Issue 18, 2023

Elemental and molecular characterization of degrading blood pools

Abstract

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 Fe2+ to primarily Fe3+, as expected for ex vivo 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 C[double bond, length as m-dash]O, and the formation of free amines and O[double bond, length as m-dash]C-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 (p < 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.

Graphical abstract: Elemental and molecular characterization of degrading blood pools

Supplementary files

Article information

Article type
Paper
Submitted
29 Jun 2023
Accepted
13 Aug 2023
First published
15 Aug 2023

Analyst, 2023,148, 4300-4309

Elemental and molecular characterization of degrading blood pools

E. Giroux, I. I. Ebralidze and T. E. Stotesbury, Analyst, 2023, 148, 4300 DOI: 10.1039/D3AN01094E

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