Separation of sperm and vaginal cells based on ploidy, MHC class I-, CD45-, and cytokeratin expression for enhancement of 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 flow cytometry based on differences in ploidy, major histocompatibility (MHC) class I, CD45 and cytokeratin expression.


METHODS
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.


RESULTS
The sorting procedure was superior to the preferential lysis method within all tested dilutions. One documented case of rape was examined with both procedures and only after cell sorting with flow cytometry was the male DNA identified.


CONCLUSIONS
We were able to show that separation of sperm and vaginal cells using cell sorting with flow cytometry may be crucial when there is only a few sperm detectable after rape.
Department of Obstetrics and Gynecology, Karl Franzens University of Graz, Graz, Austria. wolfgang.schoell@kfunigraz.ac.at
W M
WM
Schoell
M
M
Klintschar
R
R
Mirhashemi
D
D
Strunk
A
A
Giuliani
G
G
Bogensberger
B
B
Pertl
D002469:Cell Separation / Q000379:methods*; D004247:DNA / Q000032:analysis*; D016172:DNA Fingerprinting; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005805:Genes, MHC Class I / Q000276:immunology; D006801:Humans; D007633:Keratins / Q000032:analysis; D017493:Leukocyte Common Antigens / Q000276:immunology; D008297:Male; D011003:Ploidies; D016133:Polymerase Chain Reaction; D011902:Rape / Q000175:diagnosis* / Q000331:legislation & jurisprudence; D012680:Sensitivity and Specificity; D013094:Spermatozoa / Q000166:cytology*; D014621:Vagina / Q000737:chemistry / Q000166:cytology*; D014626:Vaginal Smears
D007633:Keratins; D004247:DNA; D017493:Leukocyte Common Antigens; D016650:Fluorescein-5-isothiocyanate