Quantifying visible absorbance changes and DNA degradation in aging bloodstains under extreme temperatures

Physicochemical property changes observed in a degrading bloodstain can be used to estimate its time since deposition (TSD) and provide a timestamp to the sample's age. Many of the time-dependent processes that occur as a bloodstain degrades, such as DNA fragmentation and changes in hemoglobin structure, also exhibit temperature-dependent behaviours. Previous studies have demonstrated that pairing high-resolution automated gel electrophoresis and visible absorbance spectroscopy could be used to quantify the rate of degradation of a bloodstain in relation to time and storage substrate. Our study investigates such trends with an added factor, extreme temperatures. Passive drip stains were stored in either microcentrifuge tubes or on FTA cards at either -20°C, 21°C or 40°C and tested over 11 time points spanning 15 days. For both storage substrates, the wavelength at maximum absorbance for the Soret band and the maximum absorbance of the Alpha band showed a negative trend over time suggesting that spectral shifts are informative for TSD estimates. The ratio of the maximum peak height for DNA fragments lengths of 500-1000 base pairs to 1000-5000 base pairs was the most informative DNA variable in relation to time for both substrates. Cross-validation suggested the appropriate fit of the models with the data and reasonable predictive ability. We integrated both DNA concentration and hemoglobin visible absorbance metrics using principal component analysis (PCA) into a single model. Adding the random effect of the donor to the PCA model accounted for a large portion of the variation as did storage method and temperature. Additionally, canonical correspondence showed that temperature corresponded differently to the response variables for FTA card and microcentrifuge tube samples, suggesting a substrate specific effect. This study confirms that pairing DNA concentration and hemoglobin's visible absorbance can provide insight on the effect of different environmental and storage conditions on bloodstain degradation. While the level of uncertainty surrounding TSD estimates still precludes its use in the field, this study provides a valuable framework that improves our understanding of variation surrounding TSD estimates, which will be critical to any eventual application.