Determination of the Time since Deposition of blood‐traces in a forensic context: Application of untargeted LC‐HR‐MS/MS metabolomics profiling

Abstract

Being able to attest when a bloodstain was deposited at a crime scene can be invaluable to a prosecution process, and methods to provide that information have long been desired. Determining the Time since Deposition (TsD) of a trace would allow placing a subject both in space and time to the crime scene-or prove that a trace left by that person was unrelated to it because it was deposited before or after the time the crime had occurred. To this day, no method for TsD determination has made its way into routine forensic casework, mainly because of the numerous challenges that await when trying to understand and account for all the influencing and confounding factors that affect the aging process (such as, e.g., temperature, UV-light exposure, or humidity). Here, we present an untargeted metabolomics-based study using liquid chromatography high-resolution mass spectrometry (LC-HR-MS) and data-dependent acquisition to analyze blood samples aged under two distinctly different storage conditions over 48 weeks. Global differences in age-and storage-dependent changes in blood metabolomes were described, and TsD-classification strategies based on qualitative and quantitative assessment of molecular features (MFs) have been proposed.Based on the selected criteria to best predict the TsD, the dipeptide Phenylalanylalanine (PheAla) can be considered as a promising candidate for TsD prediction. In essence, changes in the blood metabolome dynamics showed a strong association with increasing TsD, but significant differences depending on storage conditioning were observed, facilitating the need to study further the influence of individual influencing factors on TsD determination.

Untargeted Metabolomics Profiling for Determination of the Time since Deposition of Biofluids in a Forensic Context: A Proof-of-Concept for Urine, Saliva, and Semen in Addition to Blood

“…Moreover, MFs with a high contribution to the loadings for components in sPLS-DA were included as potential candidates due to their nature of being driving forces of class separation in multivariate statistical analysis. Those, as well as previously described MFs as potential candidate biomarkers for TsD, 3 are described in more detail in Table 1. Detailed graphical illustrations of the time-dependent changes of these selected MFs can be found in Figures S3−S5.…”

“…Our previous two studies focused on studying the blood proteome and metabolome for their feasibility of estimating the TsD, emphasizing the magnitude of effects the storage condition can have on the aging process and for TsD determination. ,, The study presented in this article aims to investigate the time-dependent dynamics of the metabolomes of different types of body fluids in addition to blood: saliva, semen, and urine, and whether it is possible to find biomarkers for the estimation of the TsD, irrespective of the body fluid in question. All four of them are commonly encountered in the forensic context and thus are highly relevant due to their practical implications in criminal casework and sexual assault.…”

“…All samples were left to fully dry for approximately 8 h before the first sampling point (T0, 0.3 d). All DBFS were then allowed to age by storage in a closed laboratory drawer, exposed to only minor room temperature and humidity fluctuations and without exposure to UV light and minimum air-borne contamination (identical to the controlled storage condition from our previous study). Further sample aging was stopped by transferring the samples to an −80 °C deep freezer until sample preparation was done at the following time points: T0 (∼0.3 d, after the initial drying of the body fluid spots), T1 (1 d), T2 (2 d), T3 (3 d), T4 (4 d), T5 (5 d), T6 (7 d), T7 (10 d), T8 (14 d), T9 (17 d), T10 (21 d), T11 (23 d), T12 (26 d), and T13 (28 d).…”

“…Next to UV–vis spectroscopy, fluorescence, reflectance, and Raman spectroscopy methods have been described in the literature. − In the past few years and with the advent of more holistic (or '-omics') methodologies in forensic toxicology and forensic biology, methods studying entire classes of biomolecules have been applied to study the TsD. These include genomic and transcriptomic approaches, investigating aging patterns of RNA and DNA, − proteomics methods, , gaining an understanding of time- and storage-dependent behaviors of proteins, peptides, and their modifications as well metabolomics, researching small molecules and metabolites and their TsD-patterns. ,,− …”

Alkaline Phosphatase for Estimating the Time since Deposition of Blood Fingerprints by Scanning Electrochemical Microscopy

“…Without knowing exactly how long the blood traces have been deposited at the crime scene, forensic experts are unable to draw definite conclusions about whether the person of interest is a suspect . Thus, it is urgent to determine the time since deposition (TSD) of bloodstains, which can be a tremendous help to forensic investigations to screen out crime-related evidence and potentially aid in the crime timeline reconstruction. − …”