Integrating time since deposition estimation of bloodstains into a DNA profiling workflow: A novel approach using fluorescence spectroscopy

Colin I Elliott; Denina B D Simmons; Theresa Stotesbury

doi:10.1016/j.talanta.2024.127234

Integrating time since deposition estimation of bloodstains into a DNA profiling workflow: A novel approach using fluorescence spectroscopy

Talanta. 2024 Nov 17:284:127234. doi: 10.1016/j.talanta.2024.127234. Online ahead of print.

Authors

Colin I Elliott¹, Denina B D Simmons², Theresa Stotesbury³

Affiliations

¹ Applied Bioscience Graduate Program, Faculty of Science, Ontario Tech University, 2000 Simcoe St N, Oshawa, Ontario, L1G 0C5, Canada. Electronic address: colin.elliott1@ontariotechu.net.
² Faculty of Science Ontario Tech University, 2000 Simcoe St N, Oshawa, Ontario, L1G 0C5, Canada.
³ Faculty of Science Ontario Tech University, 2000 Simcoe St N, Oshawa, Ontario, L1G 0C5, Canada. Electronic address: theresa.stotesbury@ontariotechu.ca.

PMID: 39603014
DOI: 10.1016/j.talanta.2024.127234

Abstract

Determining the time since deposition (TSD) of bloodstains is important to establish a timeline of bloodshed, while DNA profiling addresses identity (source attribution). Traditionally treated as separate processes, this study integrates TSD estimation into routine DNA profiling by analyzing typically discarded cell lysate (eluates) from spin-column-based DNA extractions. Fluorescence spectroscopy was used to analyze eluates from bloodstains deposited up to 99 weeks. Two excitation-emission matrices (EEMs) were acquired for each sample and deconvoluted using parallel factor analysis (PARAFAC) to identify individual fluorophores. For example, tryptophan demonstrated a time-dependent decrease in fluorescence. Additionally, we observed an accumulation of fluorescent oxidation products (FOX) and advanced glycation end products (AGEs) over TSD. An untargeted metabolomics high-performance liquid chromatography-mass spectrometry workflow was applied to assist with fluorophore identification. Chemometric models were used to estimate TSD from EEM fluorescence data. Boruta feature selection coupled with random forest regression outperformed all other models and achieved high accuracy, with an R² of 0.993 and root mean square error of prediction (RMSEP) of 2.83 weeks for the full 99-week period, and an R² of 0.987 and RMSEP of 2.06 weeks for the 1-year timeframe. Comparisons were also made between anticoagulant-free (AC-free) and anticoagulant-treated (AC-treated) bloodstains deposited up to 3 months. We noted differences in fluorescence based on AC treatment, with AC-free blood exhibiting higher FOX and lower AGE fluorescence than AC-treated blood. Our findings demonstrate the effectiveness and feasibility of integrating TSD estimation into routine forensic DNA extractions while maintaining high prediction accuracies.

Keywords: Blood; Cell lysate; Excitation-emission matrix (EEM); Fluorescent oxidation products (FOX); PARAFAC; Time since deposition (TSD).