Body Fluid Identification Research Articles

Development of mRNA-based body fluid identification using reverse transcription loop-mediated isothermal amplification.

Identifying body fluids from forensic samples can provide valuable evidence for criminal investigations. Messenger RNA (mRNA)-based body fluid identification was recently developed, and highly sensitive parallel identification using reverse transcription polymerase chain reaction (RT-PCR) has been described. In this study, we developed reverse transcription loop-mediated isothermal amplification (RT-LAMP) as a simple, rapid assay for identifying three common forensic body fluids, namely blood, semen, and saliva, and evaluated its specificity and sensitivity. Hemoglobin beta (HBB), transglutaminase 4 (TGM4), and statherin (STATH) were selected as marker genes for blood, semen, and saliva, respectively. RT-LAMP could be performed in a single step including both reverse transcription and DNA amplification under an isothermal condition within 60min, and detection could be conveniently performed via visual fluorescence. Marker-specific amplification was performed in each assay, and no cross-reaction was observed among five representative forensically relevant body fluids. The detection limits of the assays were 0.3nL, 30nL, and 0.3μL for blood, semen, and saliva, respectively, and their sensitivities were comparable with those of RT-PCR. Furthermore, RT-LAMP assays were applicable to forensic casework samples. It is considered that RT-LAMP is useful for body fluid identification.

Messenger RNA biomarker signatures for forensic body fluid identification revealed by targeted RNA sequencing

The recovery of a DNA profile from the perpetrator or victim in criminal investigations can provide valuable ‘source level’ information for investigators. However, a DNA profile does not reveal the circumstances by which biological material was transferred. Some contextual information can be obtained by a determination of the tissue or fluid source of origin of the biological material as it is potentially indicative of some behavioral activity on behalf of the individual that resulted in its transfer from the body. Here, we sought to improve upon established RNA based methods for body fluid identification by developing a targeted multiplexed next generation mRNA sequencing assay comprising a panel of approximately equal sized gene amplicons. The multiplexed biomarker panel includes several highly specific gene targets with the necessary specificity to definitively identify most forensically relevant biological fluids and tissues (blood, semen, saliva, vaginal secretions, menstrual blood and skin). In developing the biomarker panel we evaluated 66 gene targets, with a progressive iteration of testing target combinations that exhibited optimal sensitivity and specificity using a training set of forensically relevant body fluid samples. The current assay comprises 33 targets: 6 blood, 6 semen, 6 saliva, 4 vaginal secretions, 5 menstrual blood and 6 skin markers. We demonstrate the sensitivity and specificity of the assay and the ability to identify body fluids in single source and admixed stains. A 16 sample blind test was carried out by one lab with samples provided by the other participating lab. The blinded lab correctly identified the body fluids present in 15 of the samples with the major component identified in the 16th. Various classification methods are being investigated to permit inference of the body fluid/tissue in dried physiological stains. These include the percentage of reads in a sample that are due to each of the 6 tissues/body fluids tested and inter-sample differential gene expression revealed by agglomerative hierarchical clustering.

Enhanced sensitivity of CpG island search and primer design based on predicted CpG island position

DNA methylation has important biological roles, such as gene expression regulation, as well as practical applications in forensics, such as in body fluid identification and age estimation. DNA methylation often occurs in the CpG site, and methylation within the CpG islands affects various cellular functions and is related to tissue-specific identification. Several programs have been developed to identify CpG islands; however, the size, location, and number of predicted CpG islands are not identical due to different search algorithms. In addition, they only provide structural information for predicted CpG islands without experimental information, such as primer design. We developed an analysis pipeline package, CpGPNP, to integrate CpG island prediction and primer design. CpGPNP predicts CpG islands more accurately and sensitively than other programs, and designs primers easily based on the predicted CpG island locations. The primer design function included standard, bisulfite, and methylation-specific PCR to identify the methylation of particular CpG sites. In this study, we performed CpG island prediction on all chromosomes and compared CpG island search performance of CpGPNP with other CpG island prediction programs. In addition, we compared the position of primers designed for a specific region within the predicted CpG island using other bisulfite PCR primer programs. The primers designed by CpGPNP were used to experimentally verify the amplification of the target region of markers for body fluid identification and age estimation. CpGPNP is freely available at http://forensicdna.kr/cpgpnp/.

Body fluid identification using a targeted mRNA massively parallel sequencing approach – results of a EUROFORGEN/EDNAP collaborative exercise

In a previous study we presented an assay for targeted mRNA sequencing for the identification of human body fluids, optimised for the Illumina MiSeq/FGx MPS platform. This assay, together with an additional in-house designed assay for the Ion Torrent PGM/S5 platform, was the basis for a collaborative exercise within 17 EUROFORGEN and EDNAP laboratories, in order to test the efficacy of targeted mRNA sequencing to identify body fluids. The task was to analyse the supplied dried body fluid stains and, optionally, participants’ own bona fide or mock casework samples of human origin, according to specified protocols. The provided primer pools for the Illumina MiSeq/FGx and the Ion Torrent PGM/S5 platforms included 33 and 29 body fluid specific targets, respectively, to identify blood, saliva, semen, vaginal secretion, menstrual blood and skin. The results demonstrated moderate to high count values in the body fluid or tissue of interest with little to no counts in non-target body fluids. There was some inter-laboratory variability in read counts, but overall the results of the laboratories were comparable in that highly expressed markers showed high read counts and less expressed markers showed lower counts. We performed a partial least squares (PLS) analysis on the data, where blood, menstrual blood, saliva and semen markers and samples clustered well. The results of this collaborative mRNA massively parallel sequencing (MPS) exercise support targeted mRNA sequencing as a reliable body fluid identification method that could be added to the repertoire of forensic MPS panels.

Predicting the origin of stains from next generation sequencing mRNA data

We used our previously published NGS mRNA approach for body fluid identification to analyse 183 body fluids/tissues, including mock casework samples. The resulting data set was used to build a probabilistic model that predicts the origin of a stain. Our approach uses partial least squares followed by linear discriminant analysis to classify samples into six commonly occurring forensic body fluids. The model differs from the ones previously suggested in that it incorporates quantitative information (NGS read counts) rather than just presence/absence of markers. The suggested approach also allows for visualisation of important markers and their correlation with the different body fluids. We compared our model to previously published methods to show that the inclusion of read count information improves the prediction. Finally, we applied the model to mixed body fluid samples to test its ability to identify the individual components in a mixture.

Semen-specific miRNAs: Suitable for the distinction of infertile semen in the body fluid identification?

Non-protein coding RNA, miRNAs (microRNAs), are a class of promising molecular biomarkers for forensic body fluid identification (BFI) as their small size and tissue-specific expression manners. A number of studies have shown that semen can be distinguished from forensic-related body fluids (such as menstrual blood, venous blood, vaginal fluid, saliva, etc.) using semen-specific miRNAs through microassay screening and RT-qRCR. Infertility is becoming a global health problem, affecting 10%-15% of couples worldwide, and half of the cases are the result of male factors (Lian et al., 2009 [1]). Forensic researchers have to consider the impact of semen infertility on semen identification with a high incidence of infertility. In the present study, normal semen (NS) and four other types of infertile semen samples, including asthenospermia (AS), oligospermia (OS), azoospermia (AZ), oligospermia and asthenospermia (OSAS) semen, were collected. The expression levels of a set of semen-specific miRNA markers (miR-10a, miR-10b, miR-135a, miR-135b, miR-888 and miR-891a) were evaluated using a real-time quantitative PCR technique with a specific fluorescence-labelled TaqMan probe. The results showed the significantly high expression of these miRNAs in normal semen, and the molecules have semen specificity. Nevertheless, a distinct down-regulation in the expression of infertile samples compared with normal semen samples was observed. Moreover, differences in the results of selected optimal biomarkers between the discriminant function and two-dimensional scatter plots were also detected. The goal of the present study was to identify a small set of semen-specific miRNAs that efficiently and accurately distinguish semen (fertile and infertile) from other forensic-related body fluids. The results of the present study suggest that attention should be paid to infertile semen samples when using miRNAs to identify semen samples, for which would have a far-reaching impact on forensic identification.

DNA Methylation as a Biomarker for Body Fluid Identification

Currently, available identification techniques for forensic samples are either enzyme or protein based, which can be subjected to degradation, thus limiting its storage potentials. Epigenetic changes arising due to DNA methylation and histone acetylation can be used for body fluid identification. Markers DACT1, USP49, ZC3H12D, FGF7, cg23521140, cg17610929, chromosome 4 (25287119–25287254), chromosome 11 (72085678–72085798, 57171095–57171236, 1493401–1493538), and chromosome 19 (47395505–47395651) are currently being used for semen identification. Markers cg26107890, cg20691722, cg01774894 and cg14991487 are used to differentiate saliva and vaginal secretions from other body fluids. However, such markers show overlapping methylation pattern. This review article aimed to highlight the feasibility of using DNA methylation of certain genetic markers in body fluid identification and its implications for forensic investigations. The reviewed articles have employed molecular genetics techniques such as Bisulfite sequencing PCR (BSP), methylation specific PCR (MSP), Pyrosequencing, Combined Bisulfite Restriction Analysis (COBRA), Methylation-sensitive Single Nucleotide Primer Extension (SNuPE), and Multiplex SNaPshot Microarray. Bioinformatics software such as MATLAB and BiQ Analyzer has been used. Biological fluids have different methylation patterns and thus, this difference can be used to identify the nature of the biological fluid found at the crime scene. Using DNA methylation to identify the body fluids gives accurate results without consumption of the trace evidence and requires a minute amount of DNA for analysis. Recent studies have incorporated next-generation sequencing aiming to find out more reliable markers that can differentiate between different body fluids. Nonetheless, new DNA methylation markers are yet to be discovered to accurately differentiate between saliva and vaginal secretions with high confidence. Epigenetic changes are dynamic and it is important to find stable DNA sequences that can be used as biomarkers. Keywords: Forensic Science; DNA analysis; Methylation; body fluid; identification; Pyrosequencing; DACT1; USP49; ZC3H12D; FGF7.

Forensic DNA methylation profiling from minimal traces: How low can we go?

Analysis of human DNA methylation (DNAm) can provide additional investigative leads in crime cases, e.g. the type of tissue or body fluid, the chronological age of an individual, and differentiation between identical twins. In contrast to the genetic profile, the DNAm level is not the same in every cell. At the single cell level, DNAm represents a binary event at a defined CpG site (methylated versus non-methylated). The DNAm level from a DNA extract however represents the average level of methylation of the CpG of interest of all molecules in the forensic sample. The variance of DNAm levels between replicates is often attributed to technological issues, i.e. degradation of DNA due to bisulfite treatment, preferential amplification of DNA, and amplification failure. On the other hand, we show that stochastic variations can lead to gross fluctuation in the analysis of methylation levels in samples with low DNA levels. This stochasticity in DNAm results is relevant since low DNA amounts (1pg – 1ng) is rather the norm than the exception when analyzing forensic DNA samples.This study describes a conceptual analysis of DNAm profiling and its dependence on the amount of input DNA. We took a close look at the variation of DNAm analysis due to DNA input and its consequences for different DNAm-based forensic applications.As can be expected, the 95%-confidence interval of measured DNAm becomes narrower with increasing amounts of DNA. We compared this aspect for two different DNAm-based forensic applications: body fluid identification and chronological age determination. Our study shows that DNA amount should be well considered when using DNAm for forensic applications.

A capillary electrophoresis method for identifying forensically relevant body fluids using miRNAs.

Body fluid identification (BFID) can provide crucial information during the course of an investigation. In recent years, microRNAs (miRNAs) have shown considerable body fluid specificity, are able to be co-extracted with DNA, and their small size (18-25 nucleotides) make them ideal for analyzing highly degraded forensic samples. In this study, we designed a preliminary 8-marker system for BFID including an endogenous reference gene (let-7g) to differentiate between venous blood (miR-451a and miR-142-3p), menstrual blood (miR-141-3p and miR-412-3p), semen (miR-891a and miR-10b), and saliva (miR-205) using a capillary electrophoresis approach. This panel uses a linear primer system in order to incorporate additional miRNA markers by forming a multiplex system. The miRNA system was able to distinguish between venous blood, menstrual blood, semen, and saliva using a rudimentary data interpretation strategy. All STR amplifications from co-extracted DNA yielded complete profiles from human identification purposes.

Improving body fluid identification in forensic trace evidence-construction of an immunochromatographic test array to rapidly detect up to five body fluids simultaneously.

Body fluid identification is a substantial part of forensic trace analyses. The correct determination of the origin of a biological stain may give valuable information regarding the circumstances of a crime. A simple way to detect a body fluid in a stain is the use of immunochromatographic strip tests. They are easy to use, user-independent, quick, and cheap. Currently, however, it is only possible to analyze one body fluid at a time, requiring the analyst to make previous, possibly subjective, assumptions on the body fluid at hand. Also, identification of mixed body fluids requires the use of several tests, which results in additional sample and time consumption. To combine a simple approach with the possibility to simultaneously detect several body fluids, we constructed a combined immunochromatographic strip test array based on commercially available tests. The array rapidly detects up to five body fluids with a single analysis, and allowing for subsequent DNA extraction from the same material. With this test it was possible to identify the components of a mixture, the test was easily incorporated into standard laboratory work, and its sensitivity and specificity were shown to be comparable to those of conventional strip tests.

Forensic differentiation between peripheral and menstrual blood in cases of alleged sexual assault\u2014validating an immunochromatographic multiplex assay for simultaneous detection of human hemoglobin and D-dimer

Sexual assault is a serious offense and identification of body fluids originating from sexual activity has been a crucial aspect of forensic investigations for a long time. While reliable tests for the detection of semen and saliva have been successfully implemented into forensic laboratories, the detection of other body fluids, such as vaginal or menstrual fluid, is more challenging. Especially, the discrimination between peripheral and menstrual blood can be highly relevant for police investigations because it provides potential evidence regarding the issue of consent. We report the forensic validation of an immunochromatographic test that allows for such discrimination in forensic stains, the SERATEC PMB test, and its performance on real casework samples. The PMB test is a duplex test combining human hemoglobin and D-dimer detection and was developed for the identification of blood and menstrual fluid, both at the crime scene and in the laboratory. The results of this study showed that the duplex D-dimer/hemoglobin assay reliably detects the presence of human hemoglobin and identifies samples containing menstrual fluid by detecting the presence of D-dimers. The method distinguished between menstrual and peripheral blood in a swab from a historical artifact and in real casework samples of alleged sexual assaults. Results show that the development of the new duplex test is a substantial progress towards analyzing and interpreting evidence from sexual assault cases.

An investigation into the use of the ParaDNA® body fluid identification system in forensic examinations

Examinations carried out in forensic laboratories currently utilise chemical screening tests and microscopic analysis to locate and identify body fluids. However, there are currently no reliable tests to positively identify vaginal material, to differentiate between venous and menstrual blood and to confirm the presence of azoospermic semen. Furthermore, for cases involving allegations of oral intercourse, the attribution of a positive amylase reaction to saliva (as opposed to vaginal secretions) can be problematic.LGC has developed a system that uses the ParaDNA system together with one-tube mRNA detection chemistry for body fluid identification. This can be utilized at the laboratory examination stage, prior to DNA testing, to positively identify body fluids. A series of mRNA markers were selected to positively identify saliva, vaginal secretions, peripheral blood, menstrual blood, seminal fluid and sperm cells. We have carried out an assessment of this system in an active casework environment using mock-case samples. To investigate the sensitivity and specificity of the mRNA markers, the system was tested both by direct sampling of the body fluid stain (using the ParaDNA sampling device), and using aliquots of the retained aqueous supernatant following cell-harvesting.This paper summarises the findings obtained from the initial experiments carried out, together with the results of a further investigation into the effect of mixed body fluid samples on the detection of the individual body fluids.

Comprehensive examination of conventional and innovative body fluid identification approaches and DNA profiling of laundered blood- and saliva-stained pieces of cloths.

Body fluids like blood and saliva are commonly encountered during investigations of high volume crimes like homicides. The identification of the cellular origin and the composition of the trace can link suspects or victims to a certain crime scene and provide a probative value for criminal investigations. To erase all traces from the crime scene, perpetrators often wash away their traces. Characteristically, items that show exposed stains like blood are commonly cleaned or laundered to free them from potential visible leftovers. Mostly, investigators do not delegate the DNA analysis of laundered items. However, some studies have already revealed that items can still be used for DNA analysis even after they have been laundered. Nonetheless, a systematical evaluation of laundered blood and saliva traces that provides a comparison of different established and newly developed methods for body fluid identification (BFI) is still missing. Herein, we present the results of a comprehensive study of laundered blood- and saliva-stained pieces of cloths that were applied to a broad range of methods for BFI including conventional approaches as well as molecular mRNA profiling. The study included the evaluation of cellular origin as well as DNA profiling of blood- and saliva-stained (synthetic fiber and cotton) pieces of cloths, which have been washed at various washing temperatures for one or multiple times. Our experiments demonstrate that, while STR profiling seems to be sufficiently sensitive for the individualization of laundered items, there is a lack of approaches for BFI with the same sensitivity and specificity allowing to characterize the cellular origin of challenging, particularly laundered, blood and saliva samples.

Evaluation of genetic markers for forensic identification of human body fluids

Identification of body fluids deposited at crime scenes can often become challenging due to their small size, poor visibility to the naked eye and the effect of environmental insults. Traditionally, body fluids are identified using techniques that are enzyme-based, non-specific, require large amount of samples, prone to contaminations and not available for all body fluids. These techniques are presumptive and mostly require confirmatory tests. New techniques of body fluid identification have emerged over the last decade and are still on the rise. In this study, highly specific genetic markers (mRNA) were used to identify four commonly encountered forensic body fluids which include blood, semen, saliva and vaginal secretion. A 14-marker mRNA multiplex based assay was developed and validated. All markers were expressed in their target body fluids with no cross reactions in non-target body fluids. The unambiguous identification of the four body fluids with this assay further established its compatibility with forensic laboratory workflows.

Evaluation of the inclusion of circular RNAs in mRNA profiling in forensic body fluid identification.

The use of messenger RNA (mRNA) profiling is considered a promising method in the identification of forensically relevant body fluids which can provide crucial information for reconstructing a potential crime. However, casework samples are usually of limited quantity or have been subjected to degradation, which requires improvement of body fluid identification. Circular RNAs (circRNAs), a class of products from the backsplicing of pre-mRNAs, are shown to have high abundance, remarkable stability, and cell type-specific expression in human cells. In this study, we investigated whether the inclusion of circRNAs in mRNA profiling improve the detection of biomarkers including δ-aminolevulinate synthase 2 (ALAS2) and matrix metallopeptidase 7 (MMP7) in body fluid identification. The major circRNAs of ALAS2 and MMP7 were first identified and primer sets for the simultaneous detection of linear and circular transcripts were developed. The inclusion of circRNAs in mRNA profiling showed improved detection sensitivity and stability of biomarkers revealed by using serial dilutions, mixed samples, and menstrual bloodstains as well as degraded and aged samples. Therefore, the inclusion of circRNAs in mRNA profiling should facilitate the detection of mRNA markers in forensic body fluid identification.

Novel messenger RNAs for body fluid identification

In forensic investigations, the identification of the cellular or body fluid source of biological evidence can provide crucial probative information for the court. Messenger RNA (mRNA) profiling has become a valuable tool for body fluid and cell type identification due to its high sensitivity and compatibility with DNA analysis. However, using a single marker to determine the somatic origin of a sample can lead to misinterpretation as a result of cross-reactions. While false positives may be avoided through the simultaneous detection of multiple markers per body fluid, this approach is currently limited by the small number of known differentially expressed mRNAs.Here we characterise six novel mRNAs, partly identified from RNA-Seq, which can supplement existing markers for the detection of circulatory blood, semen (with and without spermatozoa), and menstrual fluid: HBD and SLC4A1 for blood, TNP1 for spermatozoa, KLK2 for seminal fluid, and MMP3 and STC1 for menstrual fluid. Respective expression profiles were evaluated by singleplex endpoint reverse transcription polymerase chain reaction (RT-PCR). HBD, SLC4A1, and KLK2 were specific to their target body fluids. TNP1, MMP3, and STC1 each cross-reacted with two non-target samples; however, these signals were below 350RFU, not reproducible, and likely resulted from large body fluid inputs. All candidates were more sensitive for the detection of their target body fluids than corresponding well-known mRNAs, in particular those for menstrual fluid. The increased sensitivities were statistically significant, except for KLK2. Thus, the new mRNAs introduced here are promising new targets for improved body fluid profiling.

Effect of the absence of spermatozoa on microrna-based semen identification

MicroRNA-based body fluid identification systems can play a substantial role in forensic casework. Although semen is one of the most important stains in cases of sexual assault, the effect of the absence of spermatozoa in the semen on the efficacy of microRNA-based body fluid identification has not been investigated. In this study, we aimed to investigate the influence of the absence of spermatozoa by determining the expression of candidate microRNAs for in forensically relevant body fluids using RT-qPCR. The expression levels of microRNAs relevant in semen, venous blood, saliva, and vaginal secretion were not significantly different between semen samples with normospermia and azoospermia. We concluded that the absence of spermatozoa did not influence microRNA-based body fluid identification. Therefore, microRNAs can be useful markers in sexual assaults cases, even if the perpetrator is infertile.

Reproducibility of methylated CpG typing with the Illumina MiSeq

DNA methylation patterns may be used for identification of body fluids and for age estimation of human individuals. We evaluated some of the challenges and pitfalls of studying methylated CpG sites. We compared the methylated CpG analysis of two different methods 1) massively parallel sequencing (MPS) using the Illumina MiSeq and 2) the iPLEX assay on the MassARRAY® System. On the Illumina MiSeq, the standard deviation of the fraction of methylation was under 3% between replicates, whereas the reproducibility of the MassARRAY® was very difficult to achieve. We tested the denaturation and conversion times of the Premium Bisulfite kit (Diagenode) in order to optimise the conversion rate and minimise DNA degradation. Finally, we tested the reproducibility of the methylation patterns using the Illumina MiSeq.

Extended specificity studies of mRNA assays used to infer human organ tissues and body fluids.

Messenger RNA (mRNA) profiling is a technique increasingly applied for the forensic identification of body fluids and skin. More recently, an mRNA-based organ typing assay was developed which allows for the inference of brain, lung, liver, skeletal muscle, heart, kidney, and skin tissue. When applying this organ typing system in forensic casework for the presence of animal, rather than human, tissue is an alternative scenario to be proposed, for instance that bullets carry cell material from a hunting event. Even though mRNA profiling systems are commonly in silico designed to be primate specific, physical testing against other animal species is generally limited. In this study, human specificity of the organ tissue inferring system was assessed against organ tissue RNAs of various animals. Results confirm human specificity of the system, especially when utilizing interpretation rules considering multiple markers per cell type. Besides, we cross-tested our organ and body fluid mRNA assays against the target types covered by the other assay. Marker expression in the nontarget organ tissues and body fluids was observed to a limited extent, which emphasizes the importance of involving the case-specific context of the forensic samples in deciding which mRNA profiling assay to use and when for interpreting results.

Expression difference of miR-10b and miR-135b between the fertile and infertile semen samples (p)

Non-protein coding RNAs, miRNAs (microRNAs), are a class of promising molecular biomarkers for forensic body fluid identification as the very small size and their tissue-specific expression manners. A number of studies have showed that semen can be distinguished from forensic related body fluids by the semen-specific miRNAs through microassay screening and RT-RCR. Infertility is becoming a global health problem affecting 10%–15% of couples worldwide and half of the cases are due to male factors. So high incidence of infertility forced the forensic researchers to consider the impact of the semen infertility on the semen identification. Hence, several kinds of semen from man infertility were collected into our research such as asthenospermia (AS) and azoospermia (AZ). The expression level of semen samples (fertile and infertile) and other common forensic body fluids were evaluated using two miRNA markers (miR-10b and miR-135b) by RT-PCR with TaqMan probe. Results showed that the two miRNAs expressed significantly higher in normal semen (NS) than other body fluids. Nevertheless, there was a distinct down-regulation in the expression of AS samples. The goal of our study was to ascertain whether the two miRNAs could distinguish semen (fertile and infertile) efficiently and accurately. This study reminded forensic workers should pay attention to samples in pathology when identifying body fluids, which would have a far-reaching significance for forensic application.