In forensic genetics, the accurate classification of biological material can provide important insights into activity-level propositions, such as the reconstruction of potential interactions between individuals, and therefore providing valuable information to law enforcement investigations. Forensic samples are often degraded, exposed to environmental factors, or present only in trace quantities, therefore requiring highly sensitive and specific approaches to ensure accurate results.

Traditionally, the identification of body fluids is based on microscopic, enzymatic and/or immunological presumptive assays. Although well established in forensic practice, these techniques exhibit some challenges and limitations, including issues of cross-reactivity, limited specificity, and difficulties in distinguishing between similar body fluids, such as blood versus menstrual blood.

In recent years, molecular biomarker-based approaches have emerged as promising alternatives. Particularly, mRNA expression profiling, DNA methylation (DNAm) and microbiome-based methods have shown promising results due to their body site-specific molecular signatures. Each method, however, has its own specific advantages and drawbacks.

In this study, we systematically investigated over 400 forensically relevant samples using mRNA profiling, DNAm analysis, and microbiome-based methods. The analysis of the body fluids/tissues started with an RNA/DNA co-extraction [1], and included blood, saliva, semen, vaginal secretion and menstrual blood in different amounts and on various biological trace materials. The focus was on challenging samples, such as low-input samples, mock casework samples and body fluid mixtures, while additional real casework samples were analyzed to evaluate the practical applicability. The mRNA- and DNAm-based approaches were performed with multiplex-assays developed for capillary electrophoresis [2,3], and the microbiome sequencing was done through targeted amplification and Illumina sequencing of the 16S rRNA gene V4V5 region [4]. The performance of each method was assessed with respect to specificity, sensitivity, repeatability and robustness, thereby providing deeper insights into their respective strengths and limitations. In addition to examining each method alone, we explored the interpretation accuracy when combining markers from all three approaches.

Our findings from the comparative evaluation support the use of multiple markers to improve the accuracy and reliability of body fluid identification in forensic genetics, especially when dealing with challenging biological trace material.

Authors

• Jacqueline Neubauer (Zurich Institute of Forensic Medicine, Forensic Genetics, University of Zurich, Switzerland)
• Marina Huber (Zurich Institute of Forensic Medicine, Forensic Genetics, University of Zurich, Switzerland)
• Bertalan Lendvay (Zurich Institute of Forensic Medicine, Forensic Genetics, University of Zurich, Switzerland)
• Natasha Arora (Zurich Institute of Forensic Medicine, Forensic Genetics, University of Zurich, Switzerland)
• Cordula Haas (Zurich Institute of Forensic Medicine, Forensic Genetics, University of Zurich, Switzerland)

References

[1] A. Lindenbergh et al. Forensic Science International: Genetics 6 (2012) 565–577)
[2] Wang S, et al. Forensic Science International 328 (2021) 110996
[3] Lee et al. Forensic science international. Genetics 24 (2016): 75-82
[4] Swayambhu et al. Applied and Environmental Microbiology 91 (2025) e01871-24