The detection of blood from crime scenes is a critical step in many forensic investigations as it can assist in identifying biological stains and indicate criminal activities. Presumptive tests that rely on the peroxidase-like activity of hemoglobin, such as luminol and MultiStix®, are used for screening due to their high sensitivity, but they often produce false positive reactions. Confirmatory tests, such as RSID™-Blood and HemDirect, are designed to detect the human-specific blood proteins glycophorin A or hemoglobin, respectively, using a one-step immunoassay. While these conventional methods are valued for their specificity, ease of use, and ability to detect trace amounts of blood, they consume a fraction of the valuable sample. Messenger RNA (mRNA)-profiling offers potential advantages as it can detect specific transcripts for identification of several body fluids simultaneously, including blood. Additionally, the method is integrated in the DNA-profiling process because DNA and RNA can be co-extracted, maximising the yield of a sample. However, RNA is prone to environmental degradation, leading to reduced sensitivity. In this study we challenged a novel mRNA-assay for blood detection under variable conditions and compared the results with the conventional protein-based blood detection tests: MultiStix®, HemDirect and RSID™-Blood.

Blood samples were subjected to serial dilutions (1:10 to 1:1,000,000), different environmental conditions (UV-light, heat, asphalt and soil), or they were exposed to arctic winter conditions for up to three months. For mRNA testing we used an in-house mRNA assay (BodyFluID) based on 24 markers for six different body fluids, including five markers for blood. In this assay, DNA and RNA are co-extracted using PrepFiler™ Express and the ReliaPrep™ RNA Cell Miniprep System. Both mRNA- and DNA-profiles were obtained from the same samples. Following reverse transcription, the 24 mRNA targets were amplified using QIAGEN multiplex PCR reagents and detected by capillary electrophoresis.  

The mRNA assay showed promising results with sensitivity comparable to RSID™-Blood and detection levels aligning with successful DNA profiling. However, environmental conditions, such as arctic winter weather and asphalt exposure, had a negative impact on mRNA, even in samples where complete DNA-profiles were obtained. Expectedly, MultiStix® showed the highest sensitivity and may be suitable to detect latent bloodstains, whereas HemDirect proved most robust for detecting blood in compromised samples, including those heated to 200°C. However, considering the advantage of co-extraction and the ability of detecting several body fluids simultaneously, mRNA-profiling may be favorable in certain forensic scenarios.

Authors

• Nina Mjølsnes Salvo (Centre for Forensic Genetics, Department of Medical Biology, Faculty of Health Sciences, UiT The Arctic University of Norway, Norway)
• Julie Kloster Snekkevik (Centre for Forensic Genetics, Department of Medical Biology, Faculty of Health Sciences, UiT The Arctic University of Norway, Norway)
• Ane Midtun (Centre for Forensic Genetics, Department of Medical Biology, Faculty of Health Sciences, UiT The Arctic University of Norway, Norway)
• Martine Høinæs (Centre for Forensic Genetics, Department of Medical Biology, Faculty of Health Sciences, UiT The Arctic University of Norway, Norway)
• Kirstin Janssen (Centre for Forensic Genetics, Department of Medical Biology, Faculty of Health Sciences, UiT The Arctic University of Norway, Norway)
• Gunn-Hege Olsen (Centre for Forensic Genetics, Department of Medical Biology, Faculty of Health Sciences, UiT The Arctic University of Norway, Norway)
• Thomas Berg (Centre for Forensic Genetics, Department of Medical Biology, Faculty of Health Sciences, UiT The Arctic University of Norway, Norway)