Results of the 2003–2004 GEP-ISFG collaborative study on mitochondrial DNA: Focus on the mtDNA profile of a mixed semen-saliva stain

We report here a review of the seventh mitochondrial DNA (mtDNA) exercise undertaken by the Spanish and Portuguese working group (GEP) of the International Society for Forensic Genetics (ISFG) corresponding to the period 2003–2004. Five reference bloodstains from five donors (M1–M5), a mixed stain of saliva and semen (M6), and a hair sample (M7) were submitted to each participating laboratory for nuclear DNA (nDNA; autosomal STR and Y-STR) and mtDNA analysis. Laboratories were asked to investigate the contributors of samples M6 and M7 among the reference donors (M1–M5). A total of 34 laboratories reported total or partial mtDNA sequence data from both, the reference bloodstains (M1–M5) and the hair sample (M7) concluding a match between mtDNA profiles of M5 and M7. Autosomal STR and Y-STR profiling was the preferred strategy to investigate the contributors of the semen/saliva mixture (M6). Nuclear DNA profiles were consistent with a mixture of saliva from the donor (female) of M4 and semen from donor M5, being the semen (XY) profile the dominant component of the mixture. Strikingly, and in contradiction to the nuclear DNA analysis, mtDNA sequencing results yield a more simple result: only the saliva contribution (M4) was detected, either after preferential lysis or after complete DNA digestion. Some labs provided with several explanations for this finding and carried out additional experiments to explain this apparent contradictory result. The results pointed to the existence of different relative amounts of nuclear and mtDNAs in saliva and semen. We conclude that this circumstance could strongly influence the interpretation of the mtDNA evidence in unbalanced mixtures and in consequence lead to false exclusions. During the GEP-ISFG annual conference a validation study was planned to progress in the interpretation of mtDNA from different mixtures.     

Results of the 2003-2004 GEP-ISFG collaborative study on mitochondrial DNA: focus on the mtDNA profile of a mixed semen-saliva stain

We report here a review of the seventh mitochondrial DNA (mtDNA) exercise undertaken by the Spanish and Portuguese working group (GEP) of the International Society for Forensic Genetics (ISFG) corresponding to the period 2003-2004. Five reference bloodstains from five donors (M1-M5), a mixed stain of saliva and semen (M6), and a hair sample (M7) were submitted to each participating laboratory for nuclear DNA (nDNA; autosomal STR and Y-STR) and mtDNA analysis. Laboratories were asked to investigate the contributors of samples M6 and M7 among the reference donors (M1-M5). A total of 34 laboratories reported total or partial mtDNA sequence data from both, the reference bloodstains (M1-M5) and the hair sample (M7) concluding a match between mtDNA profiles of M5 and M7. Autosomal STR and Y-STR profiling was the preferred strategy to investigate the contributors of the semen/saliva mixture (M6). Nuclear DNA profiles were consistent with a mixture of saliva from the donor (female) of M4 and semen from donor M5, being the semen (XY) profile the dominant component of the mixture. Strikingly, and in contradiction to the nuclear DNA analysis, mtDNA sequencing results yield a more simple result: only the saliva contribution (M4) was detected, either after preferential lysis or after complete DNA digestion. Some labs provided with several explanations for this finding and carried out additional experiments to explain this apparent contradictory result. The results pointed to the existence of different relative amounts of nuclear and mtDNAs in saliva and semen. We conclude that this circumstance could strongly influence the interpretation of the mtDNA evidence in unbalanced mixtures and in consequence lead to false exclusions. During the GEP-ISFG annual conference a validation study was planned to progress in the interpretation of mtDNA from different mixtures.     

Individual assignment of body fluids in mixed stains by synonymous SNP analysis

Genotyping of transcribed single nucleotide polymorphisms (SNPs) in cDNA molecules derived from tissue-specific mRNAs has a potential for linking cell types to donors in mixed stains. To test this hypothesis, a single base extension assay targeting the synonymous SNP marker rs857870 in the myeloid cell nuclear differentiation antigen (MNDA) gene was designed. In blood/semen mixtures from donors with informative MNDA genotype (opposite homozygotes), only the MNDA allele of the blood contributor was observed in cDNA, even at blood/semen ratios of 1/20, corresponding to the limit of detection for mixtures by STR profiling. In blood/saliva mixtures, the MNDA allele of the saliva donor was sporadically seen in cDNA, probably due to trace amounts of blood in the donor's saliva, suggesting that the informativeness of the assay may be limited in presence of cell types with partially overlapping mRNA profiles.     

Analysis of body fluid mixtures by mtDNA sequencing: An inter-laboratory study of the GEP-ISFG working group

The mitochondrial DNA (mtDNA) working group of the GEP-ISFG (Spanish and Portuguese Group of the International Society for Forensic Genetics) carried out an inter-laboratory exercise consisting of the analysis of mtDNA sequencing patterns in mixed stains (saliva/semen and blood/semen). Mixtures were prepared with saliva or blood from a female donor and three different semen dilutions (pure, 1:10 and 1:20) in order to simulate forensic casework. All labs extracted the DNA by preferential lysis and amplified and sequenced the first mtDNA hypervariable region (HVS-I). Autosomal and Y-STR markers were also analysed in order to compare nuclear and mitochondrial results from the same DNA extracts. A mixed stain prepared using semen from a vasectomized individual was also analysed. The results were reasonably consistent among labs for the first fractions but not for the second ones, for which some laboratories reported contamination problems. In the first fractions, both the female and male haplotypes were generally detected in those samples prepared with undiluted semen. In contrast, most of the mixtures prepared with diluted semen only yielded the female haplotype, suggesting that the mtDNA copy number per cell is smaller in semen than in saliva or blood. Although the detection level of the male component decreased in accordance with the degree of semen dilution, it was found that the loss of signal was not consistently uniform throughout each electropherogram. Moreover, differences between mixtures prepared from different donors and different body fluids were also observed. We conclude that the particular characteristics of each mixed stain can deeply influence the interpretation of the mtDNA evidence in forensic mixtures (leading in some cases to false exclusions). In this sense, the implementation of preliminary tests with the aim of identifying the fluids involved in the mixture is an essential tool. In addition, in order to prevent incorrect conclusions in the interpretation of electropherograms we strongly recommend: (i) the use of additional sequencing primers to confirm the sequencing results and (ii) interpreting the results to the light of the phylogenetic perspective.     

Advanced statistical analysis of Raman spectroscopic data for the identification of body fluid traces: Semen and blood mixtures

Conventional confirmatory biochemical tests used in the forensic analysis of body fluid traces found at a crime scene are destructive and not universal. Recently, we reported on the application of near-infrared (NIR) Raman microspectroscopy for non-destructive confirmatory identification of pure blood, saliva, semen, vaginal fluid and sweat. Here we expand the method to include dry mixtures of semen and blood. A classification algorithm was developed for differentiating pure body fluids and their mixtures. The classification methodology is based on an effective combination of Support Vector Machine (SVM) regression (data selection) and SVM Discriminant Analysis of preprocessed experimental Raman spectra collected using an automatic mapping of the sample. This extensive cross-validation of the obtained results demonstrated that the detection limit of the minor contributor is as low as a few percent. The developed methodology can be further expanded to any binary mixture of complex solutions, including but not limited to mixtures of other body fluids.     

Validation studies of the ParaDNA® Intelligence System with artificial evidence items

Short tandem repeat (STR) profiling is one of the mostly used systems for forensic applications. In certain circumstances, STR profiling is time-consuming and costly, which potentially leads to delays in criminal investigations. LGC (Laboratory of the Government Chemist, UK) Forensics has developed a robust STR profiling platform called the ParaDNA^® Intelligence Test System which can provide early tactical intelligence and aid investigators in making informed decisions on sample prioritization for detection. Here, we validated the ParaDNA intelligence test for its application in forensic cases using a range of mock evidence items following guidelines set by the Scientific Working Group on DNA Analysis Methods (SWGDAM). Specifically, we tested the sensitivity and accuracy of the ParaDNA intelligence test, as well as the success rates for detecting mock samples and for use in case scenarios. Our findings demonstrate that the ParaDNA intelligence test generates useful DNA profiles, especially for samples such as blood, saliva, and semen that contain ample DNA, indicating the benefits of including ParaDNA as a prior step in forensic STR profiling pipelines.     

Screening and identification of tissue-specific methylation for body fluid identification

Identification of body fluid stains can bring important information to crime case. Recent research in epigenome indicates that tissue-specific differentially methylated regions (tDMRs) show different DNA methylation profiles according to the type of cell or tissue, which makes it possible to identify body fluid based on analysis of DNA. This study screened and identified tDMRs from genome for forensic purpose. DNA samples from blood, saliva, semen, and vaginal fluid were analyzed by methylation sensitive represent difference analysis and Sequenom Massarray^® quantitative analysis of methylation. Six blood-specific tDMRs were obtained. Two tDMRs display blood-specific hypomethylation, and four tDMRs show blood-specific hypermethylation. These tDMRs may discriminate blood stain from other body fluids. The result indicated that tDMRs could become potential DNA markers for body fluid identification.     

Evaluation of Commercial Kits for Dual Extraction of DNA and RNA from Human Body Fluids, ,

STR typing of DNA evidence can identify the donor with a high power of discrimination but cannot identify the tissue origin of a body‐fluid stain. Using RNA to attribute a crime scene stain to a particular tissue may aid in reconstruction efforts. With blood from 10 donors, four DNA and RNA coextraction kits were evaluated by measuring yields and STR and mRNA profiles. T tests indicated some significant differences in kit performance. The Zymo Research ZR‐Duet^? kit performed best based on average DNA (41.4 ng) and mRNA (4.07 ng) yields and was the only kit to provide complete DNA/RNA profiles for all samples. The consistency of this kit was challenged by data from additional blood and saliva donors. Further testing is advised before a superior kit is unequivocally chosen. Stand‐alone DNA or RNA purification generally offers higher yield, but coextraction may still allow successful STR profiling and tissue source 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.     

Assessing the background levels of body fluids on hands

Forensic scientists are often asked to assist the court by evaluating the significance of finding body fluids on the hands of an individual; however, there is an absence of published data regarding the background levels of body fluids on hands. Whilst the scientist can use casework experience to inform the courts on the significance of the results, it would be advantageous to have data which could assist with this interpretation. This study was designed to ascertain the background levels of blood, semen, saliva, hairs/fibres and staining/debris on hands in the general population by sampling from delegates attending a scientific conference.The findings suggest that approximately one third of the population would be expected to have hairs or fibres on their hands and that females are more likely to have visible staining on their hands than males. Presumptive tests for blood and semen yielded negative results in all samples; however, almost 2 % of the samples were found to contain a very low number of sperm heads. In contrast, the majority of samples tested positive for the presence of saliva using the presumptive Phadebas® amylase test. The data supports the caution applied by forensic practitioners when evaluating the presence of saliva detected using the presumptive Phadebas® amylase test based on the lack of specificity and indicates that the RSID™-Saliva test would be more suitable to use.     

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.     

Messenger RNA profiling: a novel method for body fluid identification by real-time PCR

Conventional methods for the identification of different body fluids like blood, semen and saliva from biological stains involve immunological or enzymatic detection of certain proteins. In this study, we investigated potential RNA markers with the aim of developing Real-Time polymerase chain reaction (PCR) based methods to allow differentiation between several body fluids. Total RNA samples from artificially stained swabs and from various pieces of evidence from case work were extracted, amplified and analyzed with several RNA markers. Three assays detecting the body fluids of interest were selected: hemoglobin-alpha locus 1 (HBA), kallikrein 3 (KLK) and mucin 4 (MUC). With this approach, we demonstrate that specific Real-Time PCR assays are useful in identifying the source of the biological stain. Furthermore, RNA profiling of various body fluids was even possible on samples stored over a long period of time at ambient temperature. The stability and sensitivity of the applied method outlines a novel application for Real-Time PCR within the forensic field.     

Development and validation of molecular methodologies for identification of biological traces from crime scenes

This study aimed to develop a methodology to identify biological fluids in sexual assault cases through mRNA markers. Biological fluid samples such as blood, saliva, and semen were collected from volunteers and submitted to RT-qPCR reactions with specific primers for the biomarkers HTN3 (saliva), ALAS (blood) andTGM4 (semen). The Melting (Tm) of each biological fluid was analyzed and the result inferred a high specificity capable of differentiating such traces. Biplex systems were generated to improve trace analysis in a single qPCR reaction.     

Raman spectroscopy offers great potential for the nondestructive confirmatory identification of body fluids

Raman spectroscopy was used to compare body fluids commonly found at crime scenes in a nondestructive manner. The dry traces of semen, vaginal fluid, sweat, saliva, and blood were analyzed using confocal Raman microscopy with a 785-nm excitation. The results show that the five fluids can be differentiated from one another by visual comparison of their Raman spectra, and that the laser radiation does not damage the sample. The Raman signature of each body fluid is specific and correlates with the known composition of the fluid. Dry traces of human and canine semen exhibited distinctly different Raman signatures. Overall, this preliminary study demonstrates the great potential of Raman spectroscopy for nondestructive, confirmatory identification of body fluids for forensic purposes.