During a forensic case of investigating a suspicious pool of blood found in a school restroom, the question arose if by means of forensic molecular biology a distinction of peripheral blood, menstrual blood, or blood from childbirth or abortion from dried bloodstains was possible. In our forensic laboratory, we routinely apply a PCR-CE-based analysis of specific mRNA amplicons for the differentiation of forensically relevant body fluids, including the distinction between peripheral and menstrual blood. The mRNA transcripts used to detect menstrual blood (MMP7, MMP10, and MMP11) belong to the group of matrix metalloproteinases (MMPs), which are involved in numerous important remodeling processes of the extracellular matrix, in growth, wound healing processes, and during embryonic development. However, it is still unclear, whether these menstrual blood markers are suitable for distinguishing menstrual blood from uterine blood caused by other circumstances (e.g. abortion or childbirth).

For this reason, we aimed to develop an mRNA-based analysis using placenta specific markers. So far, a forensic pregnancy diagnostic assay based on mRNA markers has received little attention in the scientific literature. Existing studies focus on the analysis of placental expressed mRNA transcripts in maternal blood or plasma using real-time PCR. Gauvin et al. 2010 [1] specifically demonstrated the forensic utility of placental mRNA transcripts by analyzing aged and dried maternal blood to detect pregnancy.

Here, we present a multiplex panel of mRNA markers employing the PCR-CE workflow, which can be applied specifically for the analysis of unknown blood traces to determine its origin. For peripheral blood, the established and validated markers for HBB and ALAS2 are used; for menstrual blood, MMP7, MMP10, and MMP11 as described above. As placental/pregnancy specific targets, markers for previously published mRNA transcripts of the human placental lactogen (hPL) and the beta subunit of human chorionic gonadotropin (βhCG) are implemented.

To assess the assay’s sensitivity, specificity and for optimization purposes, defined quantities of various blood samples, collected from informed healthy pregnant and non-pregnant adults, were prepared and stored at room temperature for multiple drying periods to simulate realistic time delays before analysis in forensic practice.

Authors

• Jan Euteneuer (Institute of Forensic Medicine, University Hospital of Schleswig-Holstein, Germany)
• Emily Fichtner (Institute of Forensic Medicine, University Hospital of Schleswig-Holstein, Germany)
• Johanna Preuß-Wössner (Institute of Forensic Medicine, University Hospital of Schleswig-Holstein, Germany)
• Maria Seidel (Institute of Forensic Medicine, University Hospital of Schleswig-Holstein, Germany)

References

[1] Gauvin J, Zubakov D, van Rhee-Binkhorst J, Kloosterman A, Steegers E, Kayser M. Forensic pregnancy diagnostics with placental mRNA markers. Int J Legal Med. 2010 Jan;124(1):13-7. doi: 10.1007/s00414-008-0315-6. Epub 2009 Jan 16. PMID: 19148664; PMCID: PMC2795858.