An evaluation of Feulgen-acriflavine-SO2 and Hoechst 33258 for DNA cytofluorometry in tumour pathology.

The performance of Feulgen-acriflavine-SO2 and Hoechst 33258 for cytofluorometric ploidy determination is evaluated and compared. The fast initial fading of acriflavine-SO2 stained nuclei, with substantial reduction in fluorescence intensity within about 10 ms, is shown to be related to changes in DNA-ratios between cell types when measurements are performed using different excitation light intensities. Although low intensity excitation-, without such fading-, was used, the acriflavine-SO2 staining procedure showed large specimen to specimen variability regarding both mean fluorescence intensities and coefficients of variation (CV). The cells in such specimens could be shown to contain the same amount of chromophore using scanning absorbance measurements and the differing fluorescence values therefore probably represent variability in fluorescence yield. Specimens stained with Hoechst 33258 after RNase treatment showed very small deviations in mean fluorescence value between slides, and also much lower CV's than in acriflavine-SO2 stained specimens. The results indicate that with proper internal standards this procedure would allow the detection of deviations in DNA content in the order of 2%.