Monitoring the solid-state VIS profiles of degrading bloodstains

Determining the time since deposition (TSD) of bloodstained evidence can be an important process in forensic investigations. Hemoglobin is often examined as a biomolecule of interest for these purposes due to the known ex vivo oxidative changes to its structure. These time-dependent oxidative processes have previously been probed using UV–VIS spectroscopy following the resuspension of bloodstains. Our study investigated the solid-state VIS spectra of degrading bloodstains without sample pre-treatment, effectively bypassing the need for resuspension. A total of 128 bloodstains from eight biological replicates were created and stored on glass slides in four temperature conditions: −20 °C, 4 °C, 22 °C, and 45 °C (see graphical abstract, created with BioRender.com). Spectra were acquired from 380−800 nm at five time points spanning 96 h. The peak area of the methemoglobin (metHb) band displayed the largest time and temperature differences, an interesting contrast to previous literature using the Soret band for TSD. Principal Component Analysis (PCA) demonstrated that storage temperature delineated the data, with the metHb band showing the greatest contributions to PC1. Linear mixed models from the PCA data with time showed clear TSD relationships with temperature, and with minimal inter-donor variability. Overall, this work complements the UV–VIS analysis of bloodstains for TSD estimation, with the importance of noting clear differences between phases and sample preparation methods.