Since Raman spectroscopy has proven to be a rapid, nondestructive, and specific technique that is capable of identifying and analyzing bloodstains down to a single red blood cell (1), we have combined Raman spectroscopy with chemometrics to discriminate bloodstains from other body fluid traces (2–4), perform species identification (5–8), and determine phenotypic traits of human donors such as sex (9), race (10), age group (11).
It is of special importance for practical forensic purposes that the Raman spectroscopic method for bloodstain identification is confirmatory and “immune” from environmental false positives (12). The availability of portable and handheld Raman instruments makes bloodstain analysis within the first minutes of the crime scene discovery a reality. The near infrared (NIR) Raman spectra of blood, when excited at 785 nm, are dominated by a contribution from hemoglobin. When blood exits the body, it is red in appearance, but as it dries, bloodstains darken. This red-to-brown color change occurs as a result of the autoxidation of hemoglobin into oxyhemoglobin (oxyHb). As the bloodstain aging process continues, oxyHb is converted into methemoglobin (metHb). Once in this state, the hemoglobin molecule is no longer able to bind oxygen. Finally, when blood is outside the body for an extended period of time, metHb will form aggregates of hemichrome. Raman spectroscopy detects these changes in hemoglobin over time and provides the basis for determining the TSD of bloodstains. In our initial study, we developed an accurate regression model for bloodstain aging for the first week since the deposition (13). Doty and associates were able to monitor the autoxidation process of hemoglobin by following the intensity changes of the Raman bands at 377 and 420 cm-1. These bands are markers for metHb and oxyHb, respectively. Changes to these two bands began after one hour of deposition, with a relative increase in the 377 cm-1 peak and a decrease in the 420 cm-1 peaks. Using partial least squares regression (PLSR), the TSD The near infrared (NIR) Raman spectra of blood, when excited at 785 nm, are dominated by a contribution from hemoglobin. When blood exits the body, it is red in appearance, but as it dries, bloodstains darken. This red-to-brown color change occurs as a result of the autoxidation of hemoglobin into oxyhemoglobin (oxyHb). As the bloodstain aging process continues, oxyHb is converted into methemoglobin (metHb). Once in this state, the hemoglobin molecule is no longer able to bind oxygen. Finally, when blood is outside the body for an extended period of time, metHb will form aggregates of hemichrome. Raman spectroscopy detects these changes in hemoglobin over time and provides the basis for determining the TSD of bloodstains. In our initial study, we developed an accurate regression model for bloodstain aging for the first week since the deposition (13). Doty and associates were able to monitor the autoxidation process of hemoglobin by following the intensity changes of the Raman bands at 377 and 420 cm-1. These bands are markers for metHb and oxyHb, respectively. Changes to these two bands began after one hour of deposition, with a relative increase in the 377 cm-1 peak and a decrease in the 420 cm-1 peaks. Using partial least squares regression (PLSR), the TSD of bloodstains was predicted with an accuracy rate of 0.99 and when cross-validated the accuracy rate was 0.97 for up to one week. o ascertain the full capabilities of the method, we continued to monitor the Raman spectra that allowed for building a regression model to determine the TSD for up to two years (Figure 1) (14). There were evident changes to Raman spectra even after two years of aging. As a result of heme aggregation during aging, the fluorescence interference continuously decreased the quality of the Raman spectra. This is because of the changes in the electronic structure of heme as it ages (15). The intensity of the Raman bands decreased, and the peaks broadened after a prolonged (about a year or longer) TSD. These changes were tentatively attributed to the advanced degradation of hemoglobin. Using the PLSR model, Doty and associates were able to predict the TSD of bloodstains up to two years with 70% accuracy. Some might speculate that this error is too large for practical application, but the method definitively differentiates between bloodstains that were present at crime scene for a few hours, a few days, a few weeks, a few months or over a year. Just as Raman spectroscopy has proven to be a valuable tool for discriminating between body fluids, this technique has the potential to be a one-step analysis method to both identify a biological stain as blood and approximate its TSD. (Publisher Abstract)
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