Age determination through DNA methylation patterns in fingernails and toenails.

Over the past decade, age prediction based on DNA methylation has become a vastly investigated topic; many age prediction models have been developed based on different DNAm markers and using various tissues. However, the potential of using nails to this end has not yet been explored. Their inherent resistance to decay and ease of sampling would offer an advantage in cases where post-mortem degradation poses challenges concerning sample collection and DNA-extraction. In the current study, clippings from both fingernails and toenails were collected from 108 living test subjects (age range: 0-96 years). The methylation status of 15 CpGs located in 4 previously established age-related markers (ASPA, EDARADD, PDE4C, ELOVL2) was investigated through pyrosequencing of bisulphite converted DNA. Significant dissimilarities in methylation levels were observed between all four limbs, hence both limb-specific age prediction models and prediction models combining multiple sampling locations were developed. When applied to their respective test sets, these models yielded a mean absolute deviation between predicted and chronological age ranging from 5.48 to 9.36 years when using ordinary least squares regression. In addition, the assay was tested on methylation data derived from 5 nail samples collected from deceased individuals, demonstrating its feasibility for application in post-mortem cases. In conclusion, this study provides the first proof that chronological age can be assessed through DNA methylation patterns in nails.

Improving the age estimation model for toenails.

In a previous study, we have provided the first proof that chronological age can be estimated through DNA methylation (DNAm) patterns in fingernails and toenails. DNAm data of 15 CpGs located in 4 genetic markers (ASPA, EDARADD, ELOVL2 and PDE4C) were evaluated, of which variable selection yielded age prediction models with a mean absolute deviation (MAD) ranging from 7.68 to 9.36 years, depending on the sampling location. Three additional age-associated markers (KLF14, MIR29B2CHG and TRIM59) were assessed in the current study with the goal of increasing the prediction accuracy of the model initially constructed for toenails. This new and improved age estimation assay yielded an MAD of 4.82 and 5.61 years for the training and test set, respectively. The feasibility of the application for post-mortem cases was also demonstrated through testing a limited set of samples collected from deceased individuals.

Bloodstain impact pattern Area of Origin estimation using least-squares angles: A HemoVision validation study.

Bloodstain impact pattern Area of Origin (AO) estimation is an important but time-consuming process in criminal investigations. HemoVision is a software package that automates and accelerates this process. To date, however, no study has been published that evaluates HemoVision's accuracy. Moreover, HemoVision relies on an automated variant of the tangent method to estimate a pattern's AO, even though the use of front-view projections has been shown to provide biased AO estimates. Therefore, the goal of this paper is twofold. First, a novel AO estimation method is proposed, whereby AO estimation is formulated as a least-squares optimisation problem that operates in three dimensions directly, eliminating the need for front view projections. Second, ten impact patterns with known AO coordinates at both 50 cm and 100 cm with respect to the target wall are created and used to compare the proposed approach's accuracy and robustness to the manual tangent method, HemoSpat, and HemoVision's automated tangent method. Results show that for impacts at 100 cm or less to the target wall, the proposed approach achieves the lowest average error of 17.29 cm with the least uncertainty, and that it performs significantly better than the manual tangent and automated tangent methods at a 5% significance level. Moreover, it is shown to achieve errors of less than 30 cm at these distances with just nine stains, whereas the automated tangent method requires a minimum of 16 stains to achieve the same average error.

Postmortem Analysis of Opioids and Metabolites in Skeletal Tissue.

Every year, thousands of suspicious deaths are accounted for by an overdose of opioids. Occasionally all traditional matrices are unavailable due to decomposition. Skeletal tissue may pose a valid alternative. However, reference data on postmortem concentrations in bone tissue and bone marrow (BM) is sparse. Therefore, a liquid chromatography--tandem mass spectrometry method was developed and fully validated for the analysis of four opioids and two metabolites (tramadol, O-desmethyltramadol, morphine, fentanyl, norfentanyl, codeine) in bone tissue and BM. Sample preparation was performed using solid phase extraction (BM), methanolic extraction (bone) and a protein precipitation (whole blood). All validation parameters were successfully fulfilled. This method was applied to analyze 22 forensic cases involving opioids. All six opioids were proven to be detectable and quantifiable in all specimens sampled. When tramadol blood concentrations were correlated with bone concentrations, a linear trend could be detected. The same was seen between tramadol blood and BM concentration. A similar linear trend was seen when correlating codeine blood concentration with bone and BM concentration. Although some variability was detected, the same linear trend was seen for morphine. For fentanyl and norfentanyl, the sample size was too small to draw conclusions, regarding correlation. As far as the authors know this is the first-time fentanyl and norfentanyl are quantified in skeletal tissue. In conclusion, due to the absence of reference data for drugs in skeletal tissue, these findings are a step forward toward a more thorough understanding of drug concentration found in postmortem skeletal tissue.

Cell survival and DNA damage repair are promoted in the human blood thanatotranscriptome shortly after death.

RNA analysis of post-mortem tissues, or thanatotranscriptomics, has become a topic of interest in forensic science due to the essential information it can provide in forensic investigations. Several studies have previously investigated the effect of death on gene transcription, but it has never been conducted with samples of the same individual. For the first time, a longitudinal mRNA expression analysis study was performed with post-mortem human blood samples from individuals with a known time of death. The results reveal that, after death, two clearly differentiated groups of up- and down-regulated genes can be detected. Pathway analysis suggests active processes that promote cell survival and DNA damage repair, rather than passive degradation, are the source of early post-mortem changes of gene expression in blood. In addition, a generalized linear model with an elastic net restriction predicted post-mortem interval with a root mean square error of 4.75 h. In conclusion, we demonstrate that post-mortem gene expression data can be used as biomarkers to estimate the post-mortem interval though further validation using independent sample sets is required before use in forensic casework.

Belgium experience in disaster victim identification applied in handling terrorist attack at Brussels Airport 2016.

The Belgian disaster victim identification (DVI) team is involved in many investigations in our country. Indeed, this specialized team of the federal police oversees searching for and investigating criminally buried dead bodies, identification of unknown putrefied corpses, and more. The Belgian DVI team also assists with the identification of victims of mass disasters (natural, accidental, and mass murders). In this article, we consider the contributions of different teams (forensic pathology, anthropology, and odontology, federal police, and crime scene investigation) both on the scene of the attack at the Brussels National Airport (Zaventem) and in the laboratory work (autopsies, sample studies).

Skeletal tissue, a viable option in forensic toxicology? A view into post mortem cases.

Blood analysis is the golden standard in the field of forensic toxicology. However, when extended decomposition of the remains has occurred, alternative matrices are required. Skeletal tissue may provide an appropriate sample of choice since it is very resistant to putrefaction. However, today, the absence of reference data of drug concentrations in skeletal tissue poses a problem to meaningfully and reliably conduct toxicological testing on human skeletal material. The present study investigates the viability of skeletal tissue as an alternative matrix to evaluate xenobiotic consumption in legal cases. Blood, bone tissue and bone marrow of different forensic cases were screened for 415 compounds of forensic interest. Afterwards, methadone, clomipramine, citalopram and their respectively metabolites positive samples were quantified using fully validated methods. Sample preparation was carried out by SPE (whole blood and bone marrow), methanol extraction (bone sections) or protein precipitation (whole blood). All samples were analyzed using liquid chromatography coupled to a triple quad mass spectrometer. Multiple drugs were successfully identified in all sampled matrices. In bone (marrow) not as many substances were detected as in blood but it poses a valid alternative when blood is not available. Especially bone marrow showed big potential with a concordance of 80.5% with blood. Clomipramine, citalopram and their metabolites were proven to be detectable and quantifiable in all specimens sampled. Bone marrow showed the highest concentrations followed by blood and bone tissue. When citalopram blood concentrations were correlated with the bone concentrations, a linear trend could be detected. The same was seen between blood and bone marrow for citalopram concentrations. Methadone was also proven to be detectable in all specimens sampled. However, its metabolites EMDP and EDPP were absent or below the LOD in some samples. Overall, methadone concentrations were higher in bone marrow than in bone. With exception of one case, blood concentrations were higher than bone concentrations. For methadone, a linear trend could be found between blood and bone concentration. Comparing methadone concentrations in blood and bone marrow an exponential trend could be seen. In conclusion, these findings show the potential forensic value of bone and bone marrow as an alternative matrix. Aside to that, a standard protocol for the sample collection and processing is proposed.

Evaluation of three statistical prediction models for forensic age prediction based on DNA methylation.

DNA methylation is a promising biomarker for forensic age prediction. A challenge that has emerged in recent studies is the fact that prediction errors become larger with increasing age due to interindividual differences in epigenetic ageing rates. This phenomenon of non-constant variance or heteroscedasticity violates an assumption of the often used method of ordinary least squares (OLS) regression. The aim of this study was to evaluate alternative statistical methods that do take heteroscedasticity into account in order to provide more accurate, age-dependent prediction intervals. A weighted least squares (WLS) regression is proposed as well as a quantile regression model. Their performances were compared against an OLS regression model based on the same dataset. Both models provided age-dependent prediction intervals which account for the increasing variance with age, but WLS regression performed better in terms of success rate in the current dataset. However, quantile regression might be a preferred method when dealing with a variance that is not only non-constant, but also not normally distributed. Ultimately the choice of which model to use should depend on the observed characteristics of the data.

Medical Findings and Toxicological Analysis in Infant Death by Balloon Gas Asphyxia: A Case Report.

In recent years, the increasing number of asphyxiation cases due to helium inhalation is remarkable. All described cases in the literature where diagnosed as suicide. In this article, however, we describe a triple infant homicide in which helium, as balloon gas, was administered to three young children after sedation causing asphyxiation and death through the medical findings and toxicological analysis. During autopsy, in addition to standard toxicological samples, gas samples from lungs as well as lung tissue itself were directly collected into headspace vials. Besides routine toxicological analysis, which revealed toxic levels of doxylamine, qualitative analysis on gas and lung samples was performed using headspace gas chromatography-mass spectrometry. As carrier gas, the commonly used helium was replaced by nitrogen. In gas samples from lungs of all three children, no helium was found. Nevertheless, lung tissue samples were found positive on helium. Therefore, sedation followed by asphyxia due to helium inhalation can strongly be assumed as the cause of death of all three children.

Amphetamine toxicity in the emergency department.

XTC and other amphetamines are considered to be safe by the majority of partying young people who are unaware of (or unwilling to know about) the acute and chronic toxicity of these substances, and these drugs are widespread, illicit stimulants. In this article, we describe four cases of severe acute toxicity due to recreational use of amphetamines 3,4-methylene-dioxymethamphetamine, 3,4-methylenedioxyethylamphetamine, 3,4-methylenedioxyamphetamine, 4-methylthioamphetamine or p-methoxyamphetamine, with emphasis on the presenting symptoms and acute treatment in the emergency department.

Improved age determination of blood and teeth samples using a selected set of DNA methylation markers.

Age estimation from DNA methylation markers has seen an exponential growth of interest, not in the least from forensic scientists. The current published assays, however, can still be improved by lowering the number of markers in the assay and by providing more accurate models to predict chronological age. From the published literature we selected 4 age-associated genes (ASPA, PDE4C, ELOVL2, and EDARADD) and determined CpG methylation levels from 206 blood samples of both deceased and living individuals (age range: 0-91 years). This data was subsequently used to compare prediction accuracy with both linear and non-linear regression models. A quadratic regression model in which the methylation levels of ELOVL2 were squared showed the highest accuracy with a Mean Absolute Deviation (MAD) between chronological age and predicted age of 3.75 years and an adjusted R(2) of 0.95. No difference in accuracy was observed for samples obtained either from living and deceased individuals or between the 2 genders. In addition, 29 teeth from different individuals (age range: 19-70 years) were analyzed using the same set of markers resulting in a MAD of 4.86 years and an adjusted R(2) of 0.74. Cross validation of the results obtained from blood samples demonstrated the robustness and reproducibility of the assay. In conclusion, the set of 4 CpG DNA methylation markers is capable of producing highly accurate age predictions for blood samples from deceased and living individuals.

HemoVision: An automated and virtual approach to bloodstain pattern analysis.

Bloodstain pattern analysis (BPA) is a subspecialty of forensic sciences, dealing with the analysis and interpretation of bloodstain patterns in crime scenes. The aim of BPA is uncovering new information about the actions that took place in a crime scene, potentially leading to a confirmation or refutation of a suspect's statement. A typical goal of BPA is to estimate the flight paths for a set of stains, followed by a directional analysis in order to estimate the area of origin for the stains. The traditional approach, referred to as stringing, consists of attaching a piece of string to each stain, and letting the string represent an approximation of the stain's flight path. Even though stringing has been used extensively, many (practical) downsides exist. We propose an automated and virtual approach, employing fiducial markers and digital images. By automatically reconstructing a single coordinate frame from several images, limited user input is required. Synthetic crime scenes were created and analysed in order to evaluate the approach. Results demonstrate the correct operation and practical advantages, suggesting that the proposed approach may become a valuable asset for practically analysing bloodstain spatter patterns. Accompanying software called HemoVision is currently provided as a demonstrator and will be further developed for practical use in forensic investigations.

Feasibility and validation of virtual autopsy for dental identification using the Interpol dental codes.

Virtual autopsy is a medical imaging technique, using full body computed tomography (CT), allowing for a noninvasive and permanent observation of all body parts. For dental identification clinically and radiologically observed ante-mortem (AM) and post-mortem (PM) oral identifiers are compared. The study aimed to verify if a PM dental charting can be performed on virtual reconstructions of full-body CT's using the Interpol dental codes. A sample of 103 PM full-body CT's was collected from the forensic autopsy files of the Department of Forensic Medicine University Hospitals, KU Leuven, Belgium. For validation purposes, 3 of these bodies underwent a complete dental autopsy, a dental radiological and a full-body CT examination. The bodies were scanned in a Siemens Definition Flash CT Scanner (Siemens Medical Solutions, Germany). The images were examined on 8- and 12-bit screen resolution as three-dimensional (3D) reconstructions and as axial, coronal and sagittal slices. InSpace(®) (Siemens Medical Solutions, Germany) software was used for 3D reconstruction. The dental identifiers were charted on pink PM Interpol forms (F1, F2), using the related dental codes. Optimal dental charting was obtained by combining observations on 3D reconstructions and CT slices. It was not feasible to differentiate between different kinds of dental restoration materials. The 12-bit resolution enabled to collect more detailed evidences, mainly related to positions within a tooth. Oral identifiers, not implemented in the Interpol dental coding were observed. Amongst these, the observed (3D) morphological features of dental and maxillofacial structures are important identifiers. The latter can become particularly more relevant towards the future, not only because of the inherent spatial features, yet also because of the increasing preventive dental treatment, and the decreasing application of dental restorations. In conclusion, PM full-body CT examinations need to be implemented in the PM dental charting protocols and the Interpol dental codes should be adapted accordingly.

The impact of shallow burial on differential decomposition to the body: a temperate case study.

Extant literature contains a number of specific case studies on differential decomposition involving adipocere formation or desiccation, but few describe the co-occurrence of these features within a temperate climate. The case of a 65-year-old male, partially buried in a shallow grave for 7 months, is presented in which the soft tissues of the body were outwardly well preserved. The right leg was desiccated, some parts of the body were covered with adipocere (head, neck, right shoulder, upper torso and left leg) and other parts could be classified as in the early stages of decomposition. In this study the taphonomic variables resulting in differential decomposition with desiccation and adipocere formation are discussed.