The physicochemical changes occurring in biomolecules in degrading bloodstains can be used to approximate the time since deposition (TSD) of bloodstains. This would provide forensic scientists with critical information regarding the timeline of the events involving bloodshed. Our study aims to quantify the timewise degradation trends and temperature dependence found in total RNA from bloodstains without the use of amplification, expanding the scope of the RNA TSD research which has traditionally targeted mRNA and miRNA. Bovine blood with ACD‐A anticoagulant was deposited and stored in plastic microcentrifuge tubes at 21 or 4°C and tested over different timepoints spanning 1 week. Total RNA was extracted from each sample and analyzed using automated high sensitivity gel electrophoresis. Nine RNA metrics were visually assessed and quantified using linear and mixed models. The RNA Integrity Number equivalent (RINe) and DV200 were not influenced by the addition of anticoagulant and demonstrated strong negative trends over time. The RINe model fit was high (R2 = 0.60), and while including the biological replicate as a random effect increased the fit for all RNA metrics, no significant differences were found between biological replicates stored at the same temperature for the RINe and DV200. This suggests that these standardized metrics can be directly compared between scenarios and individuals, with DV200 having an inflection point at approximately 28 h. This study provides a novel approach for blood TSD research, revealing metrics that are not affected by inter‐individual variation, and improving our understanding of the rapid RNA degradation occurring in bloodstains.

Estimating the time since deposition (TSD) of a bloodstain can provide important medico-legal information for crime scene investigation. Research in this area primarily investigates the degradation of either hemoglobin or genetic material over time. In this work, we present a comprehensive meta-analysis on bloodstain TSD research. Our results are interpreted from 25 quantitative studies used to probe the effect of biomolecule studied, analytical technique used, substrate porosity, environmental conditions, and blood source on TSD estimates. There was an overall strong effect of time across studies (Fisher’s Zr = 1.66, r = 0.93), and generally, we found that the type of biomolecule studied (e.g., hemoglobin, DNA) had equal effect sizes for TSD estimation. Differences in the mean TSD effect size were also observed between substrate porosity. Interestingly, the blood source does not significantly influence the magnitude of the effect sizes in TSD estimation. Despite the clear effect of time, forensically relevant prediction of bloodstain TSD remains complicated by inter-donor variability, type of substrate and environmental conditions. We recommend that future bloodstain TSD research increase sample size, include summary statistics and standardize experimental methodologies so that we can develop a quantitative understanding of the physicochemical processes involved in whole blood degradation in ex vivo conditions.

F. Wolf (he/him) 1 | Layla MacKay (she/her) 2 | Sarah E. Haworth (she/her) 1 | Marie-Laurence Cossette (she/her) 1 | Morgan N. Dedato (she/her) 1 | Kiana B. Young (she/her) 1 | Colin I. Elliott (he/him) 2 | Rebekah A. Oomen (she/her) 3,4This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Understanding the physical, chemical and biological changes that occur during the drying of a bloodstain is important in many aspects of forensic science including bloodstain pattern analysis and time since deposition estimation. This research assesses the use of optical profilometry to analyze changes in the surface morphology of degrading bloodstains created using three different volumes (4, 11, and 20 µL) up to four weeks after deposition. We analyzed six surface characteristics, including surface average roughness, kurtosis, skewness, maximum height, number of cracks and pits, and height distributions from the topographical scans obtained from bloodstains. Full and partial optical profiles were obtained to examine long-term (minimum of 1.5 hour intervals) and short-term (5 minute intervals) changes. The majority of the changes in surface characteristics occurred within the first 35 minutes after bloodstain deposition, in agreement with current research in bloodstain drying. Optical profilometry is a non-destructive and efficient method to obtain surface profiles of bloodstains, and can be integrated easily into additional research workflows including but not limited to time since deposition estimation.