Identification of menstrual blood markers by proteomic mass spectrometry

The ability to distinguish menstrual blood from venous blood can prove important in forensic investigations, provide evidence in criminal and sexual assault cases and give context to what may have occurred during a crime. Identification of menstrual blood, however, is difficult, as it is not a single body fluid like blood, saliva and semen, but is composed of endometrial secretions and tissue fragments shed during menses as well as vaginal secretions and venous blood. This mixed nature of menstrual blood raises barriers for identifying menstrual blood markers - i.e. proteins expressed in the endometrium. First it is necessary to demonstrate that potential menstrual blood markers are not expressed in venous blood or vaginal secretion, as such a mixture alone could be falsely identified as menstrual blood. Second, because ratio of endometrial tissue fragments and secretions to venous blood and vaginal fluid are unlikely to be constant, the level of candidate markers during different times of menses are likely to vary. Third, the specific proteins shed during menses may change over time. Fourth, the length of women's cycles vary significantly, likely influencing marker levels and the ability to detect them. Finally, the presence of venous blood with multiple highly abundant proteins (e.g. hemoglobin and albumin) that inherently obscure the presence of lower abundant proteins, compounds the difficulty of detecting the lower abundant endometrial proteins, potentially reducing sensitivity. To overcome these barriers, this study employed combinatorial peptide ligand chromatography to reduce the dynamic range of menstrual blood proteins and evaluated samples obtained from 44 volunteers on each day of menses. Identified proteins were compared with venous blood samples processed in the same manner. More than 20 potential marker proteins were identified that may be useful in a confirmatory test for menstrual blood. Results were analyzed with respect to the day of menses as well as demographic attributes of the volunteers, including age, type of birth control used and ethnicity.