Methods For Body Fluid Identification Research Articles

Specific fluorescent signatures for body fluid identification using fluorescence spectroscopy

Non-invasive, rapid, on-site detection and identification of body fluids is highly desired in forensic investigations. The use of fluorescence-based methods for body fluid identification, have so far remain relatively unexplored. As such, the fluorescent properties of semen, serum, urine, saliva and fingermarks over time were investigated, by means of fluorescence spectroscopy, to identify specific fluorescent signatures for body fluid identification. The samples were excited at 81 different excitation wavelengths ranging from 200 to 600 nm and for each excitation wavelength the emission was recorded between 220 and 700 nm. Subsequently, the total emitted fluorescence intensities of specific fluorescent signatures in the UV–visible range were summed and principal component analysis was performed to cluster the body fluids. Three combinations of four principal components allowed specific clustering of the body fluids, except for fingermarks. Blind testing showed that 71.4% of the unknown samples could be correctly identified. This pilot study shows that the fluorescent behavior of ageing body fluids can be used as a new non-invasive tool for body fluid identification, which can improve the current guidelines for the detection of body fluids in forensic practice and provide the robustness of methods that rely on fluorescence.

Screening and evaluation of endogenous reference genes for miRNA expression analysis in forensic body fluid samples

MicroRNA (miRNA)-based methods for body fluid identification are promising tools in the practice of forensic science. The selection of appropriate endogenous reference genes as normalizers for the relative quantification of miRNA expression levels using quantitative reverse transcription-polymerase chain reaction (RTqPCR) is essential to avoid errors and improve the comparability of miRNA expression level data among different body fluids. In this study, small RNAs were isolated from individual donations of five forensically relevant body fluids (peripheral blood, menstrual blood, saliva, semen and vaginal secretions). Thirty-seven samples were subjected to high-throughput miRNA sequencing. By combining our results with those obtained through a literature investigation, 28 candidate RNAs were identified. Following RTqPCR validation, the candidate RNAs were preliminarily evaluated in 15 samples to exclude miRNAs with low expression and high variation. Then, the expression levels of 10 relatively stable candidate reference RNAs in 100 samples were determined and further analysed using four commonly employed programs (geNorm, NormFinder, BestKeeper and ΔCq). According to the comprehensive stability rankings of the four algorithms, miR-320a-3p was validated as the most stable endogenous reference gene among the five forensically relevant body fluids, followed by miR-484, SNORD43, miR-320c and RNU6b. Moreover, the combined application of miR-320a-3p with RNU6b could increase the normalization effect. In addition, a total of 56 mock samples placed outdoors and indoors for different times were prepared to further evaluate the stability of candidate reference RNAs, and miR-320a-3p remained the preferred reference gene. Furthermore, the relative expression levels of publicly accepted body fluid-specific miRNAs were determined in 30 samples to verify the practicality and effectiveness of the reference genes. Our results revealed a set of alternative reference genes and could promote the development and application of miRNA-based body fluid identification by determining optional reference genes for strict normalization.

Design and optimization of a 16S microbial qPCR multiplex for the presumptive identification of feces, saliva, vaginal and menstrual secretions.

Molecular methods for body fluid identification have been extensively researched in the forensic community over the last decade, mostly focusing on RNA‐based methods. Microbial DNA analysis has long been used for forensic applications, such as postmortem interval estimations, but only recently has it been applied to body fluid identification. High‐throughput sequencing of the 16S ribosomal RNA gene by previous research groups revealed that microbial signatures and abundances vary across human body fluids at the genus and/or species taxonomic level. Since quantitative PCR is still the current technique used in forensic DNA analysis, the purpose of this study was to design a qPCR multiplex targeting the 16S gene of Bacteroides uniformis, Streptococcus salivarius, and Lactobacillus crispatus that can distinguish between feces, saliva, and vaginal/menstrual secretions, respectively. Primers and probes were designed at the species level because these bacteria are highly abundant within their respective fluid. The validated 16S triplex was evaluated in DNA extracts from thirty donors of each body fluid. A classification regression tree model resulted in 96.5% classification accuracy of the population data, which demonstrates the ability of this 16S triplex to presumptively identify these fluids with high confidence at the quantification step of the forensic workflow using minimal input volume of DNA extracted from evidentiary samples.

Assessing time dependent changes in microbial composition of biological crime scene traces using microbial RNA markers

Current body fluid identification methods do not reveal any information about the time since deposition (TsD) of biological traces, even though determining the age of traces could be crucial for the investigative process. To determine the utility of microbial RNA markers for TsD estimation, we examined RNA sequencing data from five forensically relevant body fluids (blood, menstrual blood, saliva, semen, and vaginal secretion) over seven time points, ranging from fresh to 1.5 years. One set of samples was stored indoors while another was exposed to outdoor conditions. In outdoor samples, we observed a consistent compositional shift, occurring after 4 weeks: this shift was characterized by an overall increase in non-human eukaryotic RNA and an overall decrease in prokaryotic RNA. In depth analyses showed a high fraction of tree, grass and fungal signatures, which are characteristic for the environment the samples were exposed to. When examining the prokaryotic fraction in more detail, three bacterial phyla were found to exhibit the largest changes in abundance, namely Actinobacteria, Proteobacteria and Firmicutes. More detailed analyses at the order level were done using a Lasso regression analysis to find a predictive subset of bacterial taxa. We found 26 bacterial orders to be indicative of sample age. Indoor samples did not reveal such a clear compositional change at the domain level: eukaryotic and prokaryotic abundance remained relatively stable across the assessed time period. Nonetheless, a Lasso regression analysis identified 32 bacterial orders exhibiting clear changes over time, enabling the prediction of TsD. For both indoor and outdoor samples, a larger number (around 60%) of the bacterial orders identified as indicative of TsD are part of the Actinobacteria, Proteobacteria and Firmicutes. In summary, we found that the observed changes across time are not primarily due to changes associated with body fluid specific bacteria but mostly due to accumulation of bacteria from the environment. Orders of these environmental bacteria could be evaluated for TsD prediction, considering the location and environment of the crime scene. However, further studies are needed to verify these findings, determine the applicability across samples, replicates, donors, and other variables, and also to further assess the effect of different seasons and locations on the samples.

Current Methods for Body Fluid Identification Related to Sexual Crime: Focusing on Saliva, Semen, and Vaginal Fluid.

Although, DNA typing plays a decisive role in the identification of persons from blood and body fluid stains in criminal investigations, clarifying the origin of extracted DNA has also been considered an essential task in proving a criminal act. This review introduces the importance of developing precise methods for body fluid identification. Body fluid identification has long relied on enzymatic methods as a presumptive assay and histological or serological methods as a confirmatory assay. However, because the latest DNA typing methods can rapidly obtain results from very small and even old, poorly preserved samples, the development of a novel corresponding body fluid identification method is required. In particular, an immunochromatographic method has been introduced to identify saliva and semen from sexual crimes. In addition, for vaginal fluid identification, attempts have been made in the past decade to introduce a method relying on body fluid-specific mRNA expression levels. At present, the development of molecular biological methods involving microRNA, DNA methylation, and resident bacterial DNA is ongoing. Therefore, in criminal investigations, body fluid identification is an essential task for correctly applying the results of DNA typing, although further research and development are required.

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.

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.

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.

Multiplex body fluid identification using surface plasmon resonance imaging with principal component analysis

Body fluid identification is a key component in forensic casework, providing important information for the reconstruction of criminal events. Body fluid identification in combination with DNA analysis allows the linking of individuals to criminal acts and can therefore be of great importance in determining the outcome of criminal court cases. However, none of the current body fluid identification methods meet all forensic requirements, such as a high sensitivity, a high specificity and the ability to analyse multiple body fluids in a single run. In this pilot-study, we explore, for the first time, surface plasmon resonance imaging (SPRi) with antibody-based detection to serve as a novel multiplex body fluid identification assay for blood, semen, saliva, urine and sweat using minimal sample preparation. A training set consisting of ten donors per body fluid was analysed to determine whether body fluid specific response signals could be obtained. Principal component analysis (PCA) was performed as a statistical tool to cluster the body fluid samples by response signal pattern reduction and to uncover the sources of variation between the body fluids. Four principal components allowed complete clustering of all body fluid types. Blind testing of body fluid samples revealed that five out of eight unknown samples could correctly be clustered to their corresponding group, three out of eight samples were identified as inconclusive. Although optimization of the current SPRi method is required for use in the forensic field, this pilot-study demonstrates the feasibility of SPRi to differentiate five forensically relevant body fluids.

Old meets new: Comparative examination of conventional and innovative RNA-based methods for body fluid identification of laundered seminal fluid stains after modular extraction of DNA and RNA

The knowledge about the type of the body fluid/tissue that contributed to a trace can provide contextual insight into crime scene reconstruction and connect a suspect or a victim to a crime scene. Especially in sexual assault cases, it is important to verify the presence of spermatozoa. Victims often tend to clean their underwear/bedding after a sexual assault. If they later decide to report the crime to the police, in our experience, investigators usually do not send laundered items for DNA examination, since they believe that analysis after washing is no longer promising. As not only the individualization of traces on laundered items could be important in court, but also the type of biological material, we compared the potential of modular DNA and RNA extraction from the same specimen for simultaneous body fluid identification (BFI) and STR profiling of laundered items. BFI included the comparison of a broad range of conventional approaches, as wells as new molecular mRNA- and miRNA-based methods. The examination comprises the assessment of different fabrics and washing temperatures and multiple washing steps. Our results indicate that although conventional enzymatic and immunochromatographical approaches show limitations for BFI of laundered stains, the RSID test was sensitive enough to detect seminal fluid in 80% of all tested samples.Furthermore, the HY-Liter fluorescence analysis successfully detected spermatozoa, even in cloths that were washed twice. For the first time, it could be shown that a marker set of mRNAs can be applied for the identification of seminal fluid stained cotton and synthetic fiber fabrics that have been washed at 40 °C. Our experiments demonstrate that analysis of DNA and RNA of laundered items is feasible, giving the possibility to identify the perpetrator as well as the biological properties of the same laundered (seminal) stain under certain conditions.

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.

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.

High resolution melt curve analysis based on methylation status for human semen identification.

A high resolution melt curve assay to differentiate semen from blood, saliva, urine, and vaginal fluid based on methylation status at the Dapper Isoform 1 (DACT1) gene was developed. Stains made from blood, saliva, urine, semen, and vaginal fluid were obtained from volunteers and DNA was isolated using either organic extraction (saliva, urine, and vaginal fluid) or Chelex® 100 extraction (blood and semen). Extracts were then subjected to bisulfite modification in order to convert unmethylated cytosines to uracil, consequently creating sequences whose amplicons have melt curves that vary depending on their initial methylation status. When primers designed to amplify the promoter region of the DACT1 gene were used, DNA from semen samples was distinguishable from other fluids by a having a statistically significant lower melting temperature. The assay was found to be sperm-significant since semen from a vasectomized man produced a melting temperature similar to the non-semen body fluids. Blood and semen stains stored up to 5months and tested at various intervals showed little variation in melt temperature indicating the methylation status was stable during the course of the study. The assay is a more viable method for forensic science practice than most molecular-based methods for body fluid stain identification since it is time efficient and utilizes instrumentation common to forensic biology laboratories. In addition, the assay is advantageous over traditional presumptive chemical methods for body fluid identification since results are confirmatory and the assay offers the possibility of multiplexing which may test for multiple body fluids simultaneously.

Targeted multiplexed next generation RNA sequencing assay for tissue source determination of forensic samples

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 ‘activity level’ information can be obtained by a determination of the tissue or fluid source of origin of the biological material as it indicates some behavioral activity on behalf of the individual that resulted in its transfer from the body. Here, we seek to improve upon established molecular based methods for body fluid identification (m/miRNA profiling) by developing a targeted multiplexed next generation sequencing assay. The multiplexed biomarker panel includes several highly specific gene targets for each body fluid or tissue with the necessary specificity to definitevely identify all forensically relevant biological fluids and tissues (blood, semen, saliva, vaginal secretions, menstrual blood and skin). We demonstrate the sensitivity of the assay and the ability to identify body fluids in single source and with a limited number of admixed stains. The NGS-RNA body fluid identification panel not only provides ‘activity level’ information for criminal investigations, but will serve as the basis for a future assay which will also permit an association of a DNA profile to a specific body fluid or tissue through the analysis of coding region SNPs within each individual mRNA target. Thus use of this RNA-NGS system may provide forensic scientists with the routine capability of providing some activity level context to a specific individual’s DNA profile.

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

Blood, saliva, semen and vaginal secretions are the main human body fluids encountered at crime scenes. Currently presumptive tests are routinely utilised to indicate the presence of body fluids, although these are often subject to false positives and limited to particular body fluids. Over the last decade more sensitive and specific body fluid identification methods have been explored, such as mRNA analysis and proteomics, although these are not yet appropriate for routine application. This research investigated the application of ATR FT-IR spectroscopy for the detection and discrimination of human blood, saliva, semen and vaginal secretions. The results demonstrated that ATR FT-IR spectroscopy can detect and distinguish between these body fluids based on the unique spectral pattern, combination of peaks and peak frequencies corresponding to the macromolecule groups common within biological material. Comparisons with known abundant proteins relevant to each body fluid were also analysed to enable specific peaks to be attributed to the relevant protein components, which further reinforced the discrimination and identification of each body fluid. Overall, this preliminary research has demonstrated the potential for ATR FT-IR spectroscopy to be utilised in the routine confirmatory screening of biological evidence due to its quick and robust application within forensic science.

Mass spectrometry-based cDNA profiling as a potential tool for human body fluid identification

Several mRNA markers have been exhaustively evaluated for the identification of human venous blood, saliva, and semen in forensic genetics. As new candidate human body fluid specific markers are discovered, evaluated, and reported in the scientific literature, there is an increasing trend toward determining the ideal markers for cDNA profiling of body fluids of forensic interest. However, it has not been determined which molecular genetics-based technique(s) should be utilized to assess the performance of these markers. In recent years, only a few confirmatory, mRNA/cDNA-based methods have been evaluated for applications in body fluid identification. The most frequently described methods tested to date include quantitative polymerase chain reaction (qPCR) and capillary electrophoresis (CE). However these methods, in particular qPCR, often favor narrow multiplex PCR due to the availability of a limited number of fluorescent dyes/tags. In an attempt to address this technological constraint, this study explored matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) for human body fluid identification via cDNA profiling of venous blood, saliva, and semen. Using cDNA samples at 20pg input phosphoglycerate kinase 1 (PGK1) amounts, body fluid specific markers for the candidate genes were amplified in their corresponding body fluid (i.e., venous blood, saliva, or semen) and absent in the remaining two (100% specificity). The results of this study provide an initial indication that MALDI-TOF MS is a potential fluorescent dye-free alternative method for body fluid identification in forensic casework. However, the inherent issues of low amounts of mRNA, and the damage caused to mRNA by environmental exposures, extraction processes, and storage conditions are important factors that significantly hinder the implementation of cDNA profiling into forensic casework.

The development of a method of suspension RNA-FISH for forensically relevant epithelial cells using LNA probes

Messenger RNA profiling is becoming a common method for body fluid identification in forensic science but there are disadvantages when cell mixtures are present from more than one individual. A method that could identify and separate such cell mixtures would simplify downstream analysis. To do this, we have developed a novel method of RNA suspension-fluorescent in situ hybridization (RNA S-FISH) using a locked nucleic acid (LNA) probe for the keratin 10 (KRT10) mRNA that is suitable as a potential marker for epithelial cells. As sample size may be restricted in forensic samples, this method has focused on minimizing cell loss whilst maintaining signal strength. Furthermore, we have shown that it is possible to obtain full DNA profiles from 150 RNA S-FISH labeled cells isolated using laser microdissection.

Evaluation of a co-extraction method for real-time PCR-based body fluid identification and DNA typing

Body fluid identification and individual identification are an important series of tests in usual criminal investigations. Recent reports have demonstrated a new approach using DNA/RNA co-extraction methods in which RNA for body fluid identification and DNA for short tandem repeat (STR) typing are extracted simultaneously from the same sample. This study evaluated a standard co-extraction kit, the AllPrep® DNA/RNA Mini Kit, in order to demonstrate the availability of the co-extraction procedure for those real-time polymerase chain reaction-based body fluid identification methods that we have validated previously. We demonstrated that the use of the Allprep Kit, for which we adjusted the lysis temperature to 56°C to improve extraction efficiency, can simultaneously extract sufficient RNA and DNA for body fluid identification and STR analysis; however, a longer incubation at a high temperature slightly affected the ΔCt value of each target gene and appeared to be not as effective for DNA extraction from old stains as from 1-day-old stains. This method is promising for future forensic investigations because the use of this kit can reduce sample consumption for body fluid identification and DNA typing.

Body fluid identification by mass spectrometry

Standard methods for body fluid identification typically rely on detection of the functional proteins specific to or enriched in them, such as hemoglobin in blood, alkaline phosphatase and PSA in semen, or α-amylase in saliva. While these markers can be relatively specific, the multiple methods used to identify them frequently rely on nonspecific chemical, enzymatic, or antibody reactions that usually require the structural integrity of the markers and are not confirmatory because other proteins or substances can also give positive test results. Recent advances in proteomics and mass spectrometry offer the ability to simultaneously detect multiple body fluid protein markers in a single, confirmatory test. Here, multiple markers for blood, saliva, and semen are identified by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). Data demonstrate the ability to detect these body fluids at nanoliter to subnanoliter levels and to distinguish mixtures. Protein stability of mock samples assayed after 16months showed no diminution of signal. Because multiple peptides from multiple protein markers are detected and effectively sequenced by MALDI MS/MS, the assay is confirmatory. As mass spectrometry detects whatever peptides are present in a sample, no a priori knowledge of an unknown stain is necessary to perform the test.

Multiplex high resolution melt (HRM) messenger RNA profiling assays for body fluid identification

Traditional body fluid identification methods use a variety of technologically diverse techniques that do not permit the identification of all body fluids. Definitive identification of the biological material present can be crucial to a fuller understanding of the circumstances pertaining to a crime. Thus definitive molecular based strategies for the conclusive identification of forensically relevant biological fluids need to be developed. Messenger (mRNA) profiling is an example of such a molecular based approach.Current mRNA body fluid identification assays typically involve either capillary electrophoresis (CE) or quantitative RT-PCR (qRT-PCR) platforms, each with its own limitations. Both platforms require the use of expensive fluorescently labeled primers or probes. CE-based assays require separate amplification and detection steps thus increasing the time required for analysis. For qRT-PCR assays, only 3 or 4 markers can be included in a single reaction since each requires a different fluorescent dye. To simplify mRNA profiling assays and to reduce the time and cost of analysis, we have developed multiplex high resolution melt (HRM) assays that provide an identification of all forensically relevant biological fluids and tissues.