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

Abstract

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 [Becton Dickinson Immunocytometry Systems (BDIS), Westwood, MA] 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 [αt = red/(red + green) fluorescence]. Alpha t variables important for correlations with fertility and toxicant‐induced chromatin damage include mean (Xαt), standard deviation (SDαt), and cells outside the main population (COMPαt). Mean green fluorescence intensity is an important measure for DNA content and/or degree of sperm chromatin condensation. This study showed that the SCSA can be successfully run on two epiillumination‐type instruments, an Ortho ICP22A (BDIS, San Jose, CA) and Skatron ArgusTM (Tranby, Norway), and two additional orthogonal axes instruments, a Becton Dickinson FACScanTM (BDIS) and a Coulter EliteTM (Coulter Corporation, Hialeah, FL). Epiillumination instruments produced a different fluorescence distribution than orthogonal instruments, but the resulting αt values showed strong conformity and interpretation of results was the same. SCSA values obtained on the Coulter EliteTM were most similar to the Cytofluorograf 30; the FACScanTM green fluorescence distribution was narrower and allowed resolution of cell doublets. Neither orthogonal instrument has the ability to directly calculate αt values Listmode data from these instruments were transferred to an off‐line personal computer (PC) for calculation of αt values using LIST‐VIEWTM software (Phoenix Flow Systems, Inc., San Diego, CA). © 1995 Wiley‐Liss, Inc. Copyright © 1995 Wiley‐Liss, Inc.