Mass Spectrometry & Spectroscopy
A universal method for the identifi cation of body fl uid traces for forensic purposes based on Raman spectroscopy
Alexis Weber and Igor K. Lednev, SupreMEtric LLC, 7 University Pl B210 Rensselaer, NY 12144, United States Department of Chemistry, University at Albany, SUNY, 1400 Washington Avenue, Albany, NY 12222, United States
Scenes of violent crimes including homicides or physical and sexual assaults can contain tens to hundreds of stains. These stains vary in nature and can be a combination of fresh body fl uids, old body fl uids (common in cases of repeated domestic abuse), or environmental contaminates [1]. Crime scene investigators are trained to collect samples from all seemingly relevant stains. They will collect either swabs or cutting of stained areas to submit to evidence. This can lead to an overwhelming number of samples being submitted to the crime lab with the goal of obtaining a DNA profi le. However, the time commitment and cost to perform DNA analysis is high thus, it is important to conclusively identify which body fl uids are relevant to the current investigation. Due to the large forensic case load, the analysis of body fl uids must be done effi ciently. Presumptive and confi rmatory tests have been developed over the years for the identifi cation of body fl uid traces. Colorimetric reactions are the primary method used in presumptive testing assays, while immunochromatographic cards are commonly used for confi rmatory identifi cation assays. In forensic labs, phenolphthalein (blood), acid phosphatase and Christmas tree staining (semen), and Phadebas (saliva) are the most frequently used tests to identify these fl uids [2]. However, currently, there are no reliable methods for identifying vaginal fl uid, urine, or sweat. The majority of current methods have been utilised for many years despite that they have certain disadvantages. For example, they can be destructive to the sample, and each assay can only identify one body fl uid. Moreover, most of the tests used by forensic examiners provide presumptive results, meaning that a positive result only suggests the possible presence of a body fl uid.
Raman spectroscopy can be used to identify and characterise molecules in a variety of samples, including solids, liquids, and gases. By collecting multiple spectra from a sample, the resulting signatures account for the intrinsic heterogeneity of dry traces of body fl uids and variations among individuals [3]. It is a non-destructive technique that does not require sample preparation, making it a useful tool in fi elds such as chemistry, biology, and forensic science. Multidimensional spectroscopic signatures for complex biological substances are produced and able to be analysed using advanced statistical methods, called chemometrics. Chemometrics involves the use of mathematical and statistical techniques to extract meaningful information from multivariate chemical datasets. Spectral outputs, although appearing as a single line, are made up of hundreds or thousands of data points. Consequently, spectral results require more advanced analysis techniques, as compared to univariate data that involve only a single variable. To process, quantify, and classify vibrational spectroscopic data, multivariate statistical analyses are essential.
Figure 1. Energy level diagram related to IR absorption, Raman scattering and fl uorescence emission. Reprinted from Li, Zhiyun, M. Jamal Deen, Shiva Kumar, and P. Ravi Selvaganapathy. “Raman spectroscopy for in-line water quality monitoring—Instrumentation and potential.” Sensors 14, no. 9 (2014): 17275-17303., Copyright 2014, with permission from open access MDPI.
An emerging method that has shown the most promise for identifying biological samples has been vibrational spectroscopy. Vibrational spectroscopy is a non-invasive analytical technique that evaluates the vibrational energy of a molecule upon excitation [3]. It is utilised to identify compounds by their distinct vibrational energy levels, which refl ect the specifi c bonding patterns within the molecule. The two principal forms of vibrational spectroscopy are Raman spectroscopy and Infrared (IR) spectroscopy, with Raman spectroscopy having the most success. This method works by illuminating a sample with a monochromatic laser light. Most of the incident photons are scattered elastically by the sample, meaning they do not change their. However, a small fraction of photons is scattered inelastically, meaning they lose or gain energy in the process. These photons are referred to as Raman scattered photons. The energy changes or Raman shifts of the scattered photons correspond to the vibrational energy levels of the molecules in the sample. By analysing the energy shift of the Raman scattered photons compared to the incident photons, information about the vibrational modes of the sample can be obtained [3].
Work conducted by the Lednev laboratory at the University at Albany, SUNY has developed and validated many aspects of the novel methodology. Specifi cally, an automatic statistical model for the identifi cation of all main body fl uids including blood, semen, vaginal fl uid, sweat, saliva and urine was created [4-6]. The model provides 100% accuracy of the identifi cation when the stain is found on a noninterfering substrate. A primary concern in forensic science is being able to analyse samples that are on substrates that interfere with analysis [7]. In response, a statistical approach, which allows for ‘ignoring’ the substrate interference was developed. The latter approach has been validated for fresh (10-hour old) blood and semen stains on various substrates, which exhibited strong Raman and fl uorescence emission overwhelming the signal from semen [8, 9]. In addition, it was shown that Raman spectroscopy allows for differentiating peripheral and menstrual blood [10], human and animal blood [11], and offers information about the donor phenotype profi le including sex, race, and age [12-14] and time since deposition of bloodstains [15-17]. Despite this success, more investigation is required before the proposed methodology can be utilised in practical forensic applications. This comes in the form of SupreMEtric commercialising the discussed technology for forensic scientists.
Previous research has established that these signatures are unique and distinguishable, allowing for identifi cation of all main bodily fl uids. To advance that work, SupreMEtric is developing the fi rst universal method for non-destructive, confi rmatory identifi cation of bodily fl uids in trace biological stains revealed at a crime scene, overcoming the limitations of standard presumptive and confi rmatory biochemical tests. With this technology, crime scene investigators can collect samples from a crime scene under investigation using standard approaches already in use; once in the laboratory, samples are analysed a patented digital method used for determining spectroscopic signatures developed for each body fl uid type. SupreMEtric’s advanced, proprietary algorithm recognises trace signals on interfering substrates, and can confi rm the presence or absence of each body fl uid type in the sample, without altering or destroying the evidence.
INTERNATIONAL LABMATE - JULY 2023
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