We previously found telomere repeat amplification protocol (TRAP) in situ helpful in the diagnosis of malignancy in effusions, whereas varying sensitivities and specificities for malignancy were reported by investigators using extract-based TRAP.
To compare the 2 methods and to elucidate the discrepancies between them.
Twenty-three effusions were analyzed. Telomerase activity of whole cell lysate was measured with a Telo TAGGG telomerase polymerase chain reaction ELISA PLUS kit with modifications to exclude polymerase chain reaction inhibitors. TRAP in situ was performed on cytospins. An estimate of total TRAP activity in the specimen was made based on the amount of positive cells, their fluorescence intensity, and the proportion of different cell types in the specimen. The estimate was compared with the level of telomerase activity in cell lysate-based TRAP.
TRAP in situ: Thirteen of 14 malignant cases and 2 of 2 equivocal cases showed moderate/strong reactivity. Five of 7 benign effusions were negative; in 2 of 7, mesothelial cells showed weak reactivity. Cell lysate-based TRAP assay: In 4 cases no internal standard was detected, indicating the presence of polymerase chain reaction inhibitors. The relative telomerase activities were 33.1 to 72.7 with a considerable overlap between malignant (48 +/- 9, mean +/- SD) and benign (43 +/- 9) cases.
The TRAP in situ results correlated to final diagnoses, whereas the cell lysate-based TRAP assay did not differentiate between malignant and benign cases. The varying proportions of positive cells and the variation in fluorescence intensity in the TRAP in situ slides explained some of the discrepancies. The problems encountered with TRAP performed on cell lysates are partly overcome using TRAP in situ.