Attenuated total reflection Fourier-transform infrared spectroscopy and chemometric analysis for estimating time since deposition of bloodstains on fabrics under ambient conditions.

The present study expands upon previous studies by employing attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy as a nondestructive technique for estimating the time since deposition (TSD) of blood traces on common fabrics and household items. Initial analysis showed substrate contributions; however, these did not affect the amide I and II bands specific to blood proteins in infrared spectra. A comprehensive statistical analysis was conducted which was evaluated using external validation; this was done to ensure that model predictions remain reliable and to prevent overfitting, which can be introduced by internal validation methods. To identify relatively recent bloodstains, a partial least squares discriminant analysis (PLS-DA) classification model was developed to effectively distinguish between blood samples aged on cotton and polyester for ≤72 and >72 h. The external validation of these binary models yielded average prediction accuracies of 92% for bloodstains on polyester and 94% for those on cotton. A partial least squares regression (PLSR) combined with a genetic algorithm (GA) was used for building regression models with R² prediction values of 0.86 and 0.85 for polyester and cotton, respectively. This proof-of-concept study demonstrates that ATR-FTIR spectroscopy combined with advanced chemometrics enabled estimation of the time since deposition (TSD) of blood traces on cotton and polyester fabrics. Although the results are promising, the study involved a small number of donors and limited surface types; therefore, additional future research is needed to determine its broader applicability to a wider range of donors and surfaces.