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

Abstract

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.

Comprehensive modeling of bloodstain aging by multivariate Raman spectral resolution with kinetics

“…Here, it is worth noting that the fluorescence background intensity cannot be used as a practical indicator of bloodstain aging because the fluorescence intensity can be affected not only by temperature and time, but also by other factors such as contaminants and ambient light. In actual cases, the bloodstain substrate may contribute to the fluorescence background 21,29 . Additionally, photobleaching also hinders reliable evaluation of fluorescence.…”

“…Spectral changes caused by heme autoxidation have been investigated and characteristic peaks have been identified 1,11,[16][17][18] . Moreover, multivariate statistical modeling, such as partial least squares and support vector machine, has been applied to time series of Raman scattering, Fourier transform infrared spectroscopy absorption and visible absorption spectra to distinguish new/old bloodstains and predict the time elapsed since deposition [19][20][21][22] . These studies demonstrate that spectral changes reflect transformation of the chemical constituents in bloodstains during aging, and these can be mathematically correlated with the elapsed time.…”

Raman Scattering: From Structural Biology to Medical Applications

“…However, the relative input of the peaks into the whole spectrum differed for various preparations in both liquid and dried form, which was proposed to use for the identification of specified biological liquids. The same approach was demonstrated to be successful for the discrimination and identification of races, gender, species and for the determination of the blood age while using blood serum and blood [234][235][236][237].…”

Toward a novel framework for bloodstains dating by Raman spectroscopy: How to avoid sample photodamage and subsampling errors

“…Thus, this particular feature of hemoproteins vibrational spectra holds promise for adapting RS to bloodstains dating studies, as it should give an insight into formation of different Hb derivatives over time. And indeed, the time-dependent behaviour of Raman spectra of blood deposits has been already demonstrated [9][10][11]. However, despite this valuable discovery, there are still some challenges that continually hamper implementation of Raman-based methodology in standard proceedings.…”