Forensic Body Fluid Identification Research Articles

Reference genes to normalize body fluid identification data obtained using RT-qPCR

ABSTRACT The use of messenger RNA for body fluid identification has been well documented. While reference genes are commonly used for normalizing gene expression, the use of reference genes in forensic science is not straightforward. Degradation of samples, mixed body fluids, and small volumes contribute to the complexity of normalizing gene expression. To demonstrate normalization of gene expression for forensic body fluid identification, a preliminary study involving two reference genes (beta-2-microglobulin, B2M and glucose-6-phosphate dehydrogenase, G6PD) was undertaken. Novel reverse-transcription quantitative PCR (RT-qPCR) assays for circulatory blood, buccal swabs, menstrual fluid, spermatozoa, seminal fluid, and vaginal material were designed. An appropriate PCR efficiency for the B2M and G6PD reference gene assays across the body fluid panel was confirmed prior to assessing their expression levels. B2M was found to be more highly expressed across the different body fluids than G6PD. The normalization of target assay expression using both genes was investigated. However, high variation and poor sensitivity, particularly in buccal and semen samples, were observed for both, which would limit sensitivity and mixture detection in forensic casework. If CT values are the intended output rather than comparative quantification, we propose that reference gene normalization is not required and could be detrimental to interpretation.

Comparing endpoint and real-time PCR for forensic body fluid identification

ABSTRACT Identifying the body fluid source of stains can give an indication of activities, prioritize samples for DNA profiling, and provide probative evidence when the presence of DNA alone does not. Messenger RNA profiling assays using endpoint reverse-transcription polymerase-chain reaction (RT-PCR), were developed to address the issues with conventional body fluid identification and have since been used in forensic casework. However, endpoint RT-PCR has a narrow linear dynamic range (LDR), and there is currently no specific method for mRNA quantification to direct case strategy and optimize input template. Real-time reverse-transcription quantitative PCR (RT-qPCR) collects data during the amplification phase which reflects the starting quantity. The application of RT-qPCR assays as a quantification precursor to endpoint RT-PCR was investigated using previously described assays for circulatory blood, saliva, menstrual fluid, spermatozoa, seminal fluid, and vaginal material. While blood marker SLC4A1 and vaginal marker CYP2B7P showed a strong relationship, the remaining body fluid markers lacked a robust predictive relationship, likely due to differences in LDR. CYP2B7P and SLC4A1 showed high sensitivity when compared with other markers – resulting in a greater number of data points for estimation of the linear relationship. Compared with endpoint, the RT-qPCR assays had higher precision, LDR, and sensitivity.

Expanding the scope of methylation-sensitive restriction enzyme (MSRE) PCR for forensic identification of body fluids through the novel use of methylation-dependent restriction enzymes (MDRE) and the combination of autosomal and Y-chromosomal markers

Methylation-sensitive/-dependent restriction enzyme (MSRE/MDRE) PCR can be performed to detect hypomethylated or hypermethylated CpG sites. With the combined use of different tissue-specific CpG markers, MSRE/MDRE-PCR leads to tissue-specific methylation patterns (TSMPs), enabling the correlation of DNA samples to their source tissue. MSRE/MDRE assays can use the same platform as forensic STR typing and offer many advantages in the field of forensic body fluid detection. In the present study, we aimed to establish MSRE assays for the detection of blood, saliva, vaginal secretion, and semen, using markers from literature and from our own database search. We designed two different MSRE test-sets, which include two novel Y-chromosomal non-semen markers, and enable differentiation between female and male non-semen samples. Furthermore, we established an MSRE/MDRE semen approach, which includes only Y-chromosomal non-semen and semen markers. This Y-semen multiplex PCR utilizes the novel combination of the methylation-sensitive enzyme SmaI and the methylation-dependent enzyme GlaI, which enables more sensitive detection of male body fluids within male/female DNA mixtures. Our validation tests confirmed that MSRE/MDRE assays exhibit high sensitivity, similar to that of STR typing.

Partial validation of multiplexed real-time quantitative PCR assays for forensic body fluid identification

Confirmatory body fluid identification using messenger RNA (mRNA) is a well-established technique to address issues encountered with conventional testing – such as poor sensitivity, specificity, and a lack of available tests for all body fluids of interest. For over a decade, endpoint reverse-transcription polymerase chain reaction (RT-PCR) assays have been used in forensic casework for such purposes. However, in comparison with real-time quantitative RT-PCR (RT-qPCR), endpoint RT-PCR has lower sensitivity, precision, and linear dynamic range.This research details the multiplexing and partial validation of confirmatory RT-qPCR assays. We have previously described novel assays for a range of body fluid targets and identified an optimal commercial kit for their amplification. Here, multiplexing was undertaken to form three assays: circulatory blood (SLC4A1) and menstrual fluid (STC1), saliva (HTN3) and vaginal material (CYP2B7P), and spermatozoa (PRM1) and seminal fluid (KLK2), all including a synthetic internal control RNA.Partial validation of the multiplexed assays incorporated the MIQE guidelines, ISO requirements, and SWGDAM guidelines. Using receiver operating characteristic (ROC) curves, each marker was significantly different from an uninformative assay and optimal cut-offs were all above 35 cycles. All assays showed a wide LDR (ranging from 3 to 5 logs with most R2 > 0.99), and high precision (most mean CV < 1 %). STC1 showed some instances of sporadic expression in blood, semen, and vaginal material at high CT values. CYP2B7P showed off-target expression in semen and blood. The sensitivities were approximated as; saliva: 1 in 1,000 dilution of a whole buccal swab, circulatory blood: 0.01–0.1 µL blood, menstrual fluid: 1 in 10,000 dilution of a whole menstrual swab, spermatozoa: 0.001 µL semen, seminal fluid: 0.01 µL semen, and vaginal material: 1 in 1,000 dilution of a whole vaginal swab.A total of 16 mock body fluid extract mixtures and 18 swab mixtures were tested and had 100% and 99% detection of target markers below each specific cut-off, respectively. Some mixtures containing high volumes of blood and semen showed off-target CYP2B7P expression. The successful application of a probabilistic model to the RT-qPCR data was also demonstrated. Further work will involve full developmental validation.

Evaluation and Verification of a microRNA Panel Using Quadratic Discriminant Analysis for the Classification of Human Body Fluids in DNA Extracts.

There is significant interest in the use of miRNA analysis for forensic body fluid identification. Demonstrated co-extraction and detection in DNA extracts could make the use of miRNAs a more streamlined molecular body fluid identification method than other RNA-based methods. We previously reported a reverse transcription-quantitative PCR (RT-qPCR) panel of eight miRNAs that classified venous and menstrual blood, feces, urine, saliva, semen, and vaginal secretions using a quadratic discriminant analysis (QDA) model with 93% accuracy in RNA extracts. Herein, miRNA expression in DNA extracts from 50 donors of each body fluid were tested using the model. Initially, a classification rate of 87% was obtained, which increased to 92% when three additional miRNAs were added. Body fluid identification was found to be reliable across population samples of mixed ages, ethnicities, and sex, with 72-98% of the unknown samples classifying correctly. The model was then tested against compromised samples and over biological cycles, where classification accuracy varied, depending on the body fluid. In conclusion, we demonstrated the ability to classify body fluids using miRNA expression from DNA extracts, eliminating the need for RNA extraction, greatly reducing evidentiary sample consumption and processing time in forensic laboratories, but acknowledge that compromised semen and saliva samples can fail to classify properly, and mixed sample classification remains untested and may have limitations.

Weight of evidence from mRNA data: Implementation of an LR-system for forensic body fluid identification in casework

After developing an LR-system to calculate weight of evidence from mRNA data [1] we here present how this LR-system is used in forensic casework. We aim to convey the weight of the evidence in forensic reports.

A comparative study of commercial real-time reverse transcription PCR kits for forensic body fluid identification

ABSTRACT Identifying the type(s) of body fluid present in forensic casework exhibits can assist with scene reconstruction and indicate potential activity types. Confirmatory body fluid identification can be achieved using mRNA-based profiling assays. This commonly involves endpoint reverse-transcription PCR (RT-PCR) with capillary electrophoresis (CE) detection. In comparison, real-time quantitative RT-qPCR is more sensitive, quantitative, and does not require post-PCR processing. We have developed real-time RT-qPCR assays for forensic body fluid identification. This study compared the performance (sensitivity, PCR efficiency, precision) of five real-time RT-qPCR kits across circulatory blood, buccal, semen, and vaginal fluid samples with normal and extended (3-step) PCR cycling. An objective scoring system for the experimental performance parameters was considered along with other features of the commercial kits. Statistical analysis by ANOVA and post-hoc Tukey of the slope estimates, which relate to PCR efficiency, revealed that many observed differences were insignificant (p > 0.05). Sensitivity and precision were also similar across most kits and PCR cycling protocols. Using the scoring system, the five highest performing kit and cycling combinations were: TaqMan-extended, followed by the TaqPath-normal, QuantiNova-extended, QuantiNova-extended, then UltraPlex-normal. Based upon high performance, room temperature set-up, and multiplexing capability, the UltraPlex kit was selected for our body fluid identification assays.

A novel multiplex assay system based on 10 methylation markers for forensic identification of body fluids.

Identifying the source of body fluids found at a crime scene is an essential forensic step. Some methods based on DNA methylation played significant role in body fluids identification. Since DNA methylation is related to multiple factors, such as race, age, and diseases, it is necessary to know the methylation profile of a given population. In this study, we tested 19 body fluid-specific methylation markers in a Chinese Han population. A novel multiplex assay system based on the selected markers with smaller variation in methylation and stronger tissue-specific methylation were developed for the identification of body fluids. The multiplex assay were tested in 265 body fluid samples. A random forest model was established to predict the tissue source based on the methylation data of the 10 markers. The multiplex assay was evaluated by testing the sensitivity, the mixtures, and old samples. For the result, the novel multiplex assay based on 10 selected methylation markers presented good methylation profiles in all tested samples. The random forest model worked extremely well in predicting the source of body fluids, with an accuracy of 100% and 97.5% in training data and test data, respectively. The multiplex assay could accurately predict the tissue source from 0.5 ng genomic DNA, six-months-old samples and distinguish the minor component from a mixture of two components. Our results indicated that the methylation multiplex assay and the random forest model could provide a convenient tool for forensic practitioners in body fluid identification.

MRNA and microRNA stability validation of blood samples under different environmental conditions

RNA molecules, including mRNAs and microRNAs (miRNAs), have been used for forensic body fluid identification. Specific body fluids present unique mRNA expression patterns, while miRNAs identifying body fluids are mainly differentially expressed. miRNAs are thought to be more stable than mRNAs, although this lacks adequate supporting data. In this study, we addressed perceived concerns regarding the stability of miRNAs and mRNAs in blood samples. The samples used in this study involved three groups. First, environmentally-degraded blood stain samples were exposed to a range of environmental conditions over 1–360 days to degrade naturally. Second, simulated-degraded samples were prepared using RNase A or high temperature (80 °C). Furthermore, two authentic casework samples that were proven to be degraded from short tandem repeat (STR) profiles were analyzed. mRNAs and miRNAs present in the same blood samples were simultaneously detected through reverse transcriptase qPCR (RT-qPCR). Furthermore, mRNAs expression was determined by an mRNA multiplex PCR system. Our results showed that both mRNAs and miRNAs were stable in dry environments. The stability of miRNAs was relatively higher than that of mRNAs in humid environments or at high temperature. RNase A had the most serious impact on RNA stability, both mRNA profiles and miRNAs expression patterns were altered. The results of this study provide data and support to demonstrate that miRNAs represent more stable RNA molecules in body fluid identification compared to mRNAs.

The effect of infertile semen on the mRNA-based body fluid identification.

In the past decade, mRNA markers have been well demonstrated as promising molecular markers in forensic body fluid identification (BFI), and successfully used in wide applications. Several studies have assessed the performance of semen-specific mRNA markers in distinguishing semen from other common body fluids at the crime scene. Infertility has been reported as a global health problem that is affecting approximately 15% of couples worldwide. Therefore, it is important for forensic researchers to consider the impact of infertility on semen identification. This study aimed to explore the effect of semen from infertile men (hereinafter "infertile semen") on BFI and to identify semen-specific mRNAs that can efficiently and accurately distinguish normal and infertile semen samples from other body fluids. Results showed that the selected five mRNAs (KLK3, TGM4, SEMG1, PRM1, and PRM2) performed a significantly high semen specificity in normal semen. Moreover, KLK3 was slightly influenced by infertile semen samples with over 98% positive results in all semen samples. The accuracy to predict normal semen reached up to 96.6% using the discrimination function Y1 with KLK3 and PRM1. However, when the infertile semen samples were included in discrimination function (function Y2 with KLK3), the accuracy rate of semen identification (including the normal and infertile semen) was down to 89.5%. Besides, the sensitivity of multiplex assay could reach down to 50pg. Our results suggest that it is important to consider the presence of infertile semen when using mRNAs to identify semen samples, which would have a far-reaching impact in forensic identification.

Optimization of novel loop-mediated isothermal amplification with colorimetric image analysis for forensic body fluid identification.

Accurate presumptive and confirmatory test use for forensic body fluid identification is essential for gaining contextual information for crime scene investigators. Loop-mediated isothermal amplification (LAMP) is an ideal method for forensic body fluid identification because it is highly specific and generates multi-sized amplicon DNA, and successful amplification results can be read out colorimetrically. Here, we show preliminary data on a LAMP method that rapidly identifies body fluids including venous blood, semen, and saliva, based on colorimetric response and image analysis. The method is designed for easy implementation into forensic casework protocols with minimal disruption to DNA analysis. LAMP naturally increases target specificity due to the use of multiple primers for one target and mRNA targets were used for tissue and human specificity. With colorimetric detection as an inherent part of LAMP, samples that are positive or negative for any of the body fluids are readily identified by image capture and analysis, thus eliminating subjectivity. Results show by using the 3D-printed imaging system specific color ranges can be set for easy determination of body fluids. The resulting color change can be seen in <30min using a universal temperature and primer concentration for all body fluids. This simple method and imaging system allow for minimal hands-on time with objective image analysis and presents a pathway for creating a new potential method for forensic body fluid identification.

A systematic analysis of miRNA markers and classification algorithms for forensic body fluid identification.

Identifying the types of body fluids left at the crime scene can be essential to reconstructing the crime scene and inferring criminal behavior. MicroRNA (miRNA) molecule extracted from the trace of body fluids is one of the most promising biomarkers for the identification due to its high expression, extreme stability and tissue specificity. However, the detection of miRNA markers is not the answer to a yes-no question but the probability of an assumption. Therefore, it is a crucial task to develop complicated methods combining multi-miRNAs as well as computational algorithms to achieve the goal. In this study, we systematically analyzed the expression of 10 most probable body fluid-specific miRNA markers (miR-451a, miR-205-5p, miR-203a-3p, miR-214-3p, miR-144-3p, miR-144-5p, miR-654-5p, miR-888-5p, miR-891a-5p and miR-124-3p) in 605 body fluids-related samples, including peripheral blood, menstrual blood, saliva, semen and vaginal secretion. We introduced the kernel density estimation (KDE) method and six well-established methods to classify the body fluids in order to find the most optimal combinations of miRNA markers as well as the corresponding classifying method. The results show that the combination of miR-451a, miR-891a-5p, miR-144-5p and miR-203a-3p together with KDE can achieve the most accurate and robust performance according to the cross-validation, independent tests and random perturbation tests. This systematic analysis suggests a reference scheme for the identification of body fluids in an accurate and stable manner.

RNA‐based approaches for body fluid identification in forensic science

AbstractIn forensic science, the identification of body fluids present in a stain can aid in the reconstruction of crime scenes and support activity‐level information in some cases. RNA‐based methods for body fluid identification address some limitations associated with conventional testing, including a lack of specificity and sample destruction. mRNA profiling by endpoint reverse transcription polymerase chain reaction (RT‐PCR) is a confirmatory RNA method currently used by some forensic laboratories in casework due to the availability of capillary electrophoresis instruments. This method is highly specific and reliable when the RNA input is within the optimal range. When RNA input is below the limit of detection or too high, then false negatives and false positives/cross reactivity can occur, respectively. Real‐time quantitative PCR (qPCR) is quantitative, more sensitive, and efficient than endpoint PCR, and does not require post‐PCR processing. A reverse transcription (RT) step may also be included in qPCR (RT‐qPCR), enabling mRNA quantification. However, there are concerns regarding the lack of consistency and standardization of RT‐qPCR methodology and reporting of results. Additionally, the number of currently available fluorophores somewhat reduces multiplexing ability. More recently, approaches involving Piwi‐interacting biomarkers, NanoString barcodes, and RNA sequencing have been developed. This review will cover the current and developing RNA‐based techniques for forensic body fluid identification, focusing on endpoint and quantitative RT‐PCR using mRNA. The advantages and limitations of these along with alternative methods will be discussed, and how they may be further developed to advance forensic body fluid identification.This article is categorized under: Forensic Biology &gt; Body Fluid Identification Forensic Biology &gt; Applications of RNA

Identification of novel semen and saliva specific methylation markers and its potential application in forensic analysis

Differential DNA methylation in human tissues has been widely used to develop markers for body fluid identification in forensics. In the present study, identification of potential tissue specific differentially methylated regions (tDMRs) was based on mining differentially expressed genes in surrogate tissues for blood, saliva, semen and vaginal fluid. Genes specifically over expressed in one of the surrogate tissues viz: blood, salivary glands, testis, prostrate, cervix, uterus and ovary were identified from genome wide expression datasets. We hypothesized that over expression in surrogate tissues for body fluids could be correlated with differential methylation. Methylation information from two methylation datasets, NGSmethDB and ENCODE were integrated and heavily methylated gene body CpG islands (CGI) representing the body fluids were extracted. From a total of 53 potential genes the present study reports, two genes, ZNF282 and HPCAL1 which were preferentially expressed in cervix with comparatively reduced expression in other surrogate tissues. Methylated CGIs were targeted to design primers for methylation specific PCR (MSP) and bisulphite sequencing (BS). The ZNF282 CpG sites displayed semen-specific hypomethylation while HPCAL1 CpGs showed saliva-specific hypomethylation. Clone-based bisulphite sequencing also revealed significant hypomethylation in the target body fluids. To evaluate the stability of methylation profiles, the ZNF282 tDMR was tested and each body fluid was subjected to five different forensic simulated conditions (dry at room temperature, wet in an exicator, outside on the ground, sprayed with alcohol and sprayed with bleach) for 50 days. Under the condition “outside on the ground”, saliva showed a significant decrease in methylation level by bisulphite sequencing analysis over time. Complete methylation profiles were obtained only for vaginal fluid under all conditions and no differences in methylation levels were observed for this fluid after 50 days. Thus, ZNF282 and HPCAL1 tDMRs can be used as reliable semen and saliva identification markers respectively.

A new method to detect methylation profiles for forensic body fluid identification combining ARMS-PCR technique and random forest model

A set of DNA methylation markers was detected and evaluated to identify body fluids using the amplification refractory mutation system-PCR (ARMS-PCR) and random forest algorithm. In this study, four multiplex DNA methylation reactions composed of 22 promising methylation markers were used to identify regular forensic body fluids, including venous blood, saliva, semen, menstrual blood, and vaginal fluid. The ARMS-specific primers were used to amplify the candidate markers, and then the methylation values of each CpG site were detected through capillary electrophoresis (CE). The DNA methylation patterns of 22 highly informative methylation markers were consistent with previously reported results to a certain extent. To our knowledge, our study is a new method to apply the ARMS-PCR technique and random forest model to detect DNA methylation patterns and identify the type of body fluids in forensic science, thus providing a new method for forensic body fluid identification. Moreover, we proved that this method is robust, applicable and effective for identifying body fluids using the random forest model. The accuracy to predict all body fluids reached up to 0.9966. We firmly believe that this method will have a great potential in the detection of methylation profiles at the molecular level.

Effect of indoor environmental exposure on seminal microbiota and its application in body fluid identification

Semen is a common body fluid type in forensic sexual assault cases. It is of great significance to effectively identify semen for restoring the crime scene and determining the nature of the case. Nowadays, microbiome-based method shows as a promising tool for forensic body fluid identification. To explore the environmental impact on microbial community of semen and its traceability, 16S rDNA high-throughput sequencing was conducted to ten paired semen samples. Affected by exposure, the diversity of microbial community decreased generally as the genus Staphylococcus exhibited a relatively significant increase. However, the genus Staphylococcus, Corynebacterium, Corynebacterium_1 were observed in almost all 20 samples. Community barplot analysis and heatmap analysis showed composition of the predominant microbe in semen at the phyla and genus level maintained basically, so that it could distinguish from vaginal fluid and saliva regardless of environmental exposure. Based on these results, we believe the application of single microbial marker may limit in semen identification, but the method depending on microbial community might be useful for distinguishing semen even under indoor exposure.

A universal test for the forensic identification of all main body fluids including urine

A critical aspect of forensic investigation is to detect and identify body fluid stains and preserve them for DNA extraction. The types of body fluid stains commonly found at a crime scene include blood, saliva, semen, sweat, vaginal fluid, and urine. Identification of a stain can be difficult as current methods are body fluid specific and mostly destructive. In an effort to develop a universal, confirmatory and nondestructive approach which can identify and differentiate body fluids Muro et al. (2016), combined Raman spectroscopy and chemometrics to build a statistical model which could differentiate peripheral blood, saliva, semen, sweat, and vaginal fluid. However, this model did not include urine. Urine can be vital evidence in cases of correctional officers being assaulted with urine bombs by prisoners, as well as in sexual assault cases. Recent studies have also shown that DNA can be extracted from a dried urine sample. In this study, we have combined the Raman spectral dataset collected by Muro et al. with Raman spectral data collected from 27 urine samples from different donors to build calibration and validation datasets. Chemometrics was applied to build an enhanced statistical model which can identify and differentiate all main body fluids including peripheral blood, saliva, semen, sweat, vaginal fluid, and urine. This classification model offers a universal, single step, non-destructive, and robust technique with 100% accuracy for sample identification.

Development of a multiplex system for the identification of forensically relevant body fluids

Currently, mRNA profiling is widely investigated for forensic body fluid identification, while it is still required to advance the approach for those casework samples of limited quantity or low quality. The inclusion of circular RNAs (circRNAs) can facilitate the detection of mRNA markers in forensic body fluid identification. In this study, a multiplex assay for forensic body fluid identification (F18plex assay) was developed by incorporating 14 tissue-specific mRNA markers with circRNAs expression, 2 mRNA markers with high abundance and 2 housekeeping markers for the discrimination of the most common forensic body fluids, including blood, menstrual blood, saliva, vaginal secretion, semen and urine. The markers employed in the F18plex assay show similar specificity to previous reports. Additionally, even if all linear transcripts were completely erased, the expected markers in target biofluids could still be identified, which should help the discrimination of those aged biological stains. Results from sensitivity testing and the detection of mixtures demonstrate good sensitivity of the multiplex assay. Generally, full biomarker profiles could be obtained with ≥1 μl of blood, saliva, or semen, and ≥1 ng of total RNAs from menstrual blood, vaginal secretion, or urine samples, respectively, using this multiplex assay under the established conditions. Collectively, the newly established multiplex assay can assist in determining the biological origin of forensic stains.

Characterization of DNA methylation-based markers for human body fluid identification in forensics: a critical review.

Body fluid identification in crime scene investigations aids in reconstruction of crime scenes. Several studies have identified and reported differentially methylated sites (DMSs) and regions (DMRs) which differ between forensically relevant tissues (tDMRs) and body fluids. Diverse factors affect methylation patterns such as the environment, diets, lifestyle, disease, ethnicity, genetic variation, amongst others. Thus, it is important to analyse the stability of markers employed for forensic identification. Furthermore, even though epigenetic modifications are described as stable and heritable, epigenetic inheritance of potential markers for body fluid identification needs to be assessed in the long term. Here, we discuss the current status of reported DNA methylation-based markers and their verification studies. Such thorough investigation is crucial to develop a stable panel of DNA methylation-based markers for accurate body fluid identification.

Multiplex DNA methylation profiling by ARMS-PCR for body fluid identification

A set of CpG sites were detected and evaluated to identify body fluids based on DNA methylation using the amplification refractory mutation system-PCR (ARMS-PCR). In this research, two multiplex DNA methylation reactions composed of four promising CpG sites were used for the identification of forensic regular body fluids. ARMS-specific primers were used to amplify the CpG sites and the methylation values of each CpG site were detected by capillary electrophoresis. The DNA methylation profiling of four highly informative candidate CpG sites was consistent with previously reported results. It is the first time to apply the ARMS-PCR to detect DNA methylation in forensic science, which provides a new method for forensic body fluid identification. We proved this method will have great potential in detection of the methylation profiling at molecular level.