Species identification of bloodstains by ATR-FTIR spectroscopy: the effects of bloodstain age and the deposition environment

In this study, we investigated the potential of attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopy combined with advanced chemometrics for species identification of bloodstains similar to evidence obtained from real crime scenes. Two partial least squares-discriminant analysis classification models (a human-mammal-domestic fowl trilateral model and a species-specific model) were established. The models demonstrated complete separation among the three classes (human, mammal, and domestic fowl) and distinguished six species (human, rat, rabbit, dog, chicken, and duck). Validation was subsequently conducted to evaluate the robustness of these two models, which resulted in 100 and 94.2% accuracy; even human bloodstains placed in an outdoor environment for up to 107 days were successfully identified. Additionally, all bloodstains were positively identified as blood using the squared Euclidean cosine method by comparing the spectra with those of non-blood substances that had a similar appearance or easily produced false positives. These results demonstrate that ATR-FTIR spectroscopy combined with chemometrics can be a powerful tool for species identification of bloodstains.