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 de veloped on two Ortho flow cytometers, an FC200 [Becton Dickinson Immun ocytometry Systems (BDIS), Westwood, MA] and a Cytofluorograf 30 (BDIS ), both having orthogonal axes of fluorochrome excitation, emission, a nd sample how. Sperm cells are first treated with a pH 1.4 buffer to d enature DNA in situ and then stained with the metachromatic dye acridi ne orange (AO). The metachromatic fluorescence measured reflects relat ive amounts of denatured (red fluorescence) and native (green fluoresc ence) DNA present per cell. The extent of DNA denaturation is quantifi ed by the calculated parameter alpha t [alpha(t) = red/(red + green) f luorescence]. Alpha t variables important for correlations with fertil ity and toxicant-induced chromatin damage include mean (X alpha(t)) st andard deviation (SD alpha(t)), and cells outside the main population (COMP alpha(t)). Mean green fluorescence intensity is an important mea sure for DNA content and/or degree of sperm chromatin condensation. Th is study showed that the SCSA can be successfully run on two epiillumi nation-type instruments, an Ortho ICP22A (BDIS, San Jose, CA) and Skat ron Argus(TM) (Tranby, Norway), and two additional orthogonal axes ins truments, a Becton Dickinson FACScan(TM) (BDIS) and a Coulter Elite(TM ) (Coulter Corporation, Hialeah, FL). Epiillumination instruments prod uced a different fluorescence distribution than orthogonal instruments , but the resulting alpha(t), values showed strong conformity and inte rpretation of results was the same. SCSA values obtained on the Coulte r Elite(TM) were most similar to the Cytofluorograf 30; the FACScan(TM ) green fluorescence distribution was narrower and allowed resolution of cell doublets. Neither orthogonal instrument has the ability to dir ectly calculate cw, values. Listmode data from these instruments were transferred to an off-line personal computer (PC) for calculation of a lpha(t) values using LIST-VLEW(TM) software (Phoenix Flow Systems, Inc ., San Diego, CA). (C) 1995 Wiley-Liss, Inc.