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. V C 2014 Wiley Periodicals, Inc.Key Words: pleural effusion; peritoneal effusion; cytodiagnosis; molecular diagnostic techniques; telomerase; TRAP in situ Effusions represent a major diagnostic challenge in clinical cytology and a combination of microscopic morphological evaluation and immunocytochemical staining is often necessary for an adequate diagnosis. [1][2][3] In our laboratory, we also use hyaluronan content to distinguish mesotheliomas from metastatic tumors and from benign mesothelial cells, 3,4 but around 10% of the effusions still remain unsolved, which is in agreement with other studies. 5,6 The spectrum of underlying diseases in the diagnostically refractory effusions differs considerably from those in unselected effusions and the samples may even lack diagnostic cells. Thus, a positive general marker directing attention toward malignant cells in these effusions would be of great help.A candidate target for such a marker is telomerase. 7-20Telomerase provides a mechanism for cell immortalization and plays an important role in carcinogenesis. [21][22][23] Using an in situ version of the original Telomere Repeat Amplification Protocol (TRAP) assay, 24 TRAP in situ, 25 we have previously found that telomerase is a good marker for malignancy in effusions, strong nuclear reactivity being specific for malignant cells. 26,27 With this background, we posed the following question: Does TRAP in situ add di...