A rapid system for static cytofluorometry enabling the simultaneous determination of nuclear size and DNA content.

A measuring technique enabling the simultaneous determination of nuclear size and DNA content by single cell cytofluorometry is described. The system is based on a Leitz MPV cytophotometer, a Zeiss scanning stage and a microcomputer system. Cells to be measured are not positioned in the ordinary way, but are passed through the excitation light beam. In this way rapid measurements are achieved. Further, an estimate of the object diameter is calculated from the pulse shape obtained as the fluorescence is recorded continuously by using a circular measuring aperture, congruent with the excitation light field. Repeated measurements of nuclei stained with Hoechst 33258 showed a high reproducibility for DNA content with a coefficient of variation (CV) below 1% and an acceptable precision for nuclear area with a mean CV of 7%. Nuclear area estimated by the method was well correlated to area determined from photographs (r = 0.986). Disintegrated samples from eight patients with breast cancer were analyzed and compared to Feulgen-stained samples analyzed by scanning cytophotometry. Scatter diagrams illustrating DNA content and nuclear size showed essentially the same distribution with the two methods. We conclude that the system may be a rapid alternative when fluorescence together with object size are quantified in slide preparations.