Serological Biomarkers in Forensic Science

The information gathered from crime scenes have the unique ability to direct the course of forensic investigations. In terms of criminal cases, the correct categorisation of human body fluids can provide significant leads e.g., alleged sexual offences can be supported by the presence of vaginal fluid and semen, and signs of probable physical struggle can be inferred by blood at the crime scene. Methods for body fluid identification were initially developed using catalytic, enzymatic, chromatographic, and immunological procedures. Conventional methods, however, do not facilitate multiplexing and are not deoxyribonucleic acid (DNA) based. Tissue-specific differentially methylated regions/sites (tDMRs/tDMSs) are locations/sites on the human genome which display differential methylation patterns in tissues or cells, thus, enabling their use in body fluid identification. Techniques evaluating differential methylation in tDMRs have been particularly beneficial as only extracted DNA is processed, conserving additional physical evidence. Other benefits of DNA methylation-based methods include efficiency and multiplexing to evaluate multiple tissues in a single assay, therefore, is time-saving. The present study aimed to develop two DNA methylation-based assays: methylation-specific polymerase chain reaction (MSP) and methylation SNaPshot, in order to efficiently identify four different body fluids viz. blood, saliva, semen, and vaginal fluid, simultaneously and in a mixture. Methylation profiling of novel DNA methylation markers was carried out by a qualitative MSP assay. The in-house developed ZNF282 and HPCAL1 gene-based tDMRs were employed for analysis of semen and saliva, respectively. The novel MSP primers were designed to target CpG islands in genes ZNF282 and HPCAL1. Two previously reported tDMS markers for blood (cg08792630; Park et al., 2014) and vaginal fluid (cg09765089-231d; Lee et al., 2015) were modified, to design MSP primers which targeted CpG sites flanking the reported sites for blood and vaginal fluid. MSP analysis showed that saliva, semen, and vaginal fluid were correctly identified and differentiated from one another by HPCAL1, ZNF282 and cg09765089-231d markers, respectively. The complete unmethylation of HPCAL1 and ZNF282 in saliva and semen, respectively; and complete methylation in non-target body fluids demonstrated the immense potential both markers have for forensic application. Vaginal fluid marker revealed complete methylation in vaginal fluid. MSP analysis of the blood-specific MSP marker indicated that blood could neither be efficiently identified, nor differentiated from other body fluids of the study, due to the presence of both methylation and unmethylation. A total of four SNaPshot primers were designed based on MSP amplicons, to target a single site which showed differential methylation between the body fluids. All SNaPshot markers were able to identify and differentiate their respective body fluids from others in SNapShot simplex reactions, by generating green (unmethylated) or blue (methylated) peaks only in target body fluids. Excluding the saliva marker, all others displayed high levels of specificity in the multiplex SNaPshot reaction. The clear observation of either complete methylation or complete unmethylation, depicted the high specificity of the markers for criminal investigations. However, obtaining single-body fluid samples is a luxury that crime scenes do not afford. Most evidence is severely degraded, available in low quantities, and present in the form of mixtures. To assess the sensitivity of the designed multiplex MSP-SNaPshot assay, an assortment of body fluid mixtures in varying ratios were subjected to the MSP-SNaPshot assay. The high sensitivity of the semen and saliva marker was validated by the unambiguous identification of semen and saliva, even when target body fluids were minor constituents of the mixture. Vaginal fluid could not be identified when present in lower concentrations. The newly designed blood marker showed significant blood-methylation specificity and sensitivity by efficiently identifying the body fluid in low quantities. The present study reports novel MSP and SNaPshot markers for the identification of blood, saliva, semen, and vaginal fluid in single samples as well as mixtures. Future research would involve the use of the described markers, under different forensic conditions which will enhance their applicability in forensic analysis.

Detection,discrimination and aging of human tears stains using ATR-FTIR spectroscopy for forensic purposes

Analysis of body fluids for forensic purposes: From laboratory testing to non-destructive rapid confirmatory identification at a crime scene

Raman spectroscopy for forensic semen identification: Method validation vs. environmental interferences

Identification of five types of forensic body fluids based on stepwise discriminant analysis

The detection and discrimination of human body fluids using ATR FT-IR spectroscopy

Evaluation of genetic markers for forensic identification of human body fluids

Forensic sciences are a multidisciplinary science that covers all branches of science for justice. Forensic genetics is one of the sub-branches of forensic sciences. It is a branch of science that contributes to establishing a connection between crime and criminal by using all kinds of biological evidence (blood, semen, saliva, urine, vaginal fluid, hair, hair, sweat, saliva, etc.) and reaching the perpetrator with the genetic information obtained. Serological and immunological tests are used in the identification of body fluids found as evidence at the crime scene. The number of serological tests used in the past and today is limited; not all body fluids can be analyzed routinely. Many of the existing tests are not directly specific to the body fluid but are intended to identify intermediate metabolites. One such example is the urine sample. In the identification of urine, the presence of creatinine and amines in the urine is often used. In addition, these tests require the expenditure of separate consumables to identify each body fluid. For this reason, with the development of technology, in recent years, the use of spectroscopic methods that provide cost-free and easy analysis in the identification of body fluids has increased. Fourier Transform Infrared (FTIR) spectroscopy is one of the spectroscopic methods used to identify body fluids from biological samples found at the crime scene without loss of quantity and without damaging the sample, quickly and without loss of consumables. This study investigated the identification of urine stains obtained from the crime scene with FTIR spectroscopy without using serological materials. Functional groups, which we can express as identification regions, were determined as 13 different distinguishing points in the relevant molecules and their usability in forensic serological analyses was determined.

Corrigendum to ‘The detection and discrimination of human body fluids using ATR FT-IR spectroscopy’ [Forensic Sci. Int. 252 (2015) e10–e16

Vaginal fluid is one of the most common body fluids found at crime scenes. Discriminating vaginal fluid from other body fluids is important in forensic science; however, few potential protein markers have been reported to date. Proteomic methods for identifying protein markers have gained attention, although few reports have applied this technology to forensic protein markers. Therefore, to identify characteristic vaginal proteins, we examined various body fluids (nasal secretions, saliva, urine, semen, vaginal fluids, and sweat) using liquid chromatography/electrospray ionization time-of-flight mass spectrometry and peptide mass fingerprinting. We identified three components (average molecular mass values 17,237 ± 2, 18,063 ± 2, and 15,075 ± 1) detectable only in vaginal samples: two human small proline-rich protein 3 (SPRR3) isoforms and a human fatty acid-binding protein 5 (FABP5) with an acetylated (+42) N-terminal region lacking the initiator methionine residue (-131). Using ELISA, these yielded markedly high average values in vaginal fluids. The mass spectra of these proteins were not detected in infant saliva but were detected in the vaginal fluid throughout the menstrual cycle. The results of forensic analysis (detection limit, mixed body fluid samples, casework samples, and blind samples) suggest that these proteins are potential forensic markers. In conclusion, high SPRR3 and FABP5 expression levels, which may be used as potential markers for vaginal fluid identification in forensic science, were detected in vaginal fluids from healthy adults.

Identification and the time since deposition (TsD) estimation of body fluid stains from a crime scene could provide valuable information for solving the cases and are always difficult for forensics. Microbial characteristics were considered as a promising biomarker to address the issues. However, changes in the microbiota may damage the specific characteristics of body fluids. Correspondingly, incorrect body fluid identification may result in inaccurate TsD estimation. The mutual influence is not well understood and limited the codetection. In the current study, saliva, semen, vaginal secretion, and menstrual blood samples were exposed to indoor conditions and collected at eight time points (from fresh to 30 days). High-throughput sequencing based on the 16S rRNA gene was performed to characterize the microbial communities. The results showed that a longer TsD could decrease the discrimination of different body fluid stains. However, the accuracies of identification still reached a quite high value even without knowing the TsD. Correspondingly, the mean absolute error (MAE) of TsD estimation significantly increased without distinguishing the types of body fluids. The predictive TsD of menstrual blood reached a quite low MAE (1.54 ± 0.39 d). In comparison, those of saliva (6.57 ± 1.17 d), semen (6.48 ± 1.33 d), and vaginal secretion (5.35 ± 1.11 d) needed to be further improved. The great effect of individual differences on these stains limited the TsD estimation accuracy. Overall, microbial characteristics allow for codetection of body fluid identification and TsD estimation, and body fluids should be identified before estimating TsD in microbiome-based stain analyses.IMPORTANCEEmerged evidences suggest microbial characteristics could be considered a promising tool for identification and time since deposition (TsD) estimation of body fluid stains. However, the two issues should be studied together due to a potential mutual influence. The current study provides the first evidence to understand the mutual influence and determines an optimal process for codetection of identification and TsD estimation for unknown stains for forensics. In addition, we involved aged stains into our study for identification of body fluid stains, rather than only using fresh stains like previous studies. This increased the predictive accuracy. We have preliminary verified that individual differences in microbiotas limited the predictive accuracy of TsD estimation for saliva, semen, and vaginal secretion. Microbial characteristics could provide an accurate TsD estimation for menstrual blood. Our study benefits the comprehensive understanding of microbiome-based stain analyses as an essential addition to previous studies.

The identification of biological fluids at crime scenes contributes to crime scene reconstruction and provides investigative leads. Traditional methods for body fluid identification are limited in terms of sensitivity and are mostly presumptive. Emerging methods based on mRNA and DNA methylation require high quality template source. An exploitable characteristic of body fluids is their distinct microbial profiles allowing for the discrimination of body fluids based on microbiome content. Microbial DNA is highly abundant within the body, robust and stable and can persist in the environment long after human DNA has degraded. 16S rRNA sequencing is the gold standard for microbial analysis; however, NGS is costly, and requires intricate workflows and interpretation. Also, species level resolution is not always achievable. Based on the current challenges, the first objective of this study was to develop a multiplex conventional PCR assay to identify vaginal fluid and saliva by targeting species-specific 16S rRNA microbial markers. The second objective was to employ droplet digital PCR (ddPCR) as a novel approach to quantify bacterial species alone and in a mixture of body fluids. Lactobacillus crispatus and Streptococcus salivarius were selected because of high abundance within vaginal fluid and saliva respectively. While Fusobacterium nucleatum and Gardnerella vaginalis, though present in healthy humans, are also frequently found in oral and vaginal infections, respectively. The multiplex PCR assay detected L. crispatus and G. vaginalis in vaginal fluid while F. nucleatum and S. salivarius was detected in saliva. Multiplex PCR detected F. nucleatum, S. salivarius and L. crispatus in mixed body fluid samples while, G. vaginalis was undetected in mixtures containing vaginal fluid. For samples exposed at room temperature for 65 days, L. crispatus and G. vaginalis were detected in vaginal swabs while only S. salivarius was detected in saliva swabs. The limit of detection was 0.06 copies/µl for F. nucleatum (2.5 ×10−9 ng/µl) and S. salivarius (2.5 ×10−6 ng/µl). L. crispatus and G. vaginalis had detection limits of 0.16 copies/µl (2.5 ×10−4 ng/µl) and 0.48 copies/µl (2.5 ×10−7 ng/µl). All 4 bacterial species were detected in mixtures and aged samples by ddPCR. No significant differences were observed in quantity of bacterial markers in saliva and vaginal fluid. The present research reports for the first time the combination of the above four bacterial markers for the detection of saliva and vaginal fluid and highlights the sensitivity of ddPCR for bacterial quantification in pure and mixed body fluids.

Environmental microbiota from substrate may interfere with microbiome-based identification of forensically relevant body fluids: A pilot study

Assigning forensic body fluids to DNA donors in mixed samples by targeted RNA/DNA deep seqeuncing of coding region SNPs using ion torrent technology

Quantitative differential analysis of tsRNAs for forensic body fluid identification: RT-qPCR-based discrimination derived from epithelial cell fluids screening.