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 sperm and numerous vaginal cells are recovered from a swab, resulting in an extremely unfavorable ratio of male to female DNA. The goal of this study was to develop a protocol involving sperm cell sorting with now cytometry based on differences in ploidy, major histocompatibility class I, CD45, and cytokeratin expression. Method: Vaginal lavages were mixed with serially diluted ejaculate. After immunostaining and stoichiometric nuclear staining, spermatocytes were isolated 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 method within all tested conditions. In unfavorable dilutions, the male DNA could 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 microtraces of sperm detectable after rape. (Obstet Gynecol 1999;94:623-7, (C) 1999 by The American College of Obstetricians and Gynecologists.).