Forensic approach for distinguishing species and sex of multispecies bloodstains in soil and sand by vibrational spectroscopy.

Bloodstains found at a crime scene must be accurately identified to correctly interpret the forensic context and events. Bloodstains (human or animal) sometimes appear on complex substrates such as sand or soil, which complicates their collection and analysis. Traditional presumptive tests for human blood may yield false positives in the presence of animal blood. This limitation, together with the need to preserve small stains, has motivated the use of non-destructive analytical techniques, such as vibrational spectroscopy. The aim of this work was to explore the combined use of ATR-FTIR, Raman microspectroscopy, and chemometrics for detecting and identifying human, dog, and cat bloodstains on sand and soil. ATR-FTIR and Raman spectral data were used to construct orthogonal partial least squares-discriminant analysis (OPLS-DA) models to assist in differentiation. In general, ATR-FTIR spectra combined with OPLS-DA demonstrated superior species discrimination capabilities compared with Raman spectra. Sex discrimination was further investigated using both datasets. However, the discriminatory performance of Raman spectroscopy was limited. These results highlight the potential of vibrational spectroscopy combined with chemometrics as a non-destructive approach for differentiating multispecies bloodstains on complex substrates. However, given the small number of donors included in this study, the findings should be considered preliminary, and further studies with larger sample sets are needed to confirm and extend these observations.