Estimation of the age of human bloodstains by electron paramagnetic resonance spectroscopy: Long-term controlled experiment on the effects of environmental factors

In this study, we examined the efficacy and limitations of electron paramagnetic resonance (EPR) for estimating the age of human bloodstains. At 77 K, human bloodstains give four striking EPR signals in the g = 6.2 (g6), 4.3 (g4), 2.27 (H) and 2.005 (R) regions due to ferric high-spin, ferric non-heme, ferric low-spin and free radical species, respectively. We found that plotting double logarithms of the EPR intensity ratio of H/g4 versus days past bleeding gave a linear correlation up to 432 days with an error range within 25% of the actual number of days under controlled conditions. However, environmental factors such as differences of absorbent, light exposure and fluctuations of storage temperature affected the changes of these EPR-active compounds, which result in misestimation of the time since bleeding occurred. Therefore, one should take such factors into account in estimating the period since bleeding by this method.