Application of ATR-FTIR spectroscopy and chemometrics for the forensic discrimination of aged peripheral and menstrual bloodstains

Accurately distinguishing between peripheral and menstrual bloodstains at crime scenes is a major challenge in forensic practice. The time since deposition (TSD) of bloodstains at the crime scene also plays an essential role in determining the postmortem interval or injury time. In this study, we used attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy combined with chemometric methods to differentiate peripheral and menstrual bloodstains considering the TSD and menstrual phase.The results of the principal component analysis (PCA) model showed that the fresh peripheral and menstrual bloodstains were relatively easy to distinguish, while the aged samples (2 W, 4 W, and 6 W TSD) caused a certain level of spectral signature convergence in the spectral features. Subsequently, the partial least squares discriminant analysis (PLS-DA) model and the genetic algorithm combined with PLS-DA (GA-PLSDA) model were constructed considering the TSD.The accuracy of internal cross-validation and external validation of the PLS-DA model demonstrated internal and external classification abilities of 88.3 % and 90.0 % accuracy, respectively. Additionally, the results of GA-PLSDA model showed that the accuracy of external and internal validation was 89.2 % and 90.0 %, respectively.The PCA, PLS-DA, and GA-PLSDA models showed that the critical differences between peripheral and menstrual bloodstains were in protein, phosphoric acid and glucose content. This preliminary study suggests that ATR-FTIR spectroscopy combined with chemometrics may be a potential method to distinguish between peripheral and menstrual bloodstains using complex conditions and provides support for further studies.