Prognoses on DNA-based identification success rates of altered human remains using capillary electrophoresis and Next Generation Sequencing technologies

The DNA-based identification success of altered human remains relies on the condition of the collected tissue sample and the associated DNA quantity and quality. Due to tissue-specific differences in post-mortem DNA stability, sampling of the best-suited biological material is essential for successful and rapid identification. However, a large variety and partly contradicting recommendations on optimal material have been published so far. The observed insecurity in sampling strategies revealed the need for a broad and systematic approach in predicting short tandem repeat (STR) genotyping success rates in a wide range of tissue types. Therefore, the overarching aim of this thesis was to improve the DNA-based identification success of altered corpses by presenting novel recommendations and guidance for optimal tissue sampling according to the condition of the body. First, the current situation of identification processes in forensics casework was assessed by a retrospective study on the identification success of 402 altered human corpses over seven years (project I). The evaluation of medical as well as genetic reports revealed an increase in the examination of highly and profoundly decomposed corpses and challenges in molecular analyses of degraded and inhibited samples from altered remains. By comparing the number of successive and parallel PCR amplifications, the most unpredictable typing success and highest number of additional analyses were observed in muscle and bone samples. A comparison with previously published studies highlighted the challenges and insecurity in tissue sampling and the need for standardized guidelines. Furthermore, during project II, the reliability of novel DNA sequencing methods was assessed by validating the MiSeq FGx system for Next Generation Sequencing (NGS) of casework samples and optimizing the sequencing workflow for samples of altered remains. The extensive evaluation of sensitivity, concordance to currently used methods and reproducibility, among others, displayed the technology as robust and implementable in forensic routine casework. Additionally, the applicability of phenotype and biogeographic ancestry prediction was demonstrated in challenging samples of altered corpses. However, as the optimization results revealed, an additional PCR purification step, an increased pooling volume and a reduction of adapter volumes for DNA input concentrations ≥ 31.2 pg is recommended for sequencing highly degraded and inhibited samples. Finally, based on the outcomes of projects I and II, the multicentre study concludes with the presentation of novel recommendations on alteration-specific optimal tissue types for first-attempt identification of altered human remains in project III. By providing an easy and rapid scoring system, a precise assessment of the corpse alteration progress is enabled. Furthermore, the systematic approach included the comparison of DNA quantity, integrity and resulting STR profile completeness in an exceptional high number of 1698 DNA extracts from 949 samples of 19 different tissue types. Thereby, standard capillary electrophoresis as well as forthcoming NGS methods were used and the impact of DNA extraction methods was assessed. The final and first-time prognoses on genotyping success of a wide range of tissues separated for two DNA extraction methods (purifying and non-purifying) and two sets of STR loci (22 loci and 16 loci of the extended European Standard Set) provide guidance that improves the first-attempt DNA-based identification success of altered corpses

Estimation of time since death using comparative proteomic and metabolomic approaches

The success of forensic investigation very often depends on the establishment of the correct timeline of events. In the investigation of fatalities, this depends greatly on the estimation of the time since death of the victim. Current methods lead to inaccurate results and depend greatly on the experience of the investigator. Pathologists estimate the time since death based on visual inspection of the bodies as well as body temperature measurement. Only very short post-mortem intervals (PMIs) can be evaluated with some degree of certainty. This investigation used untargeted proteomic and metabolomic approaches to identify potential molecular markers (proteins, metabolites) which could help to quantify post-mortem changes and aid PMI estimation. Animal models were used in the initial stages of the project. Aged beef meat (stored at 4°C for 13 days) and rat muscle samples (intact cadavers stored at ambient temperature for 3 days) were sampled at 24 h time intervals. In the final stages of the project, human tissue samples were collected at the Forensic Anthropology Centre at Texas State University (San Marcos, Texas). Muscle samples were collected at various times post-mortem from 6 different subjects over the period of two weeks. For the proteomics experiment, 0.5g of tissue was homogenized in extraction buffer consisting of urea, thiourea and 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate (CHAPS). Protein separation was carried out using two-dimensional gel electrophoresis. Protein identification was performed using liquid chromatography-tandem mass spectrometry. For the metabolomics experiment, 0.5g of tissue was homogenized in chloroform/methanol/water solution. The extracted samples were analysed using liquid chromatography-mass spectrometry as well as gas chromatography – mass spectrometry. The investigation allowed the identification of potential biomarker candidates. The proteins of interest varied between the sampled mammals. However, myosin and actin appear as promising candidates for all three species. The metabolomics experiments yielded a large number of possible biomarker candidates. Both liquid and gas chromatography approaches were successfully applied, pointing towards various compounds. Proteogenic amino acids were identified as main compounds of interest in all species using both methods. The study has shown that both proteomic and metabolomic approaches can be successfully applied in forensic medical science and can help to find PMI markers. Using the untargeted approach gives the advantage of looking at a whole range of detected molecules and choosing the most appropriate ones for the task. Furthermore, the combination of these two approaches gives a deeper insight into the post-mortem biological processes. The biomarker candidates proposed in this study require further validation in a larger cohort of subjects

Systematic investigations on the metabolism of drugs by microorganisms colonizing corpses

Nach dem Tod werden Leichen von der normalen mikrobiellen Flora aus der Umgebung der Leiche. Diese Besiedlung ist insofern von Bedeutung, als die am Verwesungsprozess organischer Materie beteiligten Enzyme von Mikroben auch zum Metabolismus und Abbau von Wirkstoffen beitragen, die in postmortalen Proben vorkommen, und so deren Konzentration oder Metabolitenmuster verändern können. Systematische Studien zu Veränderungen von Wirkstoffkonzentrationen und Metabolitenmustern durch Leichen besiedelnde Pilze liegen bislang nicht vor. Als Eukaryoten weisen Pilze jedoch Gemeinsamkeiten mit Säugetierzellen und folglich auch mit dem Stoffwechsel von Säugetieren auf. Entsprechend wurden in den hier vorgestellten Studien Pilze von menschlichem Postmortem-Material isoliert und morphologisch und molekulargenetisch identifiziert. Mit den isolierten Stämmen und dem als Positivkontrolle verwendeten Pilz Cunninghamella elegans (C. Elegans) wurden Untersuchungen zum in vitro Metabolismus von fünf Modellwirkstoffen - Amitriptylin (AT), Metoprolol (MET), Mirtazapin (MRT), Promethazin (PMT) and Zolpidem (ZOL) - durchgeführt. Alle isolierten Pilzstämme waren in der Lage die Modellwirkstoffe mehr oder weniger stark zu metabolisieren. Einige Pilze waren sowohl in der Lage, in vitro Metaboliten der Phase I bilden, die auch beim Menschen produziert werden, als auch pilzspezifische Metaboliten, die bei Säugetieren nicht beschrieben sind. Letztere könnten potentiell als Marker für eine entsprechende Pilzbesiedlung mit postmortalem Metabolismus dienen

Olfactory stimuli associated with the different stages of vertebrate decomposition and their role in the attraction of the blowfly calliphora vomitoria (Diptera: Calliphoridae) to carcasses

Digitised version available at EThOS: British Library Electronic Theses Online Servic

Forensic Taphonomy: Investigating the Post Mortem Biochemical Properties of Cartilage and Fungal Succession as Potential Forensic Tools

Post mortem interval (PMI – the time elapsed since death and discovery) is important to medicolegal investigations. It helps to construct crucial time lines and assists with the identification of unknown persons by inclusion or exclusion of a suspect’s known movements. Accurate methodologies for establishing PMI are limited to about 48-hours. Such methods involve use of increasing levels of potassium in vitreous humour, and algor mortis. This study is two-fold. Firstly, it explores the biomolecular changes in degrading porcine cartilage buried in soil environments and its potential to determine PMI in the crucial two days to two months period. Trotters were interred in a number of graves at two distinct locations exhibiting dissimilar soil environments. Weekly disinterments (for 6 weeks) resulted in dissection for cartilage samples which were processed for protein immunoblot analyses and cell vitality assays. Results demonstrate that aggrecan, a major structural proteoglycan, produces high (230kDa) and low (38kDa) molecular weight cross-reactive polypeptides (CRPs) within cartilage extracellular matrix. The 230kDa CRP degrades in a reproducible manner irrespective of the different soil environments utilised. As PMI increases, aggrecan diminishes and degrades forming heterogeneous subpopulations with time. Immunodetection of aggrecan ceases when joint exposure to the soil environment occurs. At this time, aggrecan is metabolised by soil microbes. The molecular breakdown of cartilage proteoglycans has potential for use as a reliable indicator of PMI, irrespective of differing soil environments, beyond the 48-hours period. Likewise, vitality assays also demonstrated viable chondrocytes for as long as 35 PM days. The second component of this study examined the fungal activity associated with trotters buried below ground. Results indicate that fungal growth was considerably influenced by soil chemistry and changes in the environment. Fungal colonisation did not demonstrate temporal patterns of succession. The results of this study indicate that cartilage has the potential to prolong PMI determination well beyond the current 48- and 100-hour limitations posed by various other soft tissue methods. Moreover, the long-term post mortem viability of chondrocytes presents an opportunity to explore DNA extraction from these cells for the purpose of establishing a positive identification for unidentified remains. On the contrary, the growth and colonisation patterns of post putrefactive fungi in relation to decomposing porcine trotters proved to be futile for estimating PMI. Therefore, fungi may not be a suitable candidate for evaluating PMI during the early phase fungal activity

Hospital Implementation and Acceptance of Minimally Invasive Autopsy

To boost the rate of postmortem investigation, we introduced a minimally invasive autopsy (MIA) procedure, consisting of an MRI- and a CT-scan, combined with CT-guided biopsies, as an alternative to conventional autopsy. In this thesis we discuss the diagnostic performance and acceptation of the MIA-procedure

An investigation into the use of a commonly available fabric dye as a routine stain for tissue samples to be used as a first line, low cost, diagnostic adjunct for the diagnosis of anaphylactic death at autopsy, in a resource-challenged environment

A retrospective study of deaths attributable to anaphylaxis at the Salt River Forensic Pathology Laboratory was undertaken, with a view to determine if eosinophilia was present in tissue samples of the spleen, in accordance with previously published research. Suitable cases of non-anaphylactic death were used as controls. Use was made of two commonly available fabric dyes as alternative stains to the traditional Haematoxylin -Eosin ["H&E"]

The Cerebral Microvasculature in Schizophrenia: A Laser Capture Microdissection Study

BACKGROUND: Previous studies of brain and peripheral tissues in schizophrenia patients have indicated impaired energy supply to the brain. A number of studies have also demonstrated dysfunction of the microvasculature in schizophrenia patients. Together these findings are consistent with a hypothesis of blood-brain barrier dysfunction in schizophrenia. In this study, we have investigated the cerebral vascular endothelium of schizophrenia patients at the level of transcriptomics. METHODOLOGY/PRINCIPAL FINDINGS: We used laser capture microdissection to isolate both microvascular endothelial cells and neurons from post mortem brain tissue from schizophrenia patients and healthy controls. RNA was isolated from these cell populations, amplified, and analysed using two independent microarray platforms, Affymetrix HG133plus2.0 GeneChips and CodeLink Whole Human Genome arrays. In the first instance, we used the dataset to compare the neuronal and endothelial data, in order to demonstrate that the predicted differences between cell types could be detected using this methodology. We then compared neuronal and endothelial data separately between schizophrenic subjects and controls. Analysis of the endothelial samples showed differences in gene expression between schizophrenics and controls which were reproducible in a second microarray platform. Functional profiling revealed that these changes were primarily found in genes relating to inflammatory processes. CONCLUSIONS/SIGNIFICANCE: This study provides preliminary evidence of molecular alterations of the cerebral microvasculature in schizophrenia patients, suggestive of a hypo-inflammatory state in this tissue type. Further investigation of the blood-brain barrier in schizophrenia is warranted