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Near-infrared (NIR) Raman spectroscopy was used to measure spectra of dried human blood samples from multiple donors. Two major questions addressed in this paper involve the influence of sample heterogeneity and potential Raman spectral variations that could arise between different donors of blood. Advanced statistical analysis of spectra obtained from multiple spots on dry samples showed that dry blood is chemically heterogeneous, and its Raman spectra could be presented very well as a linear combination of a fluorescent background and two Raman spectroscopic components that are dominated by hemoglobin and fibrin, respectively. Each sample Raman spectrum contains the same major peaks, but the relative contribution of the hemoglobin and fibrin components varies with the donor. Therefore, no single spectrum could adequately represent an experimental Raman spectrum of dry blood in a quantitative way, but rather the combination of hemoglobin and fibrin spectral components could be considered to be a spectroscopic signature for blood. This proof-of-concept approach shows the potential for Raman spectroscopy to be used in forensic analysis to identify an unknown substance such as blood.

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Forensic analysis has become one of the most growing areas of analytical chemistry in recent years. The ability to determine the species of origin of a body fluid sample is a very important and crucial part of a forensic investigation. We introduce here a new technique which utilizes a modern analytical method based on the combination of Raman spectroscopy and advanced statistics to analyze the composition of blood traces from different species. Near-infrared Raman spectroscopy (NIR) was used to analyze multiple dry samples of human, canine, and feline blood for the ultimate application to forensic species identification. All of the spectra were combined into a single data matrix, and the number of principle components that described the system was determined using multiple statistical methods such as significant factor analysis (SFA), principle component analysis (PCA), and several cross-validation methods. Of the six principle components that were determined to be present, the first three, which contributed over 90% to the spectral data of the system, were used to form a three-dimensional scores plot that clearly showed significant separation between the three groups of species. Ellipsoids representing a 99% confidence interval surrounding each species group showed no overlap. This technique using Raman spectroscopy is nondestructive and quick and can potentially be performed at the scene of a crime.