Temporal Morphological Differentiation of Menstrual and Peripheral Bloodstains on a Semi-Porous Substrate: A Controlled Stereo-Microscopic Study

Background It is not a new problem that menstrual blood and peripheral blood are difficult to distinguish in a forensic context, especially when analysing an alleged sexual assault, domestic violence, or unexplained injury. In this context, the place of bloodstains may provide significant contextual detail. Methods This controlled study examined temporal morphological variations in menstrual and peripheral bloodstains, with an emphasis on stain diameter, deposited on glossy paper (a semi-porous surface) under controlled environmental conditions (22 ± 1°C, 50 ± 5% relative humidity). Stain diameters were measured at six time points after deposition: 10 minutes, 1 hour, 2 hours, 24 hours, 72 hours, and 120 hours, in triplicate, under stereo microscopy (2× magnification) with ABFO-scale calibration. Group differences were evaluated using repeated-measures ANOVA (p < 0.05), followed by post hoc Tukey tests. Results The diameter of peripheral bloodstains contracted statistically significantly over time, stabilising with minimal variability. In contrast, the diameter of menstrual bloodstains contracted initially and then stabilised, showing relative dimensional stability with systematically higher variability. The differences are explained by the biochemical composition of menstrual blood (high mucus content, endometrial debris, fibrinolytic activity, lower haematocrit), differing from that of peripheral blood (fibrinogen-mediated clotting, higher plasma protein concentration). Conclusion This non-destructive optical method is simple and complements high-end techniques such as hyperspectral imaging, Raman spectroscopy, and immunochromatographic assays (e.g., the SERATEC PMB test). It may be helpful in preliminary bloodstain pattern analysis (BPA). The use of a single substrate and controlled conditions limits these findings; nonetheless, the standardised methodology and findings provide a reproducible basis for future multi-substrate and field-applicable investigations in the forensic differentiation of blood origins.