A Raman "spectroscopic clock" for bloodstain age determination: the first week after deposition

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.