Comparative sperm chromatin structure assay measurements on epiillumination and orthogonal axes flow cytometers

The sperm chromatin structure assay (SCSA) measures the susceptibility of sperm nuclear DNA to acid-induced denaturation in situ, and was developed on two Ortho flow cytometers, an FC200 and a cytofluorograf 30 (BDIS), both having orthogonal axes of fluorochrome excitation, emission, and sample flow. Sperm cells are first treated with a pH 1.4 buffer to denature DNA in situ and then stained with the metachromatic dye acridine orange (AO). The metachromatic fluorescence measured reflects relative amounts of denatured (red fluorescence) and native (green fluorescence) DNA present per cell. The extent of DNA denaturation is quantified by the calculated parameter alpha t [{alpha}{sub t} = red/(red + green) fluorescence]. Alpha t variables important for correlations with fertility and toxicant-induced chromatin damage include mean (X{alpha}{sub t}), standard deviation (SD{alpha}{sub t}), and cells outside the main population (COMP{alpha}{sub t}). This study showed that the SCSA can be successfully run on two epiillumination-type instruments, an Ortho ICP22A and Skatron Argus {trademark}, and two additional orthogonal axes instruments, a Becton Dickinson FACScan {trademark} and a Coulter Elite {trademark}. Epiillumination instruments produced a different fluorescence distribution than orthogonal instruments, but the resulting {alpha}{sub t} values showed strong conformity and interpretation of results was the same. SCSAmore » values obtained on the Coultier Elite {trademark} were most similar to the Cytofluorograf 30; the FACScan {trademark} green fluorescence distribution was narrower and allowed resolution of cell doublets. Neither orthogonal instrument has the ability to directly calculate {alpha}{sub t} values. Listmode data from these instruments were transferred to an off-line personal computer (PC) for calculation of {alpha}{sub t} values using LIST-VIEW {trademark} software. 28 refs., 5 figs., 2 tabs.« less