Development of a procedure for the simultaneous detection of mRNA markers in menstrual blood, vaginal fluid, and peripheral blood for the purpose of discriminating between menstruation and traumatic vaginal injury in sexual assault cases.

Blood from menstruation or traumatic hemorrhage is often detected in vaginal fluid in sexual assault cases. Discriminating peripheral blood from menstrual blood in a biological sample collected from a crime scene can help to determine whether sexual assault with traumatic injury has occurred. In a previous study, novel mRNA markers for menstrual blood that are specifically expressed in the endometrium were investigated and evaluated for their detectability and specificity by quantitative RT-PCR. In the present study, we developed the MB-VF-PB multiplex RT-PCR procedure for the simultaneous detection of previously evaluated menstrual blood markers, vaginal fluid, and blood markers, with the aim of discriminating between menstruation and traumatic vaginal injury in sexual assault cases. Applying the cutoff value for the positive determination of each marker as well as criteria for the precise identification of the targeted body fluid, the MB-VF-PB multiplex system successfully discriminated menstrual blood from other forensically relevant body fluids, including vaginal fluid, peripheral blood, and mixed samples. Among the menstrual blood markers incorporated into the MB-VF-PB multiplex system, PAEP was a highly specific and robust marker and is expected to be applicable to forensic casework samples, including diluted, degraded, and contaminated menstrual blood samples. Although traumatic vaginal injury during menstruation cannot be identified, the proposed procedure could be useful for discriminating between menstruation and traumatic bleeding in the female genital tract.