Improved Target Cell Selection and Counting Method for UroVysion Fluorescence in Situ Hybridization.

ABSTRACT

BACKGROUND: The UroVysion Bladder Cancer Kit requires morphological analysis of 4', 6-diamino-2-phenylindole (DAPI)-stained nuclei to identify target cells for fluorescence in situ hybridization (FISH) signals. Reproducibility and efficiency of target cell selection and counting was evaluated by combining immunofluorescence staining of cytokeratin 7 (CK7) and proliferating cell nuclear antigen (PCNA) with DAPI staining. METHODS: The reactivities to CK7, PCNA, and DAPI were compared between those for different ratios of T24 human bladder carcinoma cells and of cells from the urine of five healthy subjects. Two technicians independently performed five replicate cell counts of urine samples from four bladder cancer patients and one healthy subject. RESULTS: The positive staining rates for CK7 and PCNA were similar to DAPI, but our method showed enhanced inter-observer repeatability and reduced operating time for signal counting. CONCLUSIONS: Our proposed method showed better reproducibility and lesser operational time for signal counting than the DAPI method alone.