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.     

Assessment of body fluid identification and DNA profiling after exposure to tropical weather conditions

Despite current advances in body fluid identification, there are few studies evaluating the effect of environmental conditions. The present work assessed the detection of body fluids, blood, semen, and saliva, through lateral flow immunochromatographic (LFI) tests, exposed to tropical weather conditions over time, also evaluating the possibility of obtaining STR (short tandem repeat) profiles and identifying mitochondrial DNA (mtDNA) polymorphisms. Blood, semen, saliva samples, and mixtures of these fluids were deposited on polyester clothes and exposed to open-air tropical weather conditions for 1 month. The test versions from LFI (SERATEC®, Germany) Lab and crime scene (CS) used for the detection – one per each body fluid type – demonstrated that it is possible to identify body fluids and their mixtures up to 14 days after deposition. At 30 days, blood and semen were detected but not saliva. Full STR profiles were obtained from 14-day-old blood samples, and partial profiles were obtained from the remaining samples. It was possible to sequence mtDNA in the samples previously analyzed for STR profiling, and haplogroups could be assigned. In conclusion, this study demonstrated for the first time the possibility of body fluid identification and DNA profiling after exposure to tropical weather conditions for 1 month and also demonstrated the value of mtDNA analysis for compromised biological evidence.     

In Situ Identification of Semen Stains on Common Substrates via Raman Spectroscopy,,

The spectroscopic identification of body fluids in situ is a major objective in forensic science. This approach offers the confirmatory, nondestructive, rapid, and on‐scene identification of various body fluids. Although Raman spectroscopy has shown tremendous promise toward this goal in prior proof‐of‐concept experiments, a significant challenge which still remains is substrate interference. Here, an approach for detecting semen stains in situ on various substrates using Raman spectroscopy is explored. Simulated semen evidence was prepared on skin, glass, and various fabrics. Raman data were accumulated from stains without any pretreatment using a common confocal mapping spectrometer using 785 nm laser excitation. The results demonstrate that the spectroscopic interferences encountered by substrates can be reduced and eliminated using a combination of existing subtraction techniques and chemometric models. Heterogeneous substrates proved most challenging, however, automatic subtraction treatment, and location of fluid hotspots was able to elucidate a clear spectroscopic signature of semen in every instance.     

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.     

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.     

Forensic Identification of Blood in the Presence of Contaminations Using Raman Microspectroscopy Coupled with Advanced Statistics: Effect of Sand,Dust, and Soil

Body fluid traces recovered at crime scenes are among the most common and important types of forensic evidence. However, the ability to characterize a biological stain at a crime scene nondestructively has not yet been demonstrated. Here, we expand the Raman spectroscopic approach for the identification of dry traces of pure body fluids to address the problem of heterogeneous contamination, which can impair the performance of conventional methods. The concept of multidimensional Raman signatures was utilized for the identification of blood in dry traces contaminated with sand, dust, and soil. Multiple Raman spectra were acquired from the samples via automatic scanning, and the contribution of blood was evaluated through the fitting quality using spectroscopic signature components. The spatial mapping technique allowed for detection of “hot spots” dominated by blood contribution. The proposed method has great potential for blood identification in highly contaminated samples.     

The Differentiation of Menstrual from Venous Blood and Other Body Fluids on Various Substrates Using ATR FT‐IR Spectroscopy

Crime scene investigators and laboratory analysts use chemical tests to detect and differentiate body fluids. Testing often requires a sample of the stain, and the chemicals may cause degradation of the fluid or interfere with subsequent tests. Colorimetric chemical tests do not differentiate between different types of the same fluid, such as venous and menstrual blood, and there is no presumptive test available to simultaneously differentiate several body fluids. In this study, we recorded ATR FT ‐IR spectra of venous and menstrual blood, semen, saliva, and breastmilk. Neat and simulated casework body fluid samples were analyzed on cotton, nylon, wood, paper, and glass substrates. Differences in fluid composition, including proteins and small molecules, resulted in spectral differences. Venous and menstrual blood is differentiated by the peak at 1039 cm^?1 attributed to phosphoric acid found in menstrual blood. Peak intensity is influenced by the porosity and weave of the substrate fabric.     

Cytochemical detection of ABH antigens in human body fluids

The use of the peroxidase-anti-peroxidase (PAP) technique has been described previously for the detection of cellular antigens and in particular ABO antigens from tissue samples (Pedal and Hülle 1984; Pedal and Baedeker 1985; Pedal et al. 1985). In this survey, the PAP method has been employed to study the detection of ABO antigens in cells from body fluids of particular interest to forensic science, namely buccal cells and vaginal cells. Also tested, but in a limited number, were mixtures of body fluids and semen samples. No false reactions were obtained from buccal cells, all samples corresponding to the ABO blood type of the donor. Preliminary results from vaginal cells, vaginal/buccal cell mixtures, and semen were encouraging but must be treated with caution due to the limited number tested. Vaginal smears contaminated with semen showed varying degrees of nonspecificity.     

The development of a method for FISH identification of forensically relevant body fluids

Messenger RNA profiling is a useful confirmatory test for body fluid identification, but there are limitations to this method including sensitivity and the difficulty in linking body fluids with the corresponding DNA profile in mixed samples. We have developed a method for RNA suspension fluorescence in situ hybridisation (RNA S-FISH) for forensic-type samples using locked nucleic acid (LNA) probes. Vaginal and buccal epithelial cells have been the primary focus, with some preliminary work performed on leucocytes and seminal round cells. We have designed probes for the KRT10 messenger RNA and the micro RNA 891a. Using these probes, we have optimised a RNA S-FISH methodology and have successfully visualised these cell types using fluorescent microscopy.     

A Rapid,Confirmatory Test for Body Fluid Identification,

We have developed a technique that allows investigators to confirm the presence of blood, semen, and/or saliva in a crime scene sample. It is a confirmatory test where multiple samples can be processed in less than an hour, and it is potentially portable, permitting samples to be processed at the crime scene. Samples at a scene giving a positive result can be further processed while those failing to do so may be ignored. There is a large and growing backlog of DNA evidence in the USA, slowing down the criminal justice system. This backlog has continued to grow despite an increase in the ability to process evidence faster. This technique uses quantum dot molecular beacons to test for tissue‐specific RNA species, identifying particular body fluids. We have demonstrated the tissue specificity of molecular beacons for blood, semen, and saliva.     

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.     

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.     

Evidence based strategy for normalization of quantitative PCR data,in forensic miRNA-analysis

Micro-RNA (miRNA) based analysis of body fluids and composition of complex crime stains has recently been introduced as a potential and powerful tool to forensic genetics. Analysis of miRNA analysis has several advantages over mRNA but reliable miRNA detection and quantification using quantitative PCR requires a solid and forensically relevant normalization strategy. In our study we evaluated a panel of 12 carefully selected reference genes for their suitability as endogenous controls in miRNA qPCR normalization in forensically relevant settings. We analyzed assay performances and variances in venous blood, semen, menstrual blood, saliva and vaginal secretion and mixtures thereof integrating highly standardized protocols with contemporary methodologies and included several well established computational algorithms.Based on these empirical results, we recommend normalization to the group of RNU24, RNU43, and RNU66, as this signature exhibits the most stable expression levels and the least expected variation among the evaluated candidate reference genes in forensically relevant body fluids. To account for the lack of consensus on how best to perform and interpret quantitative PCR experiments, our study's documentation is according to MIQE guidelines, defining the “minimum information for publication of quantitative real-time PCR experiments”.     

Examination of the correlation of groupings in blood and semen

The grouping of blood/saliva samples from a male so as to predict his semen groups is only justified if there is a strict correlation between the groupings in these body fluids. This correlation has been examined in the ABO, phosphoglucomutase (PGM1) and glyoxalase I (GLO) grouping systems in blood and semen samples collected from more than 250 individuals. Though no results proved inconsistent with this correlation, a number of semen gave inconclusive grouping results. Reasons for this are discussed as well as the relevance of the results to semen stain analysis. Semen amylase activities are also reported.     

Determination of an effective housekeeping gene for the quantification of mRNA for forensic applications

The potential application of mRNA for the identification of biological fluids using molecular techniques has been a recent development in forensic serology. Constitutively expressed housekeeping genes can assess the amount of mRNA recovered from a sample, establish its suitability for downstream applications, and provide a reference point to corroborate the identity of the fluid. qPCR was utilized to compare the expression levels of housekeeping genes from forensic-like body fluid stains to establish the most appropriate assessment of human mRNA quantity prior to profiling. Although variability was observed between fluids and individuals, results indicated that beta-2 microglobulin exhibited the highest expression for all body fluids examined and across donors. A one-way analysis of variance was performed for housekeeping gene variability between donors (at the α, 0.05, significance level), and the results indicated significant differences for semen, vaginal secretions, and menstrual blood.