Hidden Clues Within Blood: ATR-FTIR Spectroscopy and Chemometric Analysis for Determining the Ante-Mortem and Post-Mortem Origin of Blood

<title>Abstract</title> <p>The forensic identification and discrimination of various types of body fluids play a pivotal role in criminal investigations to determine how a crime has occurred. Over the years, several techniques have been employed to identify body fluids; however, Attenuated ATR-FTIR spectroscopy stands out as one of the most effective methods to analyse the biochemical properties of body fluids. The present study focuses on the use of ATR-FTIR spectroscopy, in conjunction with multivariate statistical analysis, to discriminate between ante-mortem and post-mortem blood samples. In addition, the research extends to examine how different substrates may affect the detection of these blood samples. ATR-FTIR spectroscopy is a rapid and non-destructive analytical technique, and it facilitates the fingerprinting of biological fluids by analysing their unique biochemical composition through minimal or no sample preparation. The application of chemometric tools, such as PCA, PLS-DA, and LDA, facilitates the classification of ante-mortem and post-mortem blood samples. The present study demonstrates that the combined use of ATR-FTIR spectroscopy and chemometric analysis offers a rapid, non-destructive, and reliable approach for the forensic differentiation of antemortem and postmortem blood samples with 100% accuracy. The study further evaluates the influence of different substrates and storage conditions on blood analysis. Additionally, menstrual blood samples were incorporated as an external validation dataset, and the developed model successfully discriminated menstrual blood from ante-mortem and post-mortem blood samples with complete classification accuracy, thereby strengthening its forensic applicability in complex case scenarios.</p>