A semi-automated quantitative fluorescence image analysis (QFIA) technique was developed with the Leitz TAS-Plus to detect bladder cancer using hyperploidy in urinary cells. Absolute nuclear fluorescence intensity (ANFI) (emission at 540 nm with excitation at 436 nm) of individual acridine-orangestained cells was quantitated using ( I ) QFlA and (2) simple filter microspectrofluorophotametry (SFM). Bath methods employed an internal phosphor particle standard which, when once calibrated against the DNA content of normal cells, obviates the necessity of routinely calibrating against normal cells in each sample. Results of SFM and QFlA were compared with routine Papanicolaou (Pap) cytopathology, using histopathology as the diagnostic standard in 272 samples from 67 symptomatic patients. The sensitivities for detecting lowgrade transitional-cell carcinoma were 86% for SFM, 76% far QFlA, and 33% for Pap cytology. QFlA and SFM were significantly more sensitive at detecting bladder cancer than was Pap (0.01 > p >O.OOl). Comparison of sensitivity obtained
with bladder washings and urine samples showed that noninvasively obtained urines can be used. ANFl also detected recurrent and precancerous bladder lesions and kidney, ureter, and prostate lesions. This approach may prove generally useful in quantifying biochemical and immunological probes and should be broadly applicable as a research tool for studying the relationship of biochemical markers in the pathogenesis of disease and as a test for cancer control.