Preserving bloodstain evidence: Completely non-destructive estimation of time-since-deposition with ATR-FTIR spectroscopy and chemometrics.

Determining the time since deposition (TSD) of bloodstains is critical for reconstructing events in forensic investigations. However, traditional methods often involve destructive techniques that compromise evidence integrity. This study demonstrates the feasibility of attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy combined with chemometrics for non-destructive TSD estimation of bloodstains on glass slides up to 100 days old in realistic indoor environments, including open-air (macro) and zip-lock sealed (micro) conditions. Spectral data were acquired directly from the outer ring of bloodstain samples, preprocessed using standard normal variate transformation, and analysed using partial least squares regression (PLS-R) and partial least squares discriminant analysis (PLS-DA). The environment-specific PLS-R models demonstrated strong predictive performance, achieving a coefficient of determination of approximately 0.94 and a root mean square error of around 8 days. By categorising TSD prediction into broader groups, the PLS-DA models exhibited high robustness across varying environmental conditions, attaining discriminative accuracies of up to 95 %. Notably, PLS-DA effectively distinguished bloodstains less than 30 days old from older samples, with an area under the receiver operating characteristic curve approaching 1, indicating high reliability. These findings underscore the potential of PLS modelling combined with ATR-FTIR as a non-destructive, forensic-compatible technique for bloodstain age estimation, ensuring the preservation of forensic evidence integrity.