Toward a novel framework for bloodstains dating by Raman spectroscopy: How to avoid sample photodamage and subsampling errors

Answers to questions about the time of bloodstains formation are often essential to unravel the sequence of events behind criminal acts. Unfortunately, the relevance of preserved evidence to the committed offence usually cannot be verified, because forensic experts are still incapable of providing an accurate estimate of the bloodstains’ age. An antidote to this impediment might be substituting the classical dating approach – founded on the application of calibration models – by the comparison problem addressed using likelihood ratio tests. The key aspect of this concept involves comparing the evidential data with results characterizing reference bloodstains, formed during the process of supervised ageing so as to reproduce the evidence. Since this comparison requires data that conveys information inherent to changes accompanying the process of blood decomposition, this study provided a Raman-based procedure, designated for probing into the chemistry of ageing bloodstains. To circumvent limitations experienced with single-point measurements – the risk of laser-induced degradation of hemoglobin and subsampling errors – the rotating mode of spectral acquisition was introduced. In order to verify the performance of this novel sampling method, obtained spectra were confronted with those acquired during conventional static measurements. The visual comparison was followed by analysis of data structure using regularized MANOVA, which boosted the variance between differently-aged samples while minimizing the variance observed for bloodstains deposited at the same time. Studies of relation between these variances demonstrated the superiority of novel procedure, as it provided Raman signatures that enabled a better distinction between differently-aged bloodstains.