SLIT-SCAN FLOW-CYTOMETRY FOR CONSISTENT HIGH-RESOLUTION DNA ANALYSIS OF X-CHROMOSOME AND Y-CHROMOSOME BEARING SPERM

This paper describes the application of slit-scan how cytometry for ac curate DNA analysis of X- and Y-chromosome bearing sperm. The introduc tion of the slit-scanning technique was initiated to improve the consi stency in resolution of the X and Y population from donor to donor, An optimal resolution is essential for high purity sorting of X and Y sp erm, as the difference in DNA content is small (3-4%) in most mammals. This difference is the discriminatory parameter for the flow cytometr ic sorting of the two populations. Our approach was to focus on the ro le of the sperm tail in the detection process. Slit-scan now cytometri c analysis allows the whole sperm to be spatially analyzed along the d irection of flow. Sperm were stained with Dansyl Lysine, a UV excitabl e fluorescent membrane dye, which stained the head, midpiece, and prin cipal piece, Analysis of these stained sperm showed that there was no difference between the relative number of sperm that travel headfirst or tailfirst through the detection zone of the now cytometer. The infl uence off sperm with coiled tails on DNA analysis was also investigate d. The proportion of sperm with coiled tails influences semen quality. The standard X-Y separation procedure uses Hoechst 33342, which stain s all intact sperm, both living and dead. Propidium iodide was added t o discriminate the dead sperm population, Silt-scan analysis showed th at measurement of a sample containing a high proportion of living sper m with coiled tails results in an inferior DNA histogram and reduced X -Y resolution. Sperm with coiled tails can result in a lower detected fluorescence intensity, but the reason for this is unclear. Slit-scan now cytometry allows exclusion of sperm with coiled tails from the ana lysis, resulting in a restoration of high resolution of X- and Y-chrom osome bearing sperm populations. (C) 1996 Wiley-Liss, Inc.