Body Fluid Identification Research Articles

Applications of epigenetic methylation in body fluid identification, age determination and phenotyping

In this paper, four different applications are presented in forensic epigenetics using bisulfite modified PCR, HRM, and pyrosequencing. The first application involves the development of a multiplex amplification capable of simultaneous analysis of 4 different body fluid/cell types- blood, saliva, sperm, and vaginal epithelia. We have also developed a method for the detection of sperm high-resolution melt analysis (HRM). Two other applications involve a determination of suspect age and a biomarker for determining smoking status.

Validation of a streamlined RT-qPCR method for body fluid identification

Forensic DNA STR profiling provides information on the sub-source of DNA samples. However, other source information such as the type of body fluid from which a DNA profile is derived is required to better attribute a matching DNA profile to an individual and to provide activity level evidence. A range of biological molecules such as protein, mRNA and miRNA have been previously used for body fluid identification (BFID). However, many of the published options are complex and require multiple profiling pathways or multiple replicates to obtain interpretable data.We have developed a simplified workflow to deliver an RT-qPCR based system for identification of body fluids using mRNA. The process uses a modified DNA extraction protocol to optimise co-extraction of mRNA and DNA into the same sample eluate, avoiding the requirement for a specialised extraction protocol for mRNA. To simplify the subsequent analysis, we focus on binary tests for two body fluids only, which reflect common questions relevant to different casework scenarios, for example “Does this sample contain saliva or vaginal fluid?” This approach enables the development of smaller, simpler multiplexes using highly specific one-step RT-qPCR assays, designed not to be affected by the presence of genomic DNA. These RT-qPCR multiplexes provide a large dynamic range limiting the number of replicate amplifications required.Here we detail the validation of this streamlined BFID system for identification of saliva and vaginal fluid samples which are commonly encountered in investigations of sexual offences. The study included an assessment on a wide range of sample types obtained from consenting volunteers following sexual activity, providing a robust data set demonstrating the performance of the system.

Time since deposition of biological fluids using RNA degradation

In criminal casework where sexual intercourse has taken place, body fluids are usually left behind. Body fluid identification (BFID) of the type and origin can provide important information regarding what has occurred and who from, however BFID struggles to determine time since deposition (TSD) of biological stains. According to the Crime Survey in England and Wales in 2017, it shows that the majority of sexual offences that occur are committed by people already known to the victims, roughly around 90%, with 56% of these being committed by partners or ex partners. In these cases, the defence is that their DNA will be present because they are in a relationship or were in a prior relationship. To challenge this defence, the degradation of RNA in semen stains could potentially provide information regarding when a stain was deposited. The degradation pattern of a few semen specific mRNA genes and housekeeping genes were evaluated over a 1 month period. These semen stains were extracted periodically and quantified using qPCR generating real-time levels of RNA which can then be compared to the levels of fresh semen. This study shows that different semen specific primers will degrade at different rates over a period of time and therefore could be used to determine the time since deposition of biological fluids.

The characteristics of seminal fluid and the forensic tests available to identify it

AbstractSemen body fluid identification remains a critical aspect of the forensic investigation of alleged sexual assaults. Over the years, as advancements have been made in the area of forensic science, several different methods for semen identification have been utilized. It is not unusual for several methods of semen identification to be incorporated into a single sexual assault investigation. Most methods of semen identification are based on either detecting enzymatic activities or are targeted at specific cells or antigens. Other methods have evolved utilizing fluorescence or protein expression. Presumptive tests are often used to screen or locate semen staining. Further confirmatory tests can be used to identify the staining as semen and finally the donor of the stain can be further identified through DNA analysis. For routine application of these methods, they need to be specific, sensitive, cost effective, easy to use, and nondestructive. Although new techniques are being researched, it appears that traditional methods of semen identification remain the most widely applied in forensic casework.This article is categorized under: Forensic Biology > Body Fluid Identification

Distinct spectrum of microRNA expression in forensically relevant body fluids and probabilistic discriminant approach

MicroRNA is attracting worldwide attention as a new marker for the identification of forensically relevant body fluids. A probabilistic discriminant model was constructed to identify venous blood, saliva, semen, and vaginal secretion, based on microRNA expression assessed via RT-qPCR. We quantified 15 candidate microRNAs in four types of body fluids by RT-qPCR and found that miR-144-3p, miR-451a-5p, miR-888-5p, miR-891a-5p, miR-203a-3p, miR-223-3p and miR-1260b were helpful to discriminate body fluids. Using the relative expression of seven candidate microRNAs in each body fluid, we implemented a partial least squares-discriminant analysis (PLS-DA) as a probabilistic discriminant model and distinguished four types of body fluids. Of 14 testing samples, 13 samples were correctly identified with >90% posterior probability. We also investigated the effects of microRNA expression in skin, semen infertility, and vaginal secretion during different menstrual phases. Semen infertility and menstrual phases did not affect our body fluid identification system. Therefore, the selected microRNAs were effective in identifying the four types of body fluids, indicating that probabilistic evaluation may be practical in forensic casework.

Developing a DNA methylation-based multiplex SNP assay for the identification of semen in mixed stains

In forensic investigations, especially in sexual assault cases, biological samples are often mixed stains of different types of body fluids. Highly uneven proportions DNA of male and female makes the detection for stains more difficult. Recently, DNA methylation was proposed to be a promising maker for body fluid identification, and successfully applied to mixed DNA samples. In this study, 7 semen-specific sites were selected from the dataset GSE59509. Methylation-specific polymerase chain reaction and SNaPshot technology were then used to detect the genotype of SNPs in these sites. The results showed that 7-plex SNP typing system was specific in semen samples. In mixed stains, when semen and vaginal fluid in volume ratios of 1:25, the analyses for 7-plex SNP showed that the semen derived genotype could be determined correctly. Meanwhile, unambiguous profiles can be obtained when the input DNA was 0.25 ng. In addition, the 7-plex assay may be useful in the identification of semen even in the degraded samples and complex mixtures.

Evaluation of two DNA/RNA co-extraction methods for body fluid identification in forensics

Body fluid identification has become a field of interest in forensic casework as it can add value to particular investigative scenarios. Identifying the source of the biological material is not always an upfront task using conventional methods; therefore, profiling of specific mRNA markers can provide the answer. The implementation of RNA based analyses in forensic casework must focus on the quality and sensitivity of methods, starting with nucleic acid extraction, and without loss of DNA for STR profiling. In this work, two methods for DNA and RNA co-extraction were tested and compared: a commercial kit that uses a spin, mini column methodology, and a quick, simple nucleic acid isopropanol precipitation based protocol. Both methods simultaneously extract DNA and RNA, crucial for forensic casework and were tested in semen samples. Nucleic acid quantifications as well as purity assessment ratios (OD260/OD230 and OD260/OD280) were obtained by both methods to infer on the use of extracts in downstream applications such as PCR. The performance of the two tested protocols was further evaluated by analyzing two semen mRNA specific markers, PRM1 and SEMG1. When compared to the commercially developed kit, results suggest that the literature adapted protocol allowed more carryover of contaminants absorbing at 230 nm and 280 nm as the purity ratios were below the accepted standard ranges. Negative results for mRNA profiling supported the QC results obtained by spectrophotometry. On the hand, PRM1 and SEGM1 were positive in RNA samples extracted with the commercial kit.

Semi quantitative detection of signature peptides in body fluids by liquid chromatography tandem mass spectrometry (LC–MS/MS)

This study covers a modified semi-quantitative approach to detecting signature peptides for body fluid identification. A liquid chromatography tandem mass spectrometer normally used for toxicology was adapted to detect target ion transitions for five semen or saliva specific peptides. Peptide concentrations were measured based on a mixture of synthetic peptide standards. Samples were processed using a three-hour trypsin digestion and Microcon membrane filtration. This method generates PCR compatible DNA and peptide fractions that can be typed without any further treatment. Preliminary validation tests covered stains on different substrates, semen/saliva mixtures, limit of detection, and repeatability. All signature peptides were present at different concentrations, varied amongst donors, and were tissue specific. Saliva peptides were detected at lower concentrations and had a higher limit of detection (LOD). Semen peptides had higher concentrations and were detected even as a minor component in a mixture. All semen peptides and all, but one, saliva peptides were detected on the various substrates. Semen peptide concentrations had relative standard deviations (RSDs) lower than 20%, indicating high repeatability, different from saliva where higher RSDs were observed. DNA fractions did not show signs of degradation or PCR inhibition.

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

Biological traces found at crime scenes can be analyzed to genetically identify the donor(s) but also to determine the body fluid composition of the stain. The latter can be accomplished with high specificity by mRNA profiling. In some mixed body fluid samples, it might be of probative value to directly associate each body fluid with each of the DNA donors. Associating a DNA profile to the contributing body fluid is not often possible, with the exception of simple binary mixtures that contain male and female specific body fluids. However genomic information is transferred from DNA to mRNA, and RNA-cSNPs (coding region SNPs) should be identifiable by MPS methods. Previously, we have performed proof-of-concept studies using a prototype Illumina MiSeq MPS cSNP assay to demonstrate the usefulness of this approach. Here, we have continued our work and introduce an MPS assay on the Ion S5 system comprising a set of 21 cSNPs in body fluid specific mRNA transcripts (7 blood (3 genes); 8 semen (4 genes); 6 saliva (4 genes)). Combined with optimized primer sets for body fluid identification, the assay can identify all forensically relevant body fluids and skin as well as differentiating blood, semen and saliva transcripts from different individuals.The assay has been evaluated with numerous donors of each body fluid to evaluate the specificity and the discriminatory power of the cSNPs. The findings are promising as we were able to associate donors with body fluids in mixtures of body fluids. However, more cSNPs are needed to improve the discriminatory power, particularly for vaginal secretions and menstrual blood transcripts. Assigning body fluids to DNA donors is a realistic possibility with the continued development of MPS cSNP assays.

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

The identification of body fluid (BF) stains at a crime scene is an important step of a crime scene investigation since traces of BFs are typically the main source of DNA evidence. Raman spectroscopy is a new emerging method for identifying and differentiating between BF stains. As any new analytical method developed for identification and differentiation of classes of materials, it needs to be evaluated for potential environmental interferences (EIs). This study specifically focused on proving that Raman spectroscopy is not susceptible to false positive (FP) assignments from EIs specific to blood. Twenty-four EI substances that may be misclassified as blood due to either their appearance, or known to provide a FP result with forensic crime scene tests for blood identification were analyzed in this study. The spectra of the twenty-four substances were tested against a recently developed support vector machines discriminant analysis (SVMDA) classification model used for BF differentiation. The SVMDA model identified all substances as not peripheral blood, but some of the substances were predicted as one of the other four BFs. Therefore, random forests (RF) analysis was employed to build a new, more robust, classification method to overcome this limitation. Using this approach, complete separation was achieved based on a classification probability threshold of 70%. This method can also be used in the future to test potential EI substances specific to other common BFs.

Development of a body fluid identification multiplex via DNA methylation analysis.

The goal of this study is to develop an epigenetic multiplex for body fluid identification based on tissue specific DNA methylation. A series of genetic loci capable of discerning the origin of DNA as coming from saliva, blood, vaginal epithelia, or semen were used for this application. The markers - BCAS4, CG06379435, VE_8, and ZC3H12D - were amplified together and then sequenced via pyrosequencing. Methylation values for cytosine guanine dinucleotide (CpG) sites at each locus were then measured across the four markers. In total, 124 samples were collected, and bisulfite modified to convert unmethylated DNA to uracil. This converted DNA was then amplified via multiplex PCR with reverse primers containing a biotin molecule. Biotinylated PCR products were then analyzed using pyrosequencing to generate a series of pyrograms containing 18 CpG sites. The percent methylation at each CpG site was determined, and then agglomerative hierarchical cluster analysis was used to create a model to indicate sample origin. Further analysis reduced the number of CpG sites required for optimal determination of body fluid type to five. This study demonstrates an efficient multiplexed body fluid identification process utilizing DNA methylation that can be easily implemented in forensic laboratories.

Insects as vectors of DNA in a forensic context

AbstractInsects have historically been used in criminal investigations to provide information in relation to postmortem intervals (PMIs) but the field of forensic entomology is expanding. It is now recognized that insects can act as vectors of human and mammalian DNA through the consumption of biological material, with extraneous DNA able to be extracted and genotyped from all stages of the life cycle, and insect feces and regurgitant (artifacts). To date, DNA recovered from insects have been used to inform investigations into neglect and homicide, link body parts, and to identify victims of crime. It may also potentially be used to identify assailants, confirm the food source of insects to determine their relevance in PMI calculations, determine if a crime has occurred, identify scenes of crimes, and link people to locations and other individuals. However, insects which have consumed biological material may also transfer DNA by traveling to new areas, or depositing it via their artifacts, either within crime scenes or laboratories, or at locations distant to the food source. This could contaminate forensic evidence, confound investigations, and/or falsely incriminate or exclude an individual. Therefore, it is important that there is increased awareness into both the utility of insects as vectors of forensically relevant DNA, and the potential for contamination.This article is categorized under: Forensic Biology > Forensic DNA Technologies Forensic Science in Action/Crime Scene Investigation > Crime Scene Examination Forensic Biology > Interpretation of Biological Evidence Forensic Biology > Body Fluid Identification

MicroRNA Detection in Blood, Urine, Semen, and Saliva Stains After Compromising Treatments.

Evaluation of microRNA (miRNA) expression as a potential method for forensic body fluid identification has been the subject of investigation over the past several years. Because of their size and encapsulation within proteins and lipids, miRNAs are inherently less susceptible to degradation than other RNAs. In this work, blood, urine, semen, and saliva were exposed to environmental and chemical conditions mimicking sample compromise at the crime scene. For many treated samples, including 100% of blood samples, miRNAs remained detectable, comparable to the untreated control. Sample degradation varied by body fluid and treatment, with blood remarkably resistant, while semen and saliva are more susceptible to environmental insult. Body fluid identification using relative miRNA expression of blood and semen of the exposed samples was 100% and 94%, respectively. Given the overall robust results herein, the case is strengthened for the use of miRNAs as a molecular method for body fluid identification.

Detection of microRNAs in DNA Extractions for Forensic Biological Source Identification.

Molecular-based approaches for biological source identification are of great interest in the forensic community because of a lack of sensitivity and specificity in current methods. MicroRNAs (miRNAs) have been considered due to their robust nature and tissue specificity; however, analysis requires a separate RNA extraction, requiring an additional step in the forensic analysis workflow. The purpose of this study was to evaluate miRNA detection in blood, semen, and saliva using DNA extraction methods commonly utilized for forensic casework. RT-qPCR analysis revealed that the tested miRNAs were consistently detectable across most tested DNA extraction methods, but detection was significantly reduced compared to RNA extracts in some biological fluids. DNase treatment was not necessary to achieve miRNA-specific results. A previously developed miRNA panel for forensic body fluid identification was evaluated using DNA extracts, and largely demonstrated concordance with results from samples deriving from RNA extracts of semen, blood, and saliva.

The expression of 10 candidate specific microRNA markers for human body fluid identification in animal buccal swabs

MicroRNAs (miRNAs) have been of interest in forensic science for body fluid identification with recent years. However, there is no study investigating the species specificity of miRNA markers by the SYBR Green method. Due to the conservation of miRNAs across species, miRNA markers maybe less species-specific than mRNA markers, and in forensic cases, animal buccal swabs are more likely to appear. Therefore, in this study we addressed the influence of 8 kinds of animal buccal swabs on human saliva, semen, vaginal secretion swabs and blood identification with 10 candidate specific miRNA markers by the SYBR Green quantitative PCR. Our data showed that the expression levels of the candidate specific miRNA markers miR-124a and 372 in the cat, dog, mouse and rabbit buccal swabs were in the same range as the human vaginal secretion swabs; buccal swabs from these animals also showed similar expression levels to human saliva for the candidate specific miRNA markers miR-200c, 205 and 658. These results indicated that biomaterials of buccal swabs from cats, dogs, mice and rabbits may be mistaken for human saliva or human vaginal secretion swabs, both of which could result in false positives for human body fluids. Thus, the interpretation of these miRNA profiles for human body fluid identification can be inaccurate in the presence of these animal buccal swabs. Therefore, we suggested performing species tests before human body identification with miRNA markers.

XIST and RPS4Y1 long non-coding RNA transcriptome as sex biomarkers in different body fluids

Background and objectivesSex determination of an individual based on biological fluid evidence is a critical issue in forensic science. RNA has proved valuable in the identification of body fluids and the estimation of stain age. However, it still could not provide sufficient information about their donor. This study aimed to evaluate the potential use of the long non-coding XIST and RPS4Y1 markers in the identification of sex from different body fluids.MethodsSaliva, semen, and peripheral and menstrual blood samples were obtained from apparently healthy volunteers. The expression of XIST and RPS4Y1 was assessed in these samples using real-time RT-PCR.ResultsXIST was detected in female saliva and peripheral and menstrual blood, while RPS4Y1 was detected in male blood and semen.ConclusionXIST and RPS4Y1 could be sex differentiation biomarkers in various body fluids.

A review of direct polymerase chain reaction of DNA and RNA for forensic purposes

Short tandem repeat (STR) profiling is frequently carried out on biological stains in forensic casework, and the application of messenger RNA (mRNA) body fluid identification is becoming more prevalent. Increasing constraints on analysis time, resources, an increase in the number of samples for analysis, and a large backlog of cases creates significant demand for quicker methods that direct polymerase chain reaction (PCR) can help overcome. Direct PCR bypasses DNA and/or RNA extraction, and in some cases, quantification by adding the sample or substrate punch to the amplification reaction. This method is currently used in some laboratories on reference samples. Direct PCR is also a component of portable devices, which could improve efficiency even further by producing results at the scene, and enabling triaging of samples for further testing. Portable devices are currently used for STR profiling of reference samples but not for mRNA profiling. Various concerns regarding the reliability of the method and equipment (portable devices) must first be addressed before incorporation into forensic casework. This review discusses the limitations and factors that need to be considered for direct PCR to be used for the analysis of casework samples, including being used for mRNA body fluid identification.This article is categorized under: Forensic Biology > Forensic DNA Technologies Forensic Biology > Body Fluid Identification Forensic Biology > Applications of RNA

The stability and persistence of blood and semen mRNA and miRNA targets for body fluid identification in environmentally challenged and laundered samples.

The identification of body fluids in evidentiary stains may provide investigators with probative information during an investigation. In this study, quantitative reverse transcription polymerase chain reaction (RT-qPCR) assays were performed to detect the presence of mRNA and miRNA in fresh and environmentally challenged samples. Blood, semen, and reference markers were chosen for both mRNA/miRNA testing. Samples of blood and semen were exposed to heat, humidity, and sunlight, and controlled conditions (room temperature, low humidity, and darkness) for 6 months. All mRNA targets were observed through six months under controlled conditions, but were undetected after 30 days in experimental conditions. However, miRNA targets persisted under all test conditions for the duration of the study. Additionally, cotton stained with blood or semen was laundered using a liquid detergent in various washing and drying conditions. An unstained cutting was evaluated for potential transfer. Both miRNA targets were observed in all stained samples regardless of the wash protocol used. Of the mRNA markers, HBB was detected in all bloodstained samples and PRM1 persisted in all but one semen stained sample. The unstained samples showed transfer of at least one body fluid specific miRNA marker in all samples and at least one body fluid specific mRNA in approximately half of the samples. These results support that RNA markers can be used for body fluid identification in challenging samples, and that miRNA markers may be more persistent than mRNA for blood and semen stains. However, some caution is warranted with laundered items due to possible transfer.

Characterization of tissue-specific biomarkers with the expression of circRNAs in forensically relevant body fluids.

Messenger RNA (mRNA) markers have been extensively investigated for the identification of forensically relevant body fluids and tissues based on their expression profiles among cell types. As products of the backsplicing of pre-mRNAs, circular RNAs (circRNAs) share exonic sequences with their linear counterparts. The inclusion of circRNAs in mRNA profiling is shown to facilitate the detection of biomarkers in the identification of body fluids. In this study, we identified the expression of circRNAs of 14 out of 45 biomarkers from five body fluid types using outward-facing primer sets and revealed the ratio of circular to total transcripts of biomarkers by RNase R treatment. Furthermore, our results of qPCR analysis show that the inclusion of circRNAs in the detection of biomarkers, including HBA and ALAS2 for blood; MMP7 and MMP10 for menstrual blood; HTN3 for saliva; SPINK5, SERPINB3, ESR1, and CYP2B7P1 for vaginal secretions; TGM4, KLK3, and PRM2 for semen; and SLC22A6 and MIOX for urine, does not impair the specificity of these biomarkers. Additionally, a high copy number of targets from linear transcripts could be employed to increase the detection sensitivity of TGM4 and KLK3 with a low expression level of circRNAs in urine samples. Altogether, these results will help with the development of robust multiplex assays for body fluid identification.

Investigating the potential of body fluid identification using the Oxford Nanopore MinION

ABSTRACTAs the interest in massively parallel sequencing (MPS) in forensic science continues to grow, the technology available to do such work is rapidly developing. An instrument of particular interest is the Oxford Nanopore MinION, a portable USB sequencer. In addition to shorter library preparation time, the MinION sequencer produces ultra-long reads allowing for sequencing of full-length transcripts. Direct RNA and cDNA kits eliminate the need for PCR. However, PCR-free methods require relatively high amounts of input RNA, which could be problematic with the small, degraded samples common in forensic casework. We have initially assessed the performance and feasibility of the MinION for forensic RNA using salivary gland and tongue RNA.