CELL TYPES IN EFFUSIONS Usha Kini, MD, DCP, DNB Professor of Pathology St Johns Medical College, Bangalore 560034

1. Mesothelial Cells Normal : Free-floating mesothelial cells in fluids appear singly and in clusters. Singly mesothelial cells in body fluids are round or oval and measure between 10 20 u in diameter. The cyanophilic or eosinophilic cytoplasm in opaque and homogenous and sharply demarcated. Most often the cell membrane shows a brush border appearance and where this not clearly discernable it is represented at light microscopy, by a clear zone surrounding the cell membrane and better appreciated on electron microscopic preparations. The nuclei are large and occupy about half the cell diameter and are usually centrally located. The nuclear membrane is prominent. The chromatin net work is fine with one or two small nucleoli. Mesothelial cells in fluids more commonly occur in clusters. The clusters may more than often be made up of a small number of cells, 2-4 in number and rarely be large and composed of several cells. These clusters are usually flat and consist of a single layer of uniform cells adherent to each other. The uniformity of the nuclei speaks strongly in favour of their mesothelial origin. In smaller clusters the mesothelial cells often display a moulding of the cell surfaces. Sometimes adjacent moulded mesothelial cells appear to be separated form each other by a narrow, regular, slit-like clear space (already referred to) called the window. The nature of this space is not definitely known but may represent surface structures (microvilli or blebs) observed on mesothelial cells by scanning electron microscopy. Multinucleation is a common feature and when it occurs there are usually two nuclei which are very similar to each other. One cell grasping an adjacent cell (cell cannibalism) is an often seen features of mesothelial cells. Large clusters of mesothelial cells have a :knobby contour due to individual cells protruding at the periphery. It is the presence of these peripheral knobs which is one of the hall marks of mesothelial cells, differentiating them from malignant cell clusters which in contrast have a smooth periphery.

Inflammations: In non-specific inflammatory conditions considerable reactive changes may be seen in these cells. A size variation may occur from cells which measure 9 u to giant forms >60 u. Multinucleation increases in frequency and the number of nuclei in the multinucleated cells also increases. Mitotic figures are more frequently seen, in fluids the presence of normal mitotic figuresis just as likely to be associated with benign a with malignant cells. Individual nuclei may show an increase in size and may have a more coarsely granular but still uniform chromatin pattern. Nucleoli may also enlarge and become more prominent and occasionally multiple nucleoli are seen. In benign cells, however, the nuclei retain a smooth outline, a uniform chromatin pattern with rounded nucleoli, and an adequate amount of cytoplasm. Degeneration: Degenerative changes in mesothelial cells can cause alterations in booth cytoplasm and nuclei. The cytoplasm may be completely lost and the denuded nuclei may appear suspicious. Cytoplasmic vacuoles may also be observed either as small ones or large vacuoles pushing the nucleus to one side as in a signet ring malignant cell. However, the uniform round or oval nuclear outlines, normal chromatin pattern, appearance of single or small loosely arranged groups in contrast to the compact clusters of tumour cells, establish their benign nature. In view of the varied changes which occur in mesothelial cells in response to inflammation or as a result of degeneration much caution should be exercised in identifying cells as malignant when the main features suggest a serosal origin unless unequivocal abnormalities of chromatin pattern and nuclear shape exist. The use of the term atypical mesothelial cells is often over-used by reporting cytologists. This practice should be done away as the term may mislead the clinicians in believing a particular state to be a precursor of mesothelial neoplasia. Such cells seen may in truth be just hyperplastic mesothelial cells with a dark contour to the nucleus due to poor smear preparation. 2. Macrophages (histocytes): in effusions macrophages appear as mononucleated cells similar in size to mesothelial cells (10-20 u in diameter). They usually occur singly or in loosely arranged clusters and never show cytoplasmic moulding. These cells are characterized by a foamy cytoplasm studded with minute vacuoles and a cell border that readily blends with the smear background in contrast to the sharply demarcated mesothelial cell outline or brush border. The cytoplasm of macrophages may sometimes become markedly distended with large vacuoles.

The nuclei of macrophages are usually peripheral in location and stain more densely than the nuclei of mesothelial cells. Kidney-shaped nuclei may be present in both cell types and the nuclear properties on staining are of limited value in separation of macrophages from mesothelial cells. Binucleated and multinucleated macrophages may also be observed. The latter cells are large and in no way different from foreign body giant cells. Phagocyte activity and lysosomal activity are characteristic functions of macrophages and this may be used to advantage for the identification of these cells with phagocytosed particles. Stain for enzymes such as acid phosphatase as also supravital staining with neutral red or Janus green are other methods for identifying macrophages. Formerly the presence of macrophages in the effusion was considered to reflect a chronic inflammatory process. Current evidence suggests that macrophages are ubiquitous cells which appear in effusions not only as a consequence of inflammation but also in the presence of cancer and presumably under other circumstances as well. The various differences between the mesothelial cells, macrophages and carcinoma cells are as follows: Mesothelial cells cells PAS stain PAS +ve granules at the periphery, due to neutral mucopolysaccharides ? Minimally + ve ? Minimally + ve - ve - ve usually ve Macrophages Usually ve unless containing RBCs - ve - ve +/- ve +/- ve + ve - ve - ve - ve Carcinoma +/- ve Diffuse staining +/- ve +/- ve

Alcian Blue Mucicarmine stain Iron stain Sudan Black-B

Histiocytic usually ve Markers (acid phosphatase etc) Supravital -ve Staining: neutral red or Janus green

+ve

-ve

3. Leukocytes: these commonly occur in effusions and lymphocytes may predominate in long standing effusions. It may be the chief cell type in tuberculosis, lymphocytic leukemia or lymphoma. Polymorphonuclear neutrophilic leukocytes invariably indicate an process. Such a process may also be associated with neoplasm. leukocytes may be seen in eosinophilic pleural effusion and in inflammatory processes. They may however be rarely seen Hodgkins disease. 4. Other rare cells in effusions : a) Liver cells accidental injury to liver during aspiration of fluid. b) Respiratory cells Traumatic injury to lung tissue. c) Squamous cells, fat cells & striated muscle accidental inclusions in course of tapping 5. Neoplastic cells in Effusions: It is well recognized that the diagnosis of neoplastic cells in the effusion does not depend on any single morphologic criterion or constellation of criteria. A very useful method to identify cancer cells is by attempting to recognize cells that are alien to the type of specimen. General characteristics of neoplastic cells in effusions 1. Cell size: In metastatic tumours this varies according to the tumour type. The comparison is usually made with the size of identifiable cell types in smears i.e. polymophs, lymphocytes or RBCs. Metastatic malignant tumours in effusions may be classed according to size into 3 groups large, small and medium. Large cells: Cells are significantly larger than mesothelial cells. Metastatic epidermoid and adenocarcinomas, malignant melanomas and sarcomas belong to this group. The identification of such tumours is easy. Small cells: Tumours are made up of cells much smaller than mesothelial cells. Malignant lymphomas, small cell neoplasms like neuroblastomas, Wilms tumours, or even oat cell carcinomas belong to this category. Close attention should be paid to nuclear features for identification of theses. inflammatory Eosinophilic a variety of in cases of

Medium cells: The cells are approximately of the same size as mesothelial cells. A variety of carcinomas of mammary, gastric, pancreatic or of lung origin belong to this group. This is perhaps the most important area of diagnostic error due to similarities to reactive mesothelial cells. 2. Nuclear Abnormalities: Size and shape Most malignant cells in fluids have enlarged nuclei, however, the nuclear cytoplasmic ratio varies according to tumour type. In mucusproducing carcinomas and keratinizing squamous carcinomas the cytoplasm may remain abundant and the nuclear-cytoplasmic ratio may not be conspicuously changed. In most other tumours the nuclear-cytoplasmic ratio is larger than in mesothelial cells. With regard to shape, nuclei of malignant cells are invariably round or oval. On careful scrutiny an irregular nuclear outline may be observed and coupled with nuclear hyperchromasia may help in diagnosis. Nuclear hyperchromasia and texture: In most, although not all malignant tumours the cancer cells in effusions have enlarged, markedly hyperchromatic nuclei. The chromatin appears granular and its texture is often much denser than in mesothelial cells and macrophages. In some instance, however, the nuclei are homogenous and opaque. Nucleoli: Large, irregular shaped, single or multiple nucleoli are frequently observed except in some cases. Rarely mesothelial cells also show nucleolar changes. Abnormal Mitoses: Multipolar or other abnormal mitoses are reliable identifying features of cancer cell in effusions. Multiple sex-chromatin bodies: In cancers of female patients, 3 or more sex chromatic bodies are virtually diagnostic of cancer cells. The observation may be helpful in the diagnosis of mammary and ovarian carcinomas where the morphologic abnormalities of malignant cells may not be pronounced. Mesothelial cells and histiocytes have a single sex chromatic body (except perhaps in super females, Karyotype 47, XXX). Nuclear cytoplasmic inclusions: (Nuclear clear zones; nuclear holes) Cytoplasmic invagination in nuclei are observed in a variety of cancer cells, such as cells of metastatic melanomas, pulmonary adenocarcinomas, breast, liver etc. 3. Cell shape: Bizarre or spindle-shaped cells almost always suggest a metastatic malignant tumour. Sarcomas usually show spindle-shaped cells. Other cell configurations are columnar cells resembling bronchial lining cells in bronchogenic carcinoma.

4. Cell Aggregates: Large aggregates of cells invariably suggest malignancy. Reactive mesothelial cells are usually seen in small clusters unless in a case of malignant mesothelioma. Aggregates composed of papillary projections or glandular structures are helpful identifying features. 5. Cell products: Mucus, melanin pigment, psammoma bodies, cytoplasmic cross-striations and keratin in effusions invariably signify malignancy. Special stains may demonstrate these better on smears or on cell block preparations. The various cell products seen are Mucus Melanin Keratin Adenocarcinoma Malignant melanoma Squamous cell carcinomas Mesotheliomas (epithelial) Bronchogenic carcinomas Thyroid carcinomas Pancreatic carcinomas Carcinoma of the renal pelvis Endometrial carcinoma Breast carcinomas Carcinomas of Fallopian tube

Psammoma bodies -

Immunohistochemistry could detect minute quantities of the above cell products when they are not observed on routine staining. The presence of psammoma bodies should alert the pathologist to search for epithelial ,malignant cells. 6. Applied Aspects: a) Cytogenetic studies: 1. Abnormal chromosomal numbers and configuration in cancer cells. 2. Abnormal DNA profiles also suggests carcinoma. b) Immunologic features: 1. Association of T lymphocytes with malignancy. 2. Association of macrophages and cancer cells. c) Electron microscopy to study cell type.

GENERAL GUIDELINES TO THE INTERPRETATION OF EFFUSIONS 1. Accurate clinical details must be obtained before reporting. 2. All effusions associated with cancer need not always contain malignant cells as effusions may also result due to indirect mechanisms like: a) venous obstruction caused by a neoplasm may be associated with a transudate-type effusion. b) Inflammatory changes in an organ secondary to the presence of a neoplasm may be associated with an exudates type effusion. c) The number of neoplastic cells in an effusion depends upon the extent of direct involvement of the mesothelium. Few or no cells would be seen in effusions when the neoplastic process is limited to the submesothelial tissues. 3. A protein content of <3G% is rarely seen in cancerous cases. 4. The duration of an effusion should be known as effusions of long standing may be characterized by an accumulation of poorly preserved cells of mesothelial type and macrophages which may be enlarged, darkly stained and numerous. Also unpreserved fluid collected and kept over a period of time shows similar changes. 5. A cytologic diagnosis of cancer should be avoided if: a) The morphology of cells is not clear. b) No obvious structural nuclear abnormalities are present. c) There is evidence of an inflammatory process with numerous polymorphonuclear leukocytes, macrophages and cell necrosis with no obvious evidence of malignancy. 6. A second tap yields better morphology than the first one and should be resorted to in doubtful cases. 7. Most errors are made on technically inadequate material, such as thick overstained smears, poorly prepared and stained filter preparations and inadequate specimens. 8. Mesothelial cells and macrophages may occasionally assume very abnormal appearances that may make their differentiation from cells of metastatic tumours exceedingly difficult. 9. The diagnosis of cancer in pleural, pericardial or peritoneal fluid is of great importance to the patient in predicting the course of disease. The responsibility of the pathologist in such cases is two fold:- a) to identify cancer cells (b) to identify tumour type and if possible the site of primary origin in cases of metastatic neoplasms. As a general rule it is better exercise diagnostic caution than to stamp a patient as having primary or metastatic carcinoma on insufficient evidence.

REFERENCES FOR FURTHER READING 1. Jareer Kassis,. 1, Julius Klominek,. 2, Elise C. Kohn,. 1 * Tumor microenvironment: What can effusions teach us? Diagn. Cytopathol. 2005;33:316-319 2. Natalia Pomjanski, . *, Hans Juergen Grote,., Peyrze Doganay, Viola Schmiemann, Birgit Buckstegge, Alfred Bcking,, Immunocytochemical identification of carcinomas of unknown primary in serous effusions Diagn. Cytopathol. 2005;33:309-315 3. Cytologic features of atypical mesothelial cells in peritoneal dialysis fluid. Selvaggi SM, Migdal S Diagn Cytopathol. 1990;6(1):22-6. Differential Diagnosis of Benign and Malignant Mesothelial Proliferations on Pleural Biopsies Philip T. Cagle, MD; Andrew Churg, MD, PhD Archives of Pathology and Laboratory Medicine:2005;. 129, 14211427. Argani P, Rosai J. Hyperplastic mesothelial cells in lymph nodes. Human Pathology 1998; 29 :339 -346. Significance of AgNOR Count in Differentiating Malignant Cells from Reactive Mesothelial Cells in Serous Effusions Sujathan,., S. Kannan,., K. Raveendran Pillai,., B. Chandralekha, M.D., N. Sreedevi Amma, Krishnan Nair, Acta Cytol 1996;40:724-728 7. Geisinger RK, Stanley WM, Raab SS, Silverman FJ, Abati A. Modern Cytopathology. London: Churchill Livingstone; 2004. 8 Spriggs Al, Vanhegan Rl. Cytological diagnosis of lymphoma in serous effusions. I CUn Pathol. 1981:34:1311-1325.

9. Nodit, Laurentia, McGrath, Kevin, Peel, Robert An 86-Year-Old Woman With Gastric Outlet Obstruction Archives of Pathology & Laboratory Medicine , Oct 2005