often form a single layer of cells with some atypia May be associated with psammoma bodies An important pitfall in peritoneal specimens especially washings . important to avoid a misdiagnosis of disseminated cancer Other benign papillary inclusions Other gland-like epithelial structures 53 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

55. Megakaryocyte (1) with adjacent mesothelial cell (2) in hemorrhagic pleural fluid . 2 1 Presence of some unexpected patterns and unusual structures (continued) Megakaryocytes 54 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

56. Presence of some unexpected patterns and unusual structures (continued) Megakaryocytes Large cells with big irregular lobulated hyperchromatic nuclei Not uncommon in pleural effusions- with fresh blood as a result of bleeding from the pulmonary microvasculature May be associated with effusions in- Myeloproliferative disorders or Extensive replacement of bone marrow by metastatic carcinoma . In Pap stained preparations- Potential pitfall of misinterpreting as neoplastic cells 55 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

58. Diagnostic pitfalls in cytopathology of serous cavity fluids (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 57 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

59. Study cases Unequivocal case with obviously malignant cells False positive in patient with cirrhosis- immuno unequivocal Low grade metastatic carcinoma Solitary neoplastic cells as predominan t population Chronic inflammatory cells- DD Polymorphic low grade lymphoma Polymorphic low grade lymphoma- DD chronic inflammatory cells Mesothelioma 58 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

60. Unequivocal case with obviously malignant cells Metastatic poorly differentiated adenocarcinoma, pleural fluid. Specimen predominantly show noncohesive soliatry cells (a-d). The neoplastic cells (NC inset d) are pleomorphic with unequivocal features of malignancy. Mitotic figures (MF in c) are present in concert with apoptotic cells which show apoptotic bodies (AP in d). Solitary cells (NC in d) may resemble high grade lymphoma cells. DQ stained preparation and immunocytochemistry on cell-block sections may help in cases with unknown primary. This patient had poorly differentiated adenocarcinoma of lung. (AP, apoptotic cells; MF, mitotic figure; NC, neoplastic cells) 59 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases a b d c AP MF NC

61. False positive in patient with cirrhosis- immuno unequivocal 60 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

62. Low grade metastatic carcinoma Metastatic mammary carcinoma, pleural fluid. 61 Calretinin RM NC BerEP4 RM NC NC BerEP4 NC NC NC RM g i h f e c a j b d MF RM NC RM RM RM RM RM NC RM

63. Scattered solitary neoplastic cells as predominant population with rare mesothelial cells. DD - Poorly differentiated carcinoma, melanoma, and Large cell lymphoma Pleural fluid Solitary neoplastic cells as predominan t population 62 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

64. ‘ Subtractive coordinate immunoreactivity pattern’ (SCIP) in cell block sections Metastatic lung carcinoma, (pleural fluid). The specimen show scattered solitary neoplastic cells (red arrows NC) with rare mesothelial cells (blue arrow RM). The neoplastic cells are immunoreactive for pan-cytokeratin (B) and non-immunoreactive for vimentin (A), LCA (C), and calretinin (D). Rare reactive mesothelial cells (blue arrow RM) [with immunoreactivity for calretinin (nuclear-cytoplasmic) (D) and inflammatory cells (arrowhead) [with immunoreactivity for LCA (C)] in the background can be subtracted from neoplastic cells to deduce a diagnostic coordinate immunoreactivity pattern. This is consistent with poorly cohesive singly scattered carcinoma cells, which were also non-immunoreactive for the melanoma marker. Nuclear immunoreactivity for TTF-1 (E) with a clinical history of lung mass is consistent with a lung primary. 63 HE stained cell block section 40X 100X D. Calretinin Non-immunoreactive (Mesothelial cell- immunoreactive nuclear-cytoplasmic) E. TTF-1 Immunoreactive nuclear 40X 100X B. Pan-cytokeratin Immunoreactive C. LCA (CD45) Non-immunoreactive 40X 40X 100X 100X 40X 100X A. Vimentin Non-immunoreactive NC RM NC RM NC

65. Solitary neoplastic cells as predominan t population 64 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases Scattered solitary neoplastic cells as predominant population in case with poorly differentiated non-small cell carcinoma of lung with rare mesothelial cells. a 10X b 40X c NC

66. Large B-cell lymphoma , peritoneal fluid. PAP stained Cytospin preparations (a through c) showed non-cohesive solitary cells (red arrow NC in c) with many apoptotic bodies (arrowheads in c). These cells in PAP stained preparations resemble non-cohesive poorly differentiated non-small cell carcinoma with a solitary cell pattern. They are present in the HE stained cell-block section as small groups (d). By the SCIP approach neoplastic cells could be separated out from cytokeratin 7 (A) and calretinin (B) immunoreactive mesothelial cells (blue arrow RM). After following them in serial sections, the neoplastic cells (red arrow NC) were immunoreactive for CD20 (C) and non-immunoreactive for CD3 (E). They expressed Bcl2 (D). Flow cytometry demonstrated the monoclonal nature of the CD20 immunoreactive cells. The patient had lymphoma of the small bowel. This was a consult case and a DQ stained preparation, which usually is helpful to evaluate lymphoma cells, was not available. (NC, neoplastic cell; RM, reactive mesothelial cell) ‘ Subtractive coordinate immunoreactivity Pattern’ (SCIP) in cell block sections 65 HE stained cell block Section (d) A. Cytokeratin 7 Non-immunoreactive [Mesothelial cell Immunoreactive (red arrow) Cytoplasmic] B. Calretinin Non-immunoreactive [Mesothelial cell Immunoreactive (red arrow) nuclear-cytoplasmic] D. Bcl2 Immunoreactive Cytoplasmic (red arrow) E. CD3 Non-immunoreactive C. CD 20 Immunoreactive Cytoplasmic (red arrow) PAP stained Cytospin preparation (a-c) 40X 40X 40X 40X 40X a b c d 10X 40X NC NC RM NC RM NC NC 40X

67. Chronic inflammatory cells- DD Polymorphic low grade lymphoma Chronic inflammatory cells, pleural fluid. a. With Diff-Quik stain, the typical nuclear morphology helps to interpret them as polymorphic lymphocytes which is consistent with chronic inflammatory cells. b. With PAP stain, these chronic inflammatory cells may resemble cells of lymphoma and round blue cell tumors, especially in children. (RM, reactive mesothelial cell) 66 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases a b RM RM

68. Polymorphic low grade lymphoma- DD chronic inflammatory cells Follicular lymphoma, peritoneal fluid The lymphoid population with rare reactive mesothelial cells (arrowheads RM in a,d) resemble chronic inflammatory cells (see also Figure 4.9) and cells of round blue cell tumors, especially in PAP stained preparations (d). The typical nuclear morphology in Diff-Quik stained preparation (a,b,c) helps to interpret the round cells as atypical lymphocytes (arrows b,c,e,f in a & d). The flow-cytometry demonstrated monoclonal lymphoid population. Patient had follicular lymphoma with colon mass (g). (RM, reactive mesothelial cells) PAP 100X a b RM c d e f g c b e f RM DQ 100X HE 100X 67 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

69. Mesothelioma- Immuno & radiology with history Malignant epithelioid mesothelioma pleural fluid. Mesothelioma cells show numerous large three dimensional groups of cells. The individual mesothelioma cells hardly show any variation from reactive mesothelial cells without remarkable features of malignancy. The mesothelioma cells like reactive mesothelial cells show two zone staining (red arrow 1) with peripheral vacuolation (blue arrow 2). 68 Washings, lavages, brushings, scrapings, and touch imprints Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases 1 2 a b c e f g h d

70. Thank you Milwaukee Art Museum [email_address] End Diagnostic Cytopathology of Serous Effusions IV

71. 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