A 1-year time course study of human RNA degradation in body fluids under dry and humid environmental conditions

Blood, saliva and semen are some of the forensically most relevant biological stains found at crime scenes. mRNA profiling is a reliable approach for the identification of the origin of an evidentiary trace. A stable set of markers and the knowledge about the effects of RNA degradation under different environmental conditions is necessary for the determination of an unknown biological stain. The aim of this work was to compare RNA degradation for human blood, semen and saliva at three different concentrations during a 1-year time period and exposed to dry and humid conditions. Also, this study addressed the question whether there are relevant differences in the efficiency of two RNA extraction methods.     

A comparison of methods used in the UK and Ireland for the extraction and detection of semen on swabs and cloth samples

The recent formation of a United Kingdom and Irish working group, the Body Fluids Forum (BFF), highlighted the need to investigate different working practices prior to any inter-laboratory comparison work and identification of best practice. Various dilutions of semen were seeded onto swabs and cloth samples for each BFF member laboratory to test using their standard techniques. The results showed that the detection of acid phosphatase on swabs is best achieved using direct testing rather than on an extract from the swab. Extraction methods for spermatozoa require a balance to be achieved between using a sufficient volume of water to ensure optimal release and minimal volume to ensure a concentrated extract. PSA tests were investigated and found to be more sensitive than Choline. DNA profiles were obtained from samples in which no spermatozoa had been detected during microscopic examination.     

Evaluation of vaginal mRNA markers in women from different age groups: A GeFI collaborative study

Forensic mRNA profiling assays normally include a set of vaginal-specific markers. Although it is known that vagina undergoes characteristic age-related morphological and physiological changes over a lifetime, few studies have evaluated the efficacy of proposed forensic vaginal mRNA markers in women from different age groups.In this collaborative study involving ten GeFI (Italian working group of ISFG) laboratories, a 19-plex mRNA profiling assay including three vaginal-specific markers (CYP2B7P1, MUC4, MYOZ1) was tested in a collection of vaginal swabs obtained from female volunteer donors in their reproductive years (n = 84) and postmenopausa (n = 55).Differential expression of vaginal markers in the two age categories was assessed by means of: a) overall success rate of mRNA profiling (vaginal mucosa “observed” in the tested sample according to scoring protocol) b) average peak height ratios between vaginal-specific markers within mRNA profiling replicates.Other factors potentially influencing mRNA profiling outcomes, like time interval between vaginal swab collection and analysis, concurrence of menstrual cycle and recent sexual activity at the time of sampling were also investigated.     

Examining the transfer of male DNA onto female underwear in simulated non sexual social interaction versus digital penetration to assist in the evaluation of results in casework scenarios

Experiments have been carried out by the UK and Ireland Association of Forensic Science Providers Body Fluid Forum (AFSP BFF) to determine the levels of male DNA, detected during Y-STR analysis, that may be expected on female underwear from non-sexual social interaction and digital penetration, versus non-sexual social interaction only. The data obtained strongly supports the existing assumptions made: whilst low levels of DNA may be inadvertently transferred to the inside surface of a female’s underwear during social interaction with a male, there is a low expectation of detecting a matching Y-STR profile to that male, which is suitable for statistical evaluation, unless he is a co-habitant of that female.     

QuEChERS方法在法医毒物分析领域的研究进展

在中毒或疑似中毒案件中,毒物鉴定对于提供侦查线索与犯罪证据、澄清案件性质、还原犯罪过程具有重要意义。基于液液萃取和分散固相萃取原理而建立起来的QuEChERS方法,由于其快速、简单、廉价、高效、可靠、安全等优势,正逐渐应用于法医毒物分析的样品前处理过程中。本文综述了QuEChERS方法的基本原理及其在提取和净化等过程中的优化改进,重点详述近年来QuEChERS方法在法医毒物分析领域的应用现状,并对未来发展方向进行展望。     

Application of fragment analysis based on methylation status mobility difference to identify vaginal secretions

Identifying vaginal secretions attaching or adhering to a suspect’s belongings would be beneficial for reconstructing the events that have taken place during a sexual assault. The present study describes a novel approach to identify vaginal secretions by fragment analysis using capillary electrophoresis, based on the mobility differences of PCR amplicons from bisulfite-treated DNA depending on methylation status. We targeted three genome regions including each of three vaginal secretion-specific methylated CpG sites reported previously: cg25416153, cg09765089, and cg14991487. In all three genome regions, the amplicon peaks for methylated genomic DNA (gDNA) sequences were only detected in vaginal samples, whereas samples of other body fluids (blood, saliva, semen, and deposit on skin surface) only showed amplicon peaks for unmethylated gDNA sequences. In vaginal secretions, the methylation ratio of each of the three targeted regions between samples was variable, while the ratios at the three regions in each sample were similar. Furthermore, commercial vaginal epithelial cells were completely methylated at the three regions. Therefore, vaginal secretion-specific methylation may derive from vaginal epithelial cells present in the sample.In forensic cases with a limited amount of DNA, the reproducibility of a detected peak using the present method is not high due to degradation of DNA by bisulfite treatment and subsequent stochastic PCR bias. However, it was possible to detect peaks from methylated DNA sequences by performing PCR and capillary electrophoresis in triplicate after bisulfite treatment, even when bisulfite treatment was performed using 0.5 ng of gDNA from vaginal secretions. In addition, the level of methylation at each targeted region was found to be stable in vaginal secretions stored for 1 year at room temperature. Therefore, we conclude that detection of the visual peak from vaginal secretion-specific methylated DNA sequence is useful to prove the presence of vaginal secretions. This approach has the potential to analyze multiple marker regions simultaneously, and may provide a new multiplex assay to identify various body fluids.     

Y-STRs in forensic medicine: DNA analysis in semen samples of azoospermic individuals

ABSTRACT: The incidence of rape has increased, especially in metropolitan areas, such as the city of São Paulo. In Brazil, studies about it have shown that the majority of this type of crime is committed by the relatives and persons close to the victim. This has made the crime more difficult to be denounced, as only 10% of the cases are reported to competent police authorities. Usually, cytological exams are carried out in sex crime investigations. The difficulty in showing the presence of spermatozoa is frequent, but it does not exclude the presence of male DNA. The absence of spermatozoa in material collected from rape victims can be due to several factors, including the fact that the agressor suffers from azoospermia. This condition can be the result of a successful vasectomy. As the majority of DNA in the ejaculation sample is from spermatozoa, there is much less DNA to be analyzed. This study presents the application of Y‐STRs (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, and DYS393) in DNA analysis of sperm samples from 105 vasectomized men. The study demonstrated a great variation in DNA concentration. DNA extraction and amplification was possible in all sperm samples even in the absence of spermatozoa. The same profile was observed, for each individual, from DNA extracted from blood, pre‐ and postvasectomy semen samples. The use of markers specific for Y chromosome in sex crime cases, especially in the absence of spermatozoa, is very important, mainly because in most situations there is a small quantity of the agressor's DNA in the medium and a large quantity of the victim's DNA.     

超高效液相色谱-质谱联用法测定生物检材中乌头碱

目的建立一种简单、快速测定生物检材(血液、尿液、胃内容物)乌头碱的超高效液相色谱-质谱联用法。方法样品采用乙腈沉淀处理。色谱柱为UPLC C18(2.1mm×50mm,1.7μm),流动相为乙腈-0.1%甲酸,梯度洗脱,流速为0.4m L/min,柱温40℃。采用ESI离子源,MRM模式检测。结果乌头碱在血液0.5~500ng/m L,尿液1~1000ng/m L浓度范围内线性良好(r>0.995)。乌头碱方法回收率在91.3%~110.2%之间;提取回收率在72.8%~83.5%之间;日内、日间RSD均小于14%。结论本方法简单、快速检测生物检材中乌头碱。     

生物检材中的液化石油气成分的土顶空GC/MS分析

目的 建立测定人血中液化石油气(LPG)主要成分的定性定量方法。方法 将LPG添加到血中,用综合质谱图及保留时间定性、单离子法定量分析。结果LPG的7种主要成分血中最小检出限为12.8～31.3ng;仪器最小测量限为1.02～3.36ng。结论 土顶空-GC/MS法测定人血中LPG,简便、快速、准确,适用于LPG成分的检测。     

From crime scene to courtroom: A review of the current bioanalytical evidence workflows used in rape and sexual assault investigations in the United Kingdom

Sexual assault casework requires the collaboration of multiple agency staff to formalise an investigative pipeline running from crime scene to court. While the same could be said of many other forensic investigations, few require the additional support of health care staff and the combined forensic involvement of body-fluid examiners, DNA experts and analytical chemists. The sheer amount of collaborative effort between agencies is laid out through a detailed examination of the investigative workflow from crime scene to courtroom with each step in the pipelines detailed and discussed. Beginning with a review of sexual assault legislation in the United Kingdom this article details how sexual assault investigations are initiated by police and supported by sexual assault referral centre (SARC) staff who are often the first responders providing primary healthcare and patient support to victims while simultaneously collecting and assessing forensic evidence. Detailing the myriad of evidential material that can be documented and collected at the SARC, the review identifies and categorises key forensic tests to first detect and identify body-fluids recovered from evidence through to the secondary analysis of DNA to help identify the suspect. This review also focusses on the collection and analysis of biological material used to support the allegation that the sexual activity was non-consensual and provides a breakdown of common marks and trauma as well as a review of common analytical methods used to infer Drug Facilitated Sexual Assault (DFSA). The culmination of the investigative pipeline is discussed by reviewing the Rape and Serious Sexual Assault (RASSO) workflow used by the Crown Prosecution Service before providing our thoughts on the future of forensic analysis and possible changes to the described workflows.     

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”.     

Sensitive determination of barbiturates in biological matrix by capillary electrophoresis using online large-volume sample stacking

In China, some forensic cases are caused by barbiturates. Thus, the determination of trace level barbiturates in body fluid is important for the poisoning investigation. In this study, an online large-volume sample stacking (LVSS) with polarity switching in capillary electrophoresis (CE) was applied for the sensitive determination of barbiturates. This technique involves injecting a large volume of sample into a capillary and removing the sample matrix plug out of the capillary by reversing the polarity. Quantitation limit obtained was 0.048, 0.057, 0.039, and 0.015 μg/mL for secobarbital, amobarbital, barbital, and phenobarbital (signal-to-noise ratio = 9). By using LVSS, the stacking was simply achieved at 171.7-, 169.7-, 202.7-, and 169.1-fold for the above four barbiturates. The relative standard deviation values of intraday and interday were <2.11% and 4.69%, respectively. Recoveries were ranged from 83.7 to 105.2%. Finally, the trace analysis method was applied to the analysis of real forensic specimens and has achieved satisfactory results.     

潍坊地区六种常见嗜尸性蝇类mtDNA COI区序列研究

目的解决蝇类成虫、蛹和蛆快速、准确鉴定的难题。方法随机采集潍坊地区人尸周围环境中的蝇类成虫、蛹和蛆,提取其mtDNA,经PCR扩增、产物检测与纯化、测序等,比较其序列差异。结果六种常见嗜尸性蝇类相互之间序列差异明显,同一种蝇的成虫、幼虫、蛹之间未见差异。结论mtDNACOI区序列分析可作为法医学嗜尸性蝇类种属鉴别的可靠方法。     

Quantitative analysis of multiple illicit drugs in preserved oral fluid by solid-phase extraction and liquid chromatography–tandem mass spectrometry

We present a validated method for the simultaneous analysis of basic drugs which comprises a sample clean-up step, using mixed-mode solid-phase extraction (SPE), followed by LC–MS/MS analysis. Deuterated analogues for all of the analytes of interest were used for quantitation. The applied HPLC gradient ensured the elution of all the drugs examined within 14 min and produced chromatographic peaks of acceptable symmetry. Selectivity of the method was achieved by a combination of retention time, and two precursor-product ion transitions for the non-deuterated analogues. Oral fluid was collected with the Intercept^®, a FDA approved sampling device that is used on a large scale in the US for workplace drug testing. However, this collection system contains some ingredients (stabilizers and preservatives) that can cause substantial interferences, e.g. ion suppression or enhancement during LC–MS/MS analysis, in the absence of suitable sample pre-treatment. The use of the SPE was demonstrated to be highly effective and led to significant decreases in the interferences. Extraction was found to be both reproducible and efficient with recoveries >76% for all of the analytes. Furthermore, the processed samples were demonstrated to be stable for 48 h, except for cocaine and benzoylecgonine, where a slight negative trend was observed, but did not compromise the quantitation. In all cases the method was linear over the range investigated (2–200 μg/L) with an excellent intra-assay and inter-assay precision (coefficients of variation <10% in most cases) for QC samples spiked at a concentration of 4, 12 and 100 μg/L. Limits of quantitation were estimated to be at 2 μg/L with limits of detection ranging from 0.2 to 0.5 μg/L, which meets the requirements of SAMHSA for oral fluid testing in the workplace. The method was subsequently applied to the analysis of Intercept^® samples collected at the roadside by the police, and to determine MDMA and MDA levels in oral fluid samples from a controlled study.     

Application of mRNA Expression Analysis to Human Blood Identification in Degenerated Samples that were False‐negative by Immunochromatography,,

Forensic laboratories are often faced with cases in which methamphetamine hydrochloride‐mixed blood is unable to be identified as human blood by immunochromatography against human hemoglobin A0. The application of mRNA expression analysis to samples that showed a false‐negative with immunochromatography was investigated as an alternative approach that did not depend on the antigen–antibody reaction. Real‐time PCR was used to examine the expression levels of blood markers such as glycophorin A, spectrin beta, and hemoglobin beta. Hemoglobin beta was the only marker that was specifically detected in blood, while glycophorin A was useful for determining human specificity. Hemoglobin beta showed good detection sensitivity and was detectable in 37‐year‐old blood stains. Hemoglobin beta was exclusively detectable in methamphetamine hydrochloride‐mixed blood stains. Detergents and disinfectants did not significantly influence mRNA markers. The proposed mRNA expression analysis was suitable for human blood identification as an alternative method to immunochromatography.     

Human being eaten by his own dogs: Genetic confirmation through analysis of bones recovered in a dog's stomach content

A part of a decomposed human body from an individual was found at his home, together with the decomposed bodies of his 3 dogs. The disappearance of half of the human body was hardly explained. Hypothetically the dogs, starving, could have eaten their owner after his death. All the bodies were recovered for autopsy.Small bone fragments were recovered in one dog's stomach and identified as human by anthropological analysis. DNA was extracted and cytochrome b gene analysis was made in order to determine their origin, confirmed as human.Genetic identification allowed achieving an eight mini-STR profile with MiniFiler (Applied Biosystems) identical to the bone material collected at the victim's autopsy, confirmed that the dogs had effectively eaten their owner.The results showed that it is possible to obtain nuclear DNA in samples subjected to gastric acids and the combination of different techniques allowed us to determine, in each step, the most convenient workflow for the remains identification.