Validation of a rapid method to quantify apoptosis in superficial bladder cancer.

ABSTRACT

OBJECTIVES: To derive and validate a rapid method for calculating apoptotic indices in superficial transitional cell carcinoma (TCC) as a measure of chemosensitivity to mitomycin. MATERIALS AND METHODS: Apoptotic cells, identified by light microscopy in 20 superficial TCC specimens, were expressed as an index of the total tumour cell population within defined fields. For a given field, the total cell population was estimated by (i) an exhaustive count of the total number of cells in the field and (ii) an abbreviated method in which the number of cells in a subfield was multiplied to provide an estimate of the total field number. Field and specimen estimates were compared using agreement statistics and the intra- and inter-observer reproducibility of apoptotic indices calculated. RESULTS: Cellularity and apoptotic indices obtained using method (ii) were correlated significantly with the true cell counts (P < 0.001). Agreement statistics showed that only 9.4% of counts fell outside two standard deviations (SD) from the mean in field analysis, and only 10% of counts fell outside 2 SD from the mean in specimen analysis. There was a fivefold variation in tumour cell counts among individual fields. CONCLUSIONS: The reported variation in cellularity among fields shows that the calculation of apoptosis must use the total cell population as the reference. The limits of agreement for the estimated and true cell counts are small enough to be confident that the shorter method to estimate cellularity can be used in place of counting all cells.