Surface-Specific Imaging Technique for Differentiating Menstrual and Non-Menstrual Blood Stains in Forensic Science
Introduction
Bloodstain pattern analysis (BPA) is a cornerstone of forensic science, used to reconstruct crime scenes by evaluating the size, shape, and distribution of blood stains to infer mechanisms like impact angle and force applied (Balbudhe et al., 2025). However, distinguishing between menstrual blood—originating from uterine shedding and containing endometrial tissue, mucus, and undergoing fibrinolysis—and peripheral blood from injuries, which clots via fibrinogen, has been a persistent challenge (Balbudhe et al., 2025). This distinction is critical in cases involving sexual assault, domestic violence, or trauma, where it can corroborate victim statements and clarify consent versus violence, preventing wrongful suspicions or prosecutions (Balbudhe et al., 2025).
Traditional tests, such as Teichmann or luminol, confirm the presence of blood but fail to differentiate its source (Balbudhe et al., 2025). Advanced methods like HSI, Raman spectroscopy, and the SERATEC PMB test offer molecular-specific insights but may require integration for optimal results (Kumar & Javed, 2024; Balbudhe et al., 2025). The new technique, titled "A Method for Surface-Specific Analysis of Menstrual and Non-Menstrual Blood Stains Using Imaging Techniques," addresses these gaps through a simple, non-destructive approach (Times of India, 2025). Developed by a team at KMC Mangaluru, part of Manipal Academy of Higher Education, it was published as a patent application on August 15, 2025 (Application No.: 202541066599) (KMC Mangalore, 2025).
Background on Bloodstain Analysis and Challenges
Menstrual blood is a complex mixture including red and white blood cells, plasma, vaginal secretions, cervical mucus, and endometrial tissue, leading to fibrinolysis (clot breakdown) and higher viscosity (Balbudhe et al., 2025). In contrast, peripheral blood clots rapidly due to fibrinogen. Surface types—absorbent (e.g., cotton), non-absorbent (e.g., tile), or semi-porous (e.g., glossy paper)—influence stain morphology, complicating analysis (Balbudhe et al., 2025; Egyptian Journal of Forensic Sciences, 2025). Related research includes time-dependent changes on tile and concrete, and molecular strategies like miR-451a/miR-21-5p ratios for differentiation (Egyptian Journal of Forensic Sciences, 2025; Wang et al., 2022). Additionally, studies on menstrual products highlight the need for accurate blood testing in forensics and consumer safety (The Hindu, 2023).
Detailed Methodology of the Technique
The technique employs non-destructive optical imaging to track temporal changes in blood stain morphology on semi-porous surfaces under controlled conditions (Balbudhe et al., 2025).
Sample Collection
Peripheral blood was collected via venipuncture from healthy individuals and stored in EDTA vacutainer tubes at 4°C. Menstrual blood was gathered early in the cycle from consenting volunteers, stored in uncoated EDTA vials at 4°C, and used within 24 hours to minimize variations. Volumes were standardized at 30 µL per drop, with ethical approval from the Institutional Ethics Committee (Balbudhe et al., 2025).
Surface Preparation
Glossy paper (120 gsm, polymer-coated, 30 cm x 30 cm) was selected as a semi-porous substrate, cleaned with 70% ethanol and deionized water, air-dried for 30 minutes, and inspected under UV light (365 nm). Sheets were fixed on a vibration-dampened bench (Balbudhe et al., 2025).
Analysis Procedure
Blood droplets were deposited using a micro drip infusion set (0.1 mL precision) from 55 cm height in a controlled environment (22 ± 1°C, 50 ± 5% humidity, 5000K LED lighting). Three drops each of menstrual and peripheral blood were placed on separate sheets. Changes were observed with a stereo microscope (Euromex CMEX-5PRO) and documented via Nikon DSLR 750 camera with an ABFO scale. Diameters, circularity, and edge properties were measured at 10 min, 1 h, 2 h, 24 h, 72 h, and 120 h post-deposition, in triplicate (Balbudhe et al., 2025).
Key Results and Findings
Results demonstrate distinct patterns: non-menstrual stains show consistent diameter reduction due to clotting and drying, while menstrual stains exhibit variability from viscosity, mucus, and fibrinolysis (Balbudhe et al., 2025).
| Time Point | Menstrual Blood Mean Diameter (cm, SD) | Non-Menstrual Blood Mean Diameter (cm, SD) |
|---|---|---|
| 10 min / 1 h | 8.83 (1.258) | 8.67 (0.764) |
| 2 h | 8.50 (0.866) | 8.50 (0.866) |
| 24 h | 8.33 (1.155) | 8.33 (0.577) |
| 72 h | 8.33 (1.155) | 8.17 (0.289) |
| 120 h | 8.33 (1.155) | 8.17 (0.289) |
Data from Table 5 in the study show initial similarity but increasing differences, with statistical significance in variability (Balbudhe et al., 2025). Figures 1 and 2 illustrate these changes visually.
Advantages and Limitations
Advantages: Non-destructive, preserving evidence; simple and rapid with optical measurements; high accuracy via statistical differences; versatile for surfaces like clothing or bedding; cost-effective compared to molecular tests (Balbudhe et al., 2025).
Limitations: Dependent on substrate and environmental factors (e.g., temperature, humidity); tested primarily on glossy paper; requires controlled conditions; may need integration with other methods for complex scenes (Balbudhe et al., 2025; Egyptian Journal of Forensic Sciences, 2025).
Complementary Techniques
This method integrates well with HSI, which captures spectral and spatial data for non-contact, chemical-free detection of bodily fluids, including menstrual blood in mixtures (Kumar & Javed, 2024). HSI advantages include high sensitivity and large-area analysis, though data complexity requires chemometrics (Kumar & Javed, 2024). Other complements: Raman spectroscopy for molecular specificity and SERATEC PMB for confirmation (Balbudhe et al., 2025).
"Such precision strengthens crime scene reconstruction, corroborates victim statements, and prevents misinterpretation of evidence that could otherwise lead to wrongful suspicion or prosecution." — Dr. B. Suresh Kumar Shetty
Implications for Forensic Practice and Beyond
The technique enhances investigative accuracy in criminal cases, supporting robust courtroom testimony (Balbudhe et al., 2025). It aligns with SDGs: SDG 5 (Gender Equality) for gender-specific evidence; SDG 16 (Peace, Justice) for fair justice; SDG 3 (Health) for biohazard safety (Balbudhe et al., 2025). For budding experts, it underscores the value of imaging in BPA training. Future research may extend to diverse surfaces, environments, and integration with AI for automated analysis (Balbudhe et al., 2025; Egyptian Journal of Forensic Sciences, 2025).
Conclusion
This surface-specific imaging technique represents a significant leap in forensic blood analysis, offering a practical, non-invasive tool to differentiate blood types. By addressing longstanding challenges, it promises to improve justice outcomes and inspire further innovations in the field (Balbudhe et al., 2025).
References
Balbudhe, M. S., Shetty, B. S. K., Shetty, A., & Kumar, N. A. (2025). Surface specific analysis - A comparative study of menstrual and non-menstrual bloodstains. ResearchGate. https://www.researchgate.net/publication/394650215_Surface_Specific_Analysis_-_A_Comparative_Study_of_Menstrual_and_Non-Menstrual_Bloodstains
Egyptian Journal of Forensic Sciences. (2025, July 26). Time-dependent morphological changes in bloodstains on tile and concrete surfaces. https://ejfs.springeropen.com/articles/10.1186/s41935-025-00475-9
KMC Mangalore. (2025). KMC Mangalore's post. LinkedIn. https://www.linkedin.com/posts/kmc-mangalore_kmcmangalore-facultyachievement-patentpublication-activity-7364215457047883776-H-TB
Kumar, S. R., & Javed, M. S. (2024). Innovations in forensic science: Comprehensive review of hyperspectral imaging for bodily fluid analysis. Forensic Science International, 112227. https://www.sciencedirect.com/science/article/abs/pii/S0379073824003086
The Hindu. (2023, September 3). Scientists test menstrual products with blood for the first time. https://www.thehindu.com/sci-tech/science/menstrual-products-tested-blood-misleading-advertisements/article67259290.ece
Times of India. (2025, August 23). Imaging method developed to distinguish menstrual blood stains. The Times of India. https://timesofindia.indiatimes.com/city/mangaluru/imaging-method-developed-to-distinguish-menstrual-blood-stains/articleshow/123475075.cms
Wang, L., Zhao, Y., Yang, R., & Qin, Z. (2022). A new strategy for distinguishing menstrual blood from peripheral blood by the miR-451a/miR-21-5p ratio. ResearchGate. https://www.researchgate.net/publication/357012974_A_new_strategy_for_distinguishing_menstrual_blood_from_peripheral_blood_by_the_miR-451amiR-21-5p_ratio
