Estimation of the age of human bloodstains under the simulated indoor and outdoor crime scene conditions by ATR-FTIR spectroscopy.

Estimation of the age of human bloodstains is of great importance in forensic practices, but it is a challenging task because of the lack of a well-accepted, reliable, and established method. Here, the attenuated total reflection (ATR)-Fourier transform infrared (FTIR) technique combined with advanced chemometric methods was utilized to determine the age of indoor and outdoor bloodstains up to 107 days. The bloodstain storage conditions mimicked crime scene scenarios as closely as possible. Two partial least squares regression models-indoor and outdoor models with 7-85 days-exhibited good performance for external validation, with low values of predictive root mean squared error (5.83 and 4.77) and high R2 values (0.94 and 0.96) and residual predictive deviation (4.08 and 5.14), respectively. Two partial least squares-discriminant analysis classification models were built and demonstrated excellent distinction between fresh (age ≤1 d) and older (age >1 d) bloodstains, which is highly valuable for forensic investigations. These findings demonstrate that ATR-FTIR spectroscopy coupled with advanced chemometric methods can be employed as a rapid and non-destructive tool for age estimation of bloodstains in real-world forensic investigation.