Separation of sperm and vaginal cells with flow cytometry for DNA typing after sexual assault

Background: Successful DNA typing after rape is limited when only a few spe rm and numerous vaginal cells are recovered from a swab, resulting in an ex tremely unfavorable ratio of male to female DNA. The goal of this study was to develop a protocol involving sperm cell sorting with now cytometry base d on differences in ploidy, major histocompatibility class I, CD45, and cyt okeratin expression. Method: Vaginal lavages were mixed with serially diluted ejaculate. After i mmunostaining and stoichiometric nuclear staining, spermatocytes were isola ted by fluorescence-activated cell sorting. All sorted cells were used for DNA extraction and subsequent quantitative fluorescent multiplex polymerase chain reaction. The preferential lysis was performed for comparison. Experience: The sorting procedure was superior to the preferential lysis me thod within all tested conditions. In unfavorable dilutions, the male DNA c ould be identified only after cell sorting with now cytometry. Conclusion: We were able to show that separation of sperm and vaginal cells using cell sorting with now cytometry may be crucial when there are only m icrotraces of sperm detectable after rape. (Obstet Gynecol 1999;94:623-7, ( C) 1999 by The American College of Obstetricians and Gynecologists.).