Investigating the Age of Blood Traces: How Close Are We to Finding the Holy Grail of Forensic Science?

Knowing the time since deposition (TSD) of an evidentiary bloodstain is highly desired in forensics, yet it can be extremely complicated to accurately determine in practice. Although there have been numerous attempts to solve this problem using a variety of different techniques, currently, no established, well-accepted method exists. Here, a Raman spectroscopic approach was developed for determining the age of bloodstains up to 1 week old. Raman spectroscopy, along with two-dimensional correlation spectroscopy (2D CoS) and statistical modeling, was used to analyze fresh bloodstains at ten time points under ambient conditions. The 2D CoS results indicate a high correlation between several Raman bands and the age of a bloodstain. A regression model was built to provide quantitative predictions of the TSD, with cross-validated root mean squared error and R (2) values of 0.13 and 0.97, respectively. It was determined that a "new" (1 h) bloodstain could be easily distinguished from older bloodstains, which is very important for forensic science in helping to establish the relevant association of multiple bloodstains. Additionally, all bloodstains were confirmatively identified as blood by comparing the experimentally measured spectra to multidimensional body fluid spectroscopic signatures of blood, saliva, semen, sweat, and vaginal fluid. These results demonstrate that Raman spectroscopy can be used as a nondestructive analytical tool for discriminating between bloodstains on the scale of hours to days. This approach shows promise for immediate practical use in the field to predict the TSD with a high degree of accuracy. Graphical Abstract Bloodstain aging over time illustrating naturally ocurring processes.

DNA allows for the unambiguous identification of the person from whom a biological sample was derived but provides little information about when the sample was deposited. This information only indicated that the biological material was deposited at the crime scene prior to the collection of evidence. The ability to determine the age of a biological sample would greatly benefit the forensic science community. If there were independent evidence that the biological sample was deposited at the time of the crime, then its age would reveal when the crime occurred. If the time of the crime were known through another means, then the age of the biological sample could potentially exclude the human source as a suspect. We have used real-time reverse transcriptase PCR to show that the ratio between different types of RNA (mRNA versus rRNA) changes over time in a linear fashion when dried human blood from eight individuals was examined over the course of 150 days. Although other approaches have been used in the past to estimate the age of a biological sample, our approach offers the following advantages: enhanced detectability of small samples, simultaneous isolation of DNA and RNA from the same sample, species-specific probes, and an increased window of usefulness.