The effect of environmental conditions and deposition substrate on bloodstains ageing: a spectroscopic approach to target a forensic analytical problem

Determining time since deposition of bloodstains can be a potent tool for the forensic chemist in resolving legal cases. This topic has been investigated through several analytical techniques, mainly spectroscopies, such as ultraviolet-visible (UV-Vis), Raman, mid and near infrared (MIR and NIR). In this context, the contribution of chemometrics proved to be essential to gain understanding of the subtle spectral changes ongoing during the ageing of this complex biological fluid. Until now, reduced attention has been devoted to the contribution of environmental conditions and deposition substrate which may have a significant influence on speed and kinetics of blood degradation. Therefore, understanding which factors influence this process (and to what extent) is crucial to avoid over/under estimations of bloodstain age. To study in depth the impact of such factors, bloodstains were subjected to ageing under controlled temperature (T) and humidity (RH%) in a constant climate chamber (KMF115 – BINDER GmbH, Tuttlingen, Germany), mimicking two environmental conditions with a proper day-night alternance: condition CW – day: T = 25°C, RH% = 75%; night: T = 10°C, RH% = 90%; condition HD – day: T = 35°C, RH% = 60%; night: T = 20°C, RH% = 75%. The effect of direct light exposure was investigated as well, performing the ageing sessions either with or without a window that filters the light going from the lamp to the samples. Six 20-µl bloodstains (from two different donors) were deposited on each of four different substrates, namely hydrophilic fabric (cotton), hydrophobic fabric (polyblend), metal (knife) and glass, making a total of 96 samples. Their ageing was followed for 12 days with two different analytical techniques: a handheld NIR device (MicroNIR® – Viavi Solutions Inc., Santa Rosa, California, USA), working in the spectral range 900-1700 nm; a confocal Raman microscope (InVia™ – Renishaw plc, Wotton-under-Edge, Gloucestershire, UK), acquiring in the Raman shift range 150-1900 cm-1. The obtained spectra were pre-processed for minimizing the unwanted systematic effects and subjected to chemometric analysis. Exploratory data analysis by means of principal component analysis (PCA) showed that NIR spectra are more subjected to the effect of the substrate, when compared with Raman spectra. It was observed that absorbing substrates (cotton, polyblend) yield a more efficient time trend model when analysed with NIR spectroscopy, while non-absorbing substrates (glass, metal) gave more consistent results with Raman spectroscopy. PCA also evidenced shifts between sessions carried out at different temperature and humidity, suggesting that environmental conditions may determine not only a faster/slower ageing, but also a different ageing kinetics. Then, ANOVA simultaneous component analysis (ASCA) was applied to quantify the effect of each factor (temperature, humidity, substrate, light exposure) on the ageing of bloodstains, to understand which conditions are most important to be controlled in real case scenarios. The present study confirmed that determination of time since deposition of bloodstains is strongly related to environmental conditions and deposition substrate and, therefore, that tailoring models to take into account these factors is preferable.