Faculty of Science, Ontario Tech University, Oshawa, ON, L1G 0C5, Canada.
Analyst. 2023 Sep 11;148(18):4300-4309. doi: 10.1039/d3an01094e.
Blood is a commonly encountered type of biological evidence and can provide critical information about the crime that occurred. The ability to accurately and precisely determine the time since deposition (TSD) of a bloodstain is highly sought after in the field of forensic science. Current spectral methods for determining TSD are typically developed using small volume bloodstains, we investigate the applicability to larger volume blood pools where drying and degradation mechanics are different. We explored the differences that exist between the surface and bulk of dried segments from fragments collected from 15 mL dried blood pools and identified heterogeneity using RGB colour analysis and hierarchical cluster analysis (HCA). The physical, molecular, and atomic differences between the layers were further investigated using scanning electron microscopy (SEM), X-Ray photoelectron spectroscopy (XPS), attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy, and Raman spectroscopy. SEM identified different morphology on the surface and the bulk indicative of density-dependant cellular settling. XPS revealed that iron was not present on the surface but rather was present in the bulk where the red blood cells had settled. The oxidation state of the iron was quantified over three weeks in which it transitioned from entirely Fe to primarily Fe, as expected for degradation of hemoglobin. Further, indications of amide saponification occurring at the blood-air interface were identified in the increased quantity of the C-O moiety relative to CO, and the formation of free amines and OC-ONa groups over time. ATR-FTIR and Raman spectroscopy provided insights into differences in the molecular composition of the layers, suggesting that the surface consists of more nucleic acids, lipids, and glycoproteins than the bulk, which was dominated by proteins ( < 0.001% using principal component analysis (PCA)). Additionally, spectral band trends previously reported to have applicability to the estimation of TSD were observed for the bulk portion of the blood pool as the Hb underwent predictable time dependant changes from oxyHb to metHb. PCA was performed based on all spectral data which demonstrated statistically significant differences between the surface and bulk, as well as proof-of-concept for linear TSD estimation models.
血液是一种常见的生物证据类型，可提供有关犯罪发生的关键信息。在法医学领域，准确而精确地确定血迹沉积时间（TSD）的能力是非常需要的。目前用于确定 TSD 的光谱方法通常是使用小体积的血迹开发的，我们研究了在干燥和降解机制不同的更大体积的血池中的适用性。我们从 15mL 干燥血池收集的片段中探索了干燥段表面和内部之间存在的差异，并使用 RGB 颜色分析和层次聚类分析（HCA）对其进行了识别。进一步使用扫描电子显微镜（SEM）、X 射线光电子能谱（XPS）、衰减全反射傅里叶变换红外（ATR-FTIR）光谱和拉曼光谱研究了各层之间的物理、分子和原子差异。SEM 鉴定了表面和内部的不同形态，表明细胞沉降存在密度依赖性。XPS 表明铁不在表面存在，而在红细胞沉降的内部存在。铁的氧化态在三周内进行了定量，从完全的 Fe 转变为主要的 Fe，这是血红蛋白降解的预期结果。此外，在血液-空气界面处酰胺皂化发生的迹象是在 C-O 部分相对于 C=O 的增加量以及随时间推移形成的游离胺和 O-C-ONa 基团中识别出的。ATR-FTIR 和拉曼光谱提供了有关各层分子组成差异的见解，表明表面比内部含有更多的核酸、脂质和糖蛋白，而内部主要含有蛋白质（主成分分析（PCA）<0.001%）。此外，观察到 Hb 经历了从氧合 Hb 到高铁 Hb 的可预测的时间依赖性变化，从而对血液池的大部分观察到了先前报道适用于 TSD 估计的光谱带趋势。PCA 是基于所有光谱数据进行的，表明表面和内部之间存在统计学上的显著差异，以及线性 TSD 估计模型的概念验证。
