Abstract
Quantitative, corrected fluorescence emission spectra from the nuclei of acridine orange‐stained bladder epithelial cells were obtained with a corrected spectrum microspectrofluorophotometer (MSF)5 which is calibrated with a phosphor particle fluorescence standard. The spectra demonstrated that the green fluorescence band (λmax≅530 nm) is stoichiometrically related to the amount of nucleic acids (NA) present in a given nucleus, provided this spectroscopic species has reached saturation. The shape of this spectral band is constant. On the basis of this information, we used a simple microfluorometer, (SFM), calibrated with the phosphor particle standard, to routinely measure the amount of green fluorescence emission from the nuclei of AO‐stained bladder epithelial cells. The amount of green fluorescence per normal nucleus never exceeded 0.5 phosphor particle units (ppu). In contrast, in every sample containing malignant or cancerrelated cells, one or more cells contained nuclei that exhibited more than 0.5 ppu total green nuclear fluorescence. Results obtained with the SFM on 794 AO‐stained cells from 80 samples taken from 40 patients are reported here. Samples consisted of urine specimens, bladder washings, and biopsies. One or more malignant cells from every grade 3 or 4 tumor and 17 of 17 grade 1 or 2 tumors showed markedly elevated levels of total (integrated) green nuclear fluorescence indicating the presence of DNA aneuploidy, strongly, suggestive of the presence of malignant or potentially malignant cells. Comparison with diagnoses based on H and E‐stained biopsy material and Papanicolaou‐stained cytological preparations show excellent correlation. The quantitative fluorescence measurements demonstrate a clear difference between negatives and positives with linear ordering up through Grade 4 and CIS. Grades 1 and 2 were clearly different from 3 and 4 (p<0.001). The results indicate that the quantitative fluorescence methodology reveals the presence and extent of DNA aneuploidy which may prove to be a more sensitive cytologic method for detection of low grade tumors and may also provide an objective biochemical basis for tumor grade classification. Detecting the presence of DNA aneuploidy may even serve to provide earlier detection than is possible with conventional means, but this remains to be confirmed by longitudinal studies.