Effect of environmental conditions and deposition substrate on bloodstain ageing studied by means of Raman and near infrared spectroscopies coupled with chemometrics

The accurate estimation of bloodstain age is a critical aspect of forensic investigations, providing key insights into the chronology of events on crime scenes. Numerous techniques have been proposed for time since deposition (TSD) determination, among which spectroscopic approaches offer advantages including rapidity, reproducibility, and non-destructiveness, while maintaining analytical robustness. The present study systematically investigates the influence of environmental conditions – temperature, humidity, and illumination – and substrate type on bloodstain ageing kinetics using near-infrared (NIR) and Raman spectroscopies. Blood samples were deposited on four substrates (cotton, polyblend, glass, and metal) and aged under four controlled environmental conditions over a 12 day period, with repeated spectral measurements at fourteen time points. Partial least squares (PLS) regression was then employed to extract features representative of temporal evolution. Finally, regularised multivariate analysis of variance (RMANOVA) was used to quantify the effects of factors and their interactions. Substrate type emerged as the primary determinant of spectral variability, significantly affecting both NIR and Raman signals, while environmental conditions notably modulated ageing kinetics, with the combination of temperature and humidity exerting a greater influence on Raman measurements and illumination predominantly affecting NIR spectra. Interactions between factors were also significant, highlighting the complexity of bloodstain degradation. Comparisons with a dummy factor representing donor variability indicated that substrate and environmental conditions outweigh biological variability. These findings underscore the necessity of accounting for deposition surface and environmental conditions when developing predictive models for bloodstain dating, and suggest that robust dating may be achievable even with unknown donors, provided that experimental conditions are properly replicated.