Examination and identification with current tests are prone to false-positives, do not test for all fluids simultaneously and can be destructive to the sample. Further, distinguishing the location or a more specific identity of the body fluid such as menstrual blood or venous blood, can give probative value for the case at hand (Fleming and Harbison, 2010). Recently, other possible methods of identifying body fluids have been researched in order to resolve these issues, while maintaining the ideals of examination testing. Two methods analyzed for improvements to body fluid identification involve Raman Spectroscopy and mRNA Profiling. Analyzing the strengths and limitations of these new methods in comparison to ones that are current can help forensic biologists make identification of body fluids even more efficient.
Raman Spectroscopy, a common analytical instrument in forensic analysis, has been investigated as a possible presumptive and confirmation method for body fluids. Since little to no sample preparation is needed, it’s non-destructive to the sample, has a portable form, and possesses high sensitivity, these advantages support its potential to identify body fluids.
A series of proof of concept experiments were initially performed on a variety of body fluids including blood, semen, vaginal fluid, and sweat to identify whether these samples can be distinguished using Near-Infrared Raman Spectroscopy. Body fluid differentiation was performed using the following markers: fibrin in dried blood, albumin and acid phosphatase for semen, and lactic acid and uric acid for sweat (Virkler & Ledev, 2009), (Virkler & Ledev, 2010), (Virkler & Ledev, 2010). In addition, amino acids and other components, proved to be useful in comparison despite their heterogeneous nature of each fluid. Then linear statistical models were used to determine the validity of these markers. These experiments combined served as a great indicator of the future utilizing of this method for body fluid identification.
Experimentation using Raman mapping of neat body fluid samples was performed before employing Raman Spectroscopy by researchers at the University of Albany. Raman mapping operates by scanning multiple areas of the sample and allows for intra-sample variation due to the heterogeneous nature of the fluids and biochemical composition between individuals (Muro et al., 2016). The spectra obtained were combined into a dataset and used as a standard for the particular body fluids examined and analyzed using various statistical software. Validation was performed to ensure the rate of accuracy and the effectiveness of the calibration. Internally, the cross-validation was 97.7% and externally performed at 100% accuracy (Muro et al., 2016). This experiment showed great promise, however, 16 spectra were misclassified, which would have major implications in reporting casework samples. Also, although the sample is preserved throughout the experiment, casework samples are often found in diluted concentrations on various porous and non-porous substrates. Additionally, mixtures of different body fluids and samples containing multiple profiles can be seen as well. This may present a problem with using Raman Spectroscopy. A study with a larger sample size would also need to be performed to further test the validity of this experiment, since 75 samples were used across all five body fluids analyzed.
Research performed by Boyd et al., tested diluted blood samples to identify the sensitivity of Raman Spectroscopy. The dilutions were planted onto fabrics and reconstituted for examination. This experiment showed that dilutions of 1:250 were detected, while greater dilutions resulted in weak bands (Boyd et al., 2011). Therefore, Raman Spectroscopy has comparable sensitivity to current screening methods such as phenolphthalein and leucomalachite green (Boyd et al., 2011). Although reconstitution can be destructive, current methods of human blood confirmation testing also use similar forms of this method, such as ABA card Hematrace. This allows the possibility of using the same fabric sample for both presumptive and confirmatory testing, lessening the amount of damage to the evidence and promoting efficiency in completing casework samples.
Researchers also investigated if Raman Spectroscopy could be used to differentiate between human and animal blood samples. Calibration was initially conducted using Raman mapping as well and averaged for each of the species tested, then standard deviation was used to confirm that this calibration procedure could be used for the experimental portion of the research. In performing a blind test, all samples were identified correctly as being human or non-human blood. However, the researchers identified that gorillas and chickens have blood biochemistry similar to humans and has the possibility of yielding false positives (McLaughlin et al., 2014). This strategy provides potential in casework to discriminate between human or animal blood samples before sending them for DNA analysis. Also, this method allows the possibility to do presumptive and confirmatory analysis simulatenously.
Great progress has been made in utilizing Raman Spectroscopy as a possible method for both presumptive and confirmatory testing, possibly eliminating current examination of two-test method. However, there are a plethora of questions and limitations to the experiments performed. Specifically, detecting dilute amounts of body fluids on various substrates should be investigated. Additionally, Raman mapping, although only used for calibration, and Benchtop Raman Spectroscopy can only be performed in a laboratory setting. Also, the statistical analyses performed on these experiments were cumbersome and tedious, limiting the efficiency of completing sample examination. To resolve some of the limitations faced, portable Raman can be utilized in the field and can provide a nondestructive and highly sensitive means of identifying the particular body fluid in a sample.
Since gene expression patterns are tissue specific at a certain point in time, mRNA profiling was considered a possible means of body fluid identification (Vennemann and Koppelkamm, 2010). Although mRNA was previously perceived to be unstable and subject to degradation, research has found that it is much more durable (Zhao et al., 2017) (Fleming and Harbison, 2010). Implementing this methodology would allow for the co-extraction of a of RNA to identify the body fluid present, as well as, generate a DNA profile from the same stain, providing great efficiency without destroying the sample for identification and individualization.
Researchers from the Institute of Environmental Science and Research Ltd. in New Zealand, developed a multiplex RT-PCR assay using specific genetic markers to identify saliva, semen, seminal fluid, menstrual blood and venous blood. The cross-specificity of using these markers were also analyzed. The sensitivity was also determined to be 1 microliter however, a minimum amount for menstrual blood and saliva was not able to be determined since these were collected on swabs (Fleming and Harbison, 2010). The researchers also explored mixtures of multiple profiles within one sample. Due to the intra-variability of nucleated cells in body fluids across individuals, it was difficult to determine the major and minor profiles (Fleming and Harbison, 2010).
A multiplex RT-PCR assay with specific genetic markers was also compiled by researchers in Korea in the hopes of developing a more specific and efficient combination of the genetic markers to use for m-RNA profiling. Although different m-RNA markers were used to identify the body fluids than the experiment performed in New Zealand, validation of the assay and STR profiles were processed to show how these assays can be used to gain quality profiles for individualization. After determining the markers by using gene expression profiling, validation was performed by using RT-PCR and qRT-PCR. Contrary to Fleming and Harbison in New Zealand, the researchers observed degradation and poor quality of the RNA samples. However, by using a kit to repair damaged RNA samples, enough cRNA was produced to continue the examination (Park et al.,2013). Additionally, disagreement in the markers among the body fluid samples was seen. For example, in some of the samples the expected markers were not found, while in other cases a marker of a different body fluid was observed (Park et al.,2013). In this experiment, the sample sizes used for each of the body fluids were small and inconsistent. This could have led to the discrepancies observed. Additionally, this experiment utilized neat swabs and aliquots of the body fluid which does not highlight the reality of working with diluted quantities on various substrates.
A similar study analyzed 32 genetic markers and validated the detection of specific body fluids in accordance with the SWGDAM guidelines. The samples collected were exposed to environmental conditions for 60 days before testing. The researchers determined that 14 markers showed the most promise in identifying a specific body fluid, which were developed into an assay and validated. Despite the possibility of false negatives occurring with degraded mRNA samples, the assay was highly specific and sensitive in detecting body fluids with as low as a 1:3000 dilution and 1:5000 dilution for vaginal secretions (Afolabi et al., 2017). However, this study and the one performed by Park et al., used body fluids collected on swabs. Although dilute samples often seen in forensic casework, using this assay to detect body fluids across more porous and nonporous substrates, and differentiating mixtures of fluids or multiple profiles, would be helpful to understand the potential for crime scene samples.
In a study performed in China, m-RNA profiling was used to identify bloodstains on 30 and 50-year-old samples. The stains were extracted, exposed to RT-PCR, and quantified before employing capillary electrophoresis. Seven specific markers and housekeeping genes were used to identify the bloodstain. HBA and HBB, subunits of hemoglobin, were the most stable in the aged stains and recommended to be primary markers along with B2M (beta-2-microglobulin) as a housekeeping gene, and others to ensure sensitivity. The researchers found that this extends the detection window of HBB detection from 23-year-old samples to up to 50 years. (Zhao et al.,2017). From the extraction process used, full STR profiles were able to be obtained from the 30-year-old samples while a partial profile was obtained for the 50-year-old sample (Zhao et al., 2017). This can allow labs to reduce backlogs by utilizing this technique and develop a possible lead and identify the source of stains from cold cases. Although this research only experimented with bloodstains, it shows the possibility of applying similar procedures to other body fluids in cold cases and other degraded samples.
m-RNA profiling has great potential to be used for identification of body fluid since it can differentiate based on tissue specific gene expression and provide STR profiles using the same sample. Utilizing this method also shows the possibility for confirmation testing in conjunction with the current screening tests (Fleming and Harbison, 2010). In order to solidify that this method can be relied on for crime scene samples, research with larger sample sizes, extractions from various substrates, and working with mixtures must be analyzed. Further, providing guidelines that can aid forensic biologists with interpreting mixtures, the number of genetic and housekeeping markers and needed per body fluid, would be ideal (Afolabi et al., 2017).
Establishing robust methods that are clear, specific and sensitive, are necessary in determination the most likely sequence of events (Jakubowska et al., 2013). Current methods are limited by false-positives, specificity to the presence of one type of body fluid, and can be destructive to the sample. Therefore, both Raman Spectroscopy and m-RNA profiling are great prospective methodologies that can resolve these issues or be used in conjunction with current methods in testing for all body fluids simultaneously and using the same sample for identification and individualization. Integration of these methods can greatly promote efficiency with casework and reduce false-positives in experimentation.