Value of Ki67 immunostain in identification of malignancy in serous effusions

The distinction between malignant and benign serous effusions continues to be a challenging and a frequent problem to cytopathologists. Recently, immunostains employing various antibodies have improved the diagnostic accuracy of malignant effusions. We investigated the usefulness of Ki67 (MIB1) antigen immunostaining in the evaluation and diagnosis of malignant serous effusions. Cell block sections from a total of 54 cases of serous effusions cytologically diagnosed as malignant (28), suspicious (6), and benign (20) were immunostained with MIB1 monoclonal antibody to the Ki67 nuclear proliferation antigen according to the avidin‐biotin immunoperoxidase method. The patients were 30 women and 24 men with an average age of 58 yr. Ki67 (MIB1) immunostain labeling index (LI) values were higher than 20% in 23 of 28 (82%) cytologically malignant, in 3 of 6 (50%) suspicious, and in 1 of 20 (5%) benign/reactive. Further investigation revealed histologic, radiologic, and/or clinical evidence of malignancy in the 3 suspicious (but not in the benign/reactive) cases with Ki67 LI values higher than 20%. Correlation between Ki67 LI (>20%) and cytologic effusion type (benign, suspicious, or malignant) was statistically significant (P < 0.0001). Ki67 immunostaining has value as an adjunct testing to cytomorphology and other immunostains in distinguishing benign from malignant effusions. The addition of Ki67 immunostaining to conventional cytology appears more sensitive than cytomorphology alone and may assist in arriving at accurate diagnoses in suspicious cases with inconclusive cytomorphologic features. Diagn. Cytopathol. 1999;20:24–28. © 1999 Wiley‐Liss, Inc.     

Immunohistochemical localization of survivin in serous tumors of the ovary.

The aim of this study was to determine the immunohistochemical distribution of survivin in benign, borderline, and malignant serous tumors of the ovary. Survivin was localized by an indirect immunoperoxidase method in 42 cases of serous tumors of the ovary (15 cystadenomas, 15 borderline tumors, and 12 cystadenocarcinomas). Nuclear staining and cytoplasmic staining were separately scored. Cytoplasmic staining was detected in 27% of adenomas/borderline tumors and in 58% of carcinomas. Nuclear staining was detected in 87% of adenomas/borderline tumors but in only 42% of carcinomas. Although the differences in the intensity of cytoplasmic staining between adenomas and borderline tumors versus carcinomas were not significant, the differences in the intensity of nuclear staining between low-grade versus malignant tumors were significant. These findings suggest that survivin is widely expressed in benign, borderline, and malignant serous tumors but that nuclear localization of survivin is more common in benign or borderline tumors than in malignant serous tumors of the ovary. The molecular mechanisms that determine the subcellular distribution of this protein may reflect the role of survivin in the regulation of apoptosis during the processes of malignant transformation.     

Flow cytometric immunophenotyping of serous effusions and peritoneal washings: comparison with immunocytochemistry and morphological findings

AIM: To evaluate immunophenotyping by means of flow cytometry as a complementary method for the detection of malignant cells in serous effusions and peritoneal washings. MATERIAL AND METHODS: Frozen samples of 49 fresh serous effusions and peritoneal washings were analysed by flow cytometry, using monoclonal antibodies against CD45, Ber-EP4, and N-cadherin. Results were compared with smear and cell block morphology, as well as immunocytochemistry on paraffin wax embedded cell blocks. RESULTS: Seventeen specimens were cytologically diagnosed as malignant, whereas 25 were interpreted as benign. The remaining seven specimens were diagnosed as indeterminate or suspicious for malignancy. Ber-EP4 positive cells were detected in 16 of the 17 cytologically malignant effusions, as well as in five of seven suspicious cases and five of 25 specimens with benign cytology. In the latter group, three specimens showed atypical or malignant cell groups that were missed in routine morphological evaluation. In two additional samples, obtained from patients with benign and borderline ovarian tumours, Ber-EP4 positive cells showed benign or mildly atypical features, and were interpreted as exfoliated benign or borderline malignant epithelial cells of tubal origin, or as endosalpingiosis. All five Ber-EP4 positive indeterminate specimens showed atypical or malignant cells on re-evaluation, and were Ber-EP4 positive in four of five cases using immunohistochemistry in cell block sections. Large numbers of CD45 positive and relatively few N-cadherin positive cells were detected in most specimens with the use of flow cytometry, when compared with morphological evaluation. CONCLUSIONS: Flow cytometry is a rapid and highly effective method for the evaluation of effusions and peritoneal washings. The detection of Ber-EP4 positive cells using flow cytometry is strongly indicative of the presence of carcinoma cells in effusions and peritoneal washings. Although false positives are relatively infrequent, all specimens should be carefully evaluated morphologically to prevent the diagnosis of benign epithelial clusters as malignant.     

Role of DNA flow cytometry and image cytometry on effusion fluid

The objective of the study was to assess the value of DNA flow cytometry (FCM) and image cytometry (ICM) as an adjunct to routine diagnostic cytology. In this prospective study, 100 consecutive effusion fluids were studied for routine cytology, DNA FCM, and in selected cases, ICM. One half of the centrifuged fluid sample was used for routine cytology and the remaining portion was used for DNA FCM. Nuclear area, nuclear diameter, nuclear perimeter, nuclear convex perimeter, nuclear roundess, and nuclear convex area were measured on at least 100 cells by ICM in cytologically malignant or DNA aneuploid cases along with control cases. Clinical follow-up was done in all cases. There were 22 cytologically malignant cases and 78 cytologically benign cases. Among the 22 cytologically malignant cases, there were 11 aneuploid and diploid cases each by DNA FCM. Out of 78 cytologically benign cases, six (7.7%) were aneuploid by DNA FCM. Smears of these cases showed predominantly reactive mesothelial cells, but the DNA histograms showed hypodiploid (one), hyperdiploid (three), tetraploid (one), and hypertetraploid (one) aneuploidy. Follow-up of these cases showed clinical or histologic features of malignancy except in one case of tetraploid aneuploidy, which did not show any features of malignancy and responded well to antitubercular therapy. Therefore, out of 27 malignant effusions, DNA FCM picked up 16 cases and routine cytology detected 22 cases. Sensitivity and specificity of DNA FCM were thus 59.25% and 98.63%, respectively. There was a statistically significant difference (Student's unpaired t-test, P < 0.05) between cytologically malignant cases and control benign cases in all the nuclear morphometric parameters except for nuclear roundness. There was, however, no statistically significant difference of nuclear morphometric parameters between cytologically benign vs. DNA aneuploid cases and control benign cases. DNA FCM is a useful adjunct for routine diagnostic cytology. Visual diagnostic cytology and morphometric digital microscopy miss some cases of malignancy which can be detected by DNA flow cytometry. Diagn. Cytopathol. 2000;22:81-85.     

Role of computerized morphometric analysis in diagnosis of effusion specimens

The present study was undertaken to compare morphometric analysis of cells in 100 effusion samples, with unaided cytological diagnosis. All the cases in the form of Leishman's stained smears were subjected to computerized-interactive morphometry (CIM) by usage of Image analysis software. On routine cytological examination, 44 cases were benign, 26 were malignant, and 30 cases were placed in "atypical" category. On morphometry, values for mean nuclear area (MNA) and mean cytoplasmic area (MCA) in benign cases were found to be 58.39 +/- 12.22 microm(2) and 185.70 +/- 43.69 microm(2), while for malignant cases were 120.12 +/- 16.28 microm(2) and 274.65 +/- 61.01 microm(2), respectively (P < 0.001, P < 0.001, highly significant). Significant differences were also noted among benign, atypical, and malignant cases for MNA (P < 0.001). The differences between atypical and malignant cases for N/C (nuclear/cytoplasmic) ratio were also found to be statistically significant (P < 0.001). Finally, 46 cases were found to be benign, 39 malignant, and 15 in the category of atypical cases. CIM helps in better and quicker sorting for malignant cells. MNA and N/C ratio are the most significant parameters in differentiating various cells in effusions. MCA constitutes as another useful parameter. Salient features of CIM, including their applications in the light of current study, are discussed.     

Tumour associated antigens in diagnosis of serous effusions.

The use of tumour associated antigens in the diagnosis of serous effusions was studied in 76 patients with benign and 200 patients with malignant disease. Tissue polypeptide antigen (TPA), alpha fetoprotein, and CA 125 were found to be of little value. At cut off points of 3 ng/ml, 10 U/ml, and 30 U/ml, respectively, carcinoembryonic antigen (CEA), biliary glycoprotein I (BGP I), and CA 19-9 discriminated between benign and malignant serous effusions with a sensitivity of between 24% and 67%. The immunocytochemical staining for these markers resulted in malignant cells being detected in 18% to 33% of cases. Various combinations of conventional cytological examination, effusion fluid tumour marker determination, and immunocytochemical analysis identified malignant cells in serous effusions in up to 72% of cases; conventional cytology alone detected tumour cells in only 30%.     

Telomere repeat amplification protocol (TRAP) in situ reveals telomerase activity in three cell types in effusions: malignant cells, proliferative mesothelial cells, and lymphocytes.

Telomerase Repeat Amplification Protocol (TRAP) in situ was performed on cytospin preparations from 65 effusions from the serous cavities (45 pleural and 19 ascitic fluids and one pericardial fluid) submitted for routine diagnosis and the results were correlated to cytological morphology. Three types of cells with nuclear fluorescence were identified: malignant cells, hyperplastic mesothelial cell and lymphocytes. Of 38 cytologically malignant effusions, 12 showed strong reactivity in all malignant cells, three strong reactivity in part of the malignant population, whereas 12 showed moderate reactivity in the whole and five in part of the malignant population, respectively. In five malignant effusions weak reactivity was found in all (one case) and in scattered (four cases) malignant cells. Two effusions contained telomerase-negative malignant cells. Two pleural and two ascitic fluids contained proliferative mesothelial cells with weak or, in one case, moderate reactivity. Lymphocytes usually showed weak telomerase activity. Telomerase was expressed in almost all malignant tumours metastatic to serous cavities. Heterogeneity in tumour populations was demonstrated, which may have diagnostic implications, especially in cytology. Weak or moderate reactivity was found in lymphocytes and in some mesothelial proliferations and may explain the low specificity for malignancy sometimes obtained with the TRAP extract method. The weak reactivity found in lymphocytes may reduce the specificity when the extract method is used but causes no diagnostic problem with the TRAP in situ method.     

Use of antisera to epithelial membrane antigen for the cytodiagnosis of malignancy in serous effusions.

A new human antigen, designated epithelial membrane antigen (EMA), has recently been described on surface membranes of a wide variety of normal epithelium but not on connective tissue cells. The antigen is only weakly expressed on normal or reactive mesothelium. Increased expression of the antigen has been observed in most neoplasms of epithelial origin and in malignant mesothelioma. We have investigated the possibility of using this difference in the expression of the antigen to distinguish between mesothelial cells and malignant cells in cytological smears of serous effusions. This distinction cannot always be made on morphological grounds alone and problems of differential diagnosis are encountered in about 15% of all specimens of serous effusions sent for cytological examination. Using antisera to EMA we have applied an indirect immunoalkaline phosphatase technique to alcohol-fixed smears prepared from serous effusions and have found that intense staining of the antigen is confined to effusions from patients in whom there is either clinical or cytological evidence of malignancy. The technique proved to be especially useful in cytologically equivocal cases, where there were problems of differential diagnosis.     

Alpha 1-protease inhibitor in serous effusions: differences in lectin crossed immuno-affinoelectrophoresis precipitin profiles between benign and malignant disease

Differences in the lectin crossed immuno-affinoelectrophoresis (LCIE) precipitin profile of the antiprotease alpha 1-protease inhibitor (alpha 1-PI) have been detected between serous effusions of benign and malignant origin. Extra proteins with antigenicity towards anti-human alpha 1-PI appear in both malignant pleural effusions and malignant ascitic fluid caused by ovarian cancer that are not detected in effusions of benign origin. These compounds may be protease-antiprotease complexes caused by tumour excretion of proteases into the serous effusions.     

Cytopathology of malignant mesothelioma: a stepwise logistic regression analysis.

Twenty-four cytologic features, previously reported to be useful in the distinction of malignant mesothelioma, adenocarcinoma, and benign mesothelial proliferation in serous effusions were assessed. Forty-four cases of malignant mesotheliomas, 46 cases of metastatic adenocarcinomas, and 30 cases of benign mesothelial proliferations were examined for these parameters. When these cytologic features were subjected to a stepwise logistic regression analysis, five features were selected to distinguish malignant mesothelioma from adenocarcinoma. These were true papillary aggregates, multinucleation with atypia, cell-to-cell apposition, acinus-like structures, and balloon-like vacuolation, the latter two features being characteristic of adenocarcinoma. The four variables selected to distinguish malignant mesothelioma from benign mesothelial proliferations were nuclear pleomorphism, macronucleoli, cell-in-cell engulfment, and monolayer cell groups, the latter being a feature of benign proliferations. Using these selected variables, the logistic model correctly predicted 95.4% of cases of malignant mesothelioma versus 100% of adenocarcinoma and 100% of malignant mesotheliomas versus 90% of benign mesothelial proliferations. The results of regression analysis suggest that many of the previously described cytologic features are not important diagnostic discriminators.     

Megakaryocytes in pleural and peritoneal fluids: prevalence, significance, morphology, and cytohistological correlation.

Over a period of 22 years, 4844 pleural and peritoneal fluids from 3279 patients were examined cytologically. Megakaryocytes were found in the fluids from five patients. The clinical diagnoses in the five patients were agnogenic myeloid metaplasia, chronic myeloid leukaemia, and lymphocytic lymphoma. All of these patients had persistent serous effusions. Megakaryocytes in serous fluids occurred in three forms: (1) a large type with abundant cytoplasm and multilobed nuclei, (2) a smaller type with a high nucleocytoplasmic ratio and unlobed nuclei, and (3) anucleate cytoplasmic masses. Foci of agnogenic myeloid metaplasia found on the serous surfaces at necropsy of two patients contained megakaryocytes similar to those in the corresponding effusions. The clinical course of our patients confirmed that the presence of megakaryocytes in serous fluids signifies an advanced haematopoietic malignancy.     

Role of DNA flow cytometry and immunocytochemical analysis in diagnosis of malignant effusions

Body cavity fluid examination sometimes presents a diagnostic challenge in cytology practice. This study was undertaken to evaluate efficacy of cytomorphology, epithelial membrane antigen immunocytochemistry (EMA-ICC) and DNA flow cytometry (FCM) in detection of malignant cells in effusions. One hundred effusions (55 pleural, 44 ascitic, and 1 pericardial fluid) were studied by cytology, EMA, and FCM. There were 29 malignant and 71 benign cases. On cytology, 28 of 29 malignant cases were diagnosed. With no false positives, the sensitivity and specificity was 96.55% and 100% respectively. FCM detected aneuploidy in 85.71% of cytologically malignant and 4.17% of cytologically benign effusions. EMA was positive in 75% of cytologically malignant and 4.17% of cytologically benign cases. EMA had lower sensitivity than cytology; 75.86% versus 96.55%. Sensitivity and specificity of FCM was 86.21%, and 97.18% respectively. FCM had lower sensitivity than cytology; 86.21% versus 96.55%. Sensitivity increased to 100% (P < 0.05) when the combinations of cytology plus EMA or cytology plus ploidy were applied compared to cytology alone (96.55%). Also, the combination of cytology plus EMA had higher sensitivity than EMA alone (100% versus 75.86%, P < 0.05) and combined cytology plus ploidy had higher sensitivity than ploidy alone (100% versus 86.21%, P < 0.05). The study demonstrates the usefulness of EMA-ICC and DNA FCM as adjuncts to cytology to diagnose malignancy in effusions. FCM in combination with ICC can be further developed to reduce number of false-negative cases on cytology and add objectivity to cytologically doubtful or equivocal cases.     

Telomerase activity analyzed with TRAP in situ provides additional information in effusions remaining equivocal after immunocytochemistry and hyaluronan analysis

Cytology is central in the diagnosis of malignancy in effusions. Ancillary techniques, mainly immunocytochemistry, have considerably improved the sensitivity but some 10% of all cases remain equivocal and require the addition of new diagnostic modalities. We have previously shown that strong nuclear telomerase activity determined with Telomere Repeat Amplification Protocol (TRAP) in situ is specific for malignant cells and could be a candidate for an additional test. Thirty effusions remaining diagnostically equivocal after the use of immunocytochemistry and the determination of the hyaluronan content were reviewed and their TRAP in situ reactivity was related to the definitive diagnoses based on all available data. There were seven effusions from patients with definitive benign diagnoses and 23 effusions from patients with definitive malignant diagnoses. Strong telomerase activity was seen only in effusions from patients with definitive malignant diagnosis, all effusions from patients with benign disease lacking strong telomerase activity, whereas eight of the malignant cases, including three cases of epithelial mesothelioma, showed strong reactivity. Strong nuclear TRAP in situ reactivity was demonstrated only in effusions from patients with verified malignant disease. Although the study is small, it suggests that TRAP in situ activity provides diagnostic information in about one‐third of effusions remaining cytologically equivocal after the use of current ancillary techniques. The most striking diagnostic improvement appears to be gained in epithelial mesotheliomas. Diagn. Cytopathol. 2014;42:1051–1057. © 2014 Wiley Periodicals, Inc.     

Detection of monocyte/macrophage cell populations in effusions: a comparative study using flow cytometric immunophenotyping and immunocytochemistry

The objective of the present study was to compare the efficiency of immunophenotyping using flow cytometry (FCM) and immunocytochemistry (ICC) in the detection of macrophages in serous effusions. Cytoblock sections from 90 effusions were stained for the monocyte/macrophage marker CD14, using ICC. Fresh-frozen samples of all cases were analyzed for CD14 expression, using FCM. Epithelial, lymphoid, and mesothelial cell populations were identified using antibodies against Ber-EP4, CD45, and N-cadherin, respectively. Results were compared with clinical parameters and morphological diagnosis. Thirty-nine specimens were cytologically diagnosed as malignant, containing tumor cells of nonhematologic origin, whereas 46 were interpreted as benign. Two additional specimens were diagnosed as indeterminate or suspicious for malignancy, and 3 specimens contained lymphoma cells. CD14-positive cells were detected in 85/90 (94%) of effusions using FCM, and in all 90 specimens using ICC. The percentage of CD14-positive cells was highly variable, but in some specimens was as high as 76% using FCM and 85% using ICC. A good association was observed between the two methods in the detection of CD14-positive cells (P < 0.001). The presence of macrophages in effusions showed an association with female gender, using both FCM (P = 0.002) and ICC (P = 0.011), but none with effusion site, patient age, clinical and cytological diagnosis, or presence of Ber-EP4-positive cells (P > 0.05). The presence of Ber-EP4-positive cells showed a strong association with the cytological diagnosis of malignancy (P < 0.001). In conclusion, macrophages are a significant cell population in effusions, of both benign and malignant etiology, due to both their size and their possible confusion with cancer cells. Both FCM and ICC aid in the recognition of these cells, and thus provide an effective tool for the identification of different cell populations in effusions.     

Nuclear and cell area measurements in the cytological evaluation of pleural effusions: a study of subjective assessments and morphometric measurements

Morphometric measurements of cellular area, nuclear area and nuclear:cytoplasmatic ratio were performed on single cells in pleural effusions from 15 patients with effusion caused by bronchial, ovarian, or mammary carcinoma. The results were compared with corresponding measurements on mesothelial cells in pleural effusions from 15 patients without malignant disease. Significant differences were found between the mean values from cellular area, nuclear area, and nuclear:cytoplasmatic ratios in mesothelial cells from benign effusion versus malignant cells from effusions associated with metastatic growth. Such morphometric measurements are, however, of little value in routine diagnostic work as more than 90% of the cells in the two groups showed similar values.     

The cytopathology of serous effusions: the cavalry has arrived—with reinforcements

This paper addresses well-known problem areas in the cytodiagnosis of serous effusions, relating to the distinction between adenocarcinoma, malignant mesothelioma and highly reactive mesothelial cells. The cytomorphology of these three entities has been scrutinized extensively by light microscopy; the majority of serous effusions are readily placed in one of the three categories based on astute assessment of cytological features and architecture of cell groupings. Nevertheless, a small, apparently irreducible number of cases remains in which overlapping cytological details make definitive diagnosis impossible. This occurs both histologically and cytologically. Adjunctive techniques such as histochemistry and electron microscopy were used in the past; however, it was not until the introduction of immunochemical analysis that diagnosis of exuberant serous lesions became more objective. The early generation of markers was directed primarily at the identification of adenocarcinoma, negative staining of both malignant mesothelioma and reactive mesothelial cells being accepted by exclusion. It is only in the last 4–5 years that positive markers of mesothelium have become commercially available and widely used. Chief amongst these agents are Calretinin and the Cadherins, which have brought a clarity, not previously anticipated, to bear on diagnosis in serous lesions, both histologically and cytologically. Future techniques such as measurement of ploidy and proliferation markers are addressed. It can now be stated that, with judicious use, definitive diagnosis of serous effusions can be accomplished cytopathologically in the majority of cases.     

Short and long term behaviour of pleural effusion cultures. (Report of 200 cases) (author's transl)]

Pleural effusions from 200 patients with benign or malignant pleural involvement have been cultured in vitro and observed from 48 hours to 9 months. The morphology and "social behavior" of the different types of cells are described. Mesothelial cells settle down quickly and come into contact with each other by long cytoplasmic processes. The tendency of epithelial malignant cells is to clump and grow in clusters. These "balls" of varying size are freed into the culture medium. Similar clusters may be found in benign effusions showing mesothelial hyperplasia. In case of malignant primary tumor of the pleura, the morphology of cultured cells is similar to that of mesothelial cells. In 3 cases, typical malignant epithelial cells have allowed us to discard the diagnosis of malignant mesothelioma. Discrepancies between conventional cytology and cell culture were found in 13 cases: in 6 cases, positive for malignant cells using conventional cytology, cell culture was negative. In 7 cases with negative routine techniques, cell culture disclosed malignant cells. Large multinucleated syncytia were observed in 15 cultures. They suggest a cytopathic effect similar to that seen in myxovirus infected cells. Correlation with the etiology of pleural effusions in these cases is described.     

Differential expression of CD44s and CD44v3-10 in adenocarcinoma cells and reactive mesothelial cells in effusions

The detection of malignant cells in serous effusions obtained from patients diagnosed with cancer marks the presence of metastatic disease and is associated with a poor outcome. The purpose of this study was to evaluate the role of CD44s and CD44v isoforms in the distinction between mesothelial cells and malignant epithelial cells in effusions. Fifty-nine fresh pleural and peritoneal effusions were studied. These consisted of 41 specimens from patients with known gynecological neoplasms, 9 from patients diagnosed with breast adenocarcinoma, and 9 effusions from patients with various nongynecological malignancies or tumors of unknown origin. Forty-three effusions contained malignant/atypical epithelial cells, and 16 effusions were diagnosed as reactive. Three effusions contained exclusively malignant cells. Specimens were stained with anti-CD44s, v3, v5, v6, v7 and v3-10. The presence of staining in cancer cells, benign mesothelial cells and lymphocytes was evaluated. CD44s immunoreactivity was seen in 10 of 43 (23%) cases in malignant/atypical epithelial cells and in 53 of 56 (94%) cases in benign cells. In contrast, CD44v3-10 was seen in 23 of 43 (55%) cases in malignant/atypical epithelial cells and in 3 of 56 (6%) cases in benign cells. We advocate the use of CD44s and CD44v3-10 immunostaining in diagnostic evaluation of difficult serous effusions. Received: 30 August 1999 / Accepted: 8 November 1999     

Immunocytochemical panel for the identification of malignant cells in serous effusions.

The cytologic diagnosis of malignancy in serous effusions can be challenging. An immunocytochemical (ICC) panel using commercially available antibodies (to carcinoembryonic antigen [CEA], epithelial membrane antigen [EMA], B72.3, Leu-M1, cytokeratin [CK], leukocyte common antigen [LCA], S-100 protein, and vimentin) was applied to cell blocks fixed in methyl Carnoy's solution that were from 55 consecutive pleural, peritoneal, and pericardial fluid specimens. The results were correlated with data from clinical records and routine cytologic studies. Final cytologic diagnoses included 26 of adenocarcinoma and 1 of mesothelioma. The remaining 28 cases were considered to be benign (reactive) proliferations. EMA, CEA, B72.3, and Leu-M1 were present in 96%, 77%, 58%, and 42% of adenocarcinomas, respectively. These determinants were absent in the mesothelioma and the reactive effusions, although anti-CEA yielded strong background staining of inflammatory cells. The CK markers identified malignant cells in 93% of cases, but consistently stained mesothelial cells as well. Antivimentin strongly labeled mesothelial cells in all cases, with weak to absent staining of malignant cells. In 3 of 26 carcinoma cases (12%), the ICC panel identified malignant cells that were not recognized initially on routine cytologic examination. In 1 of 26 cases (4%), the panel was falsely negative. Use of this approach can improve the diagnostic accuracy of cytologic examination of serous fluids. The ICC panel is especially helpful when atypical mesothelial proliferation is present, or in cases that are clinically suspect for malignancy, but cytologically negative because there are only a few malignant cells, or those that are cytologically bland.