Academic literature on the topic 'Q Science (General) ; QH301 Biology ; RA1001 Forensic Medicine. Medical jurisprudence. Legal medicine'

This research was conducted to provide empirical evidence to supplement advice available to the forensic community for the collection of muscle tissue for forensic analysis. This type of collection is normally carried out to determine the identity of individuals following mass disasters, such as plane crashes or natural disasters. DNA degradation was assessed in two model organisms, pig and rabbit (with human DNA as a control), over various time points. Rabbit recombination activating gene (RAG 1) was aligned to identify conserved regions in pig, rabbit and human. Primers were designed and optimised to create a 4-plex PCR multiplex that can amplify 70 bp, 194 bp, 305 bp and 384 bp in three species. The 4-plex multiplex was found to work efficiently in all three species down to 0.3 ng of DNA template. The multiplex was used to assess whether DNA degradation can be predicted by accumulated degree-days (ADD), which provides a measure of both time and temperature. A series of field studies were performed to assess DNA persistence in pig and rabbit soft muscle tissues using a combination of whole animals, suspended muscle tissues (insect activity free) and muscle fragments. Field studies were carried out in: August-September 2009; February-May 2010; May-June 2010; June-July 2010 and September-November 2010. Soft muscle tissue samples were collected at different ADD. 4-plex multiplex results showed that DNA was more persistent in pig tissues compared to rabbit tissues. In the September 2010 experiments, full multiplex amplification was obtained from rabbit until 137 ADD (whole carcases) and 210 ADD (body fragments and suspended tissues), while in the August 2009 experiments, full multiplex amplification was obtained until 112 ADD (whole carcases and body fragments) and until 141 ADD (suspended tissues). In the June 2010 experiments, full multiplex amplification was possible until 64 ADD. Pig whole carcases which were placed in the field in February 2010, showed multiplex amplification until day 90 (603 ADD), followed by September 2010 (until day 44 (490 ADD)) and May 2010 (until day 27 (338 ADD)). During the September 2010 project, body fragments produced full amplification until muscles were collected (342 ADD), while in case of whole carcases and suspended tissues; the amplification was possible until 490 ADD. There was complete failure of amplification of 305 bp and 384 bp in pig whole carcases after 342 ADD, while in suspended tissues, the amplification of 305 bp and 384 bp was possible until 420 ADD. The statistical analysis showed that amplification success of larger amplicons (194 bp, 305 bp and 384 bp) reduces with increase in ADD in pig and rabbit whole carcases, body fragments and suspended tissues while 70 bp was more persistence. The results showed that there was no significant difference in DNA persistence between whole carcases verses suspended tissues (Z=0.57, p>0.05) and whole carcases verses body fragments (Z=1.71, p>0.05), There was however a significant difference (Z=2.31, p<0.05) in DNA persistence in suspended tissues and body fragments with increase in ADD. The results from field experiments suggested that muscle tissues, if available, should be collected for DNA profiling, since even if degraded, a profile can be obtained. The results also suggested that the isolation of tissues from insect activity as quickly as possible (even if immediate storage is not possible) may be beneficial for DNA persistence. Seasonal variation in DNA persistence was observed due to maggot mass growth which increases carcase decomposition and ultimately effect on DNA persistence. Controlled incubation experiments were also performed at 27 °C, 37 °C and 47 °C until 21 days to assess DNA persistence, as these temperatures were not available under field conditions. The results showed that the amplification of 70 bp was more persistent compared to larger amplicons (194 bp, 305 bp and 384 bp). The drop-out in amplification of larger amplicons occurred more rapidly in samples incubated under laboratory conditions compared to the field samples. The statistical analysis showed species, ADD and temperature have strong effect (p<0.05) on DNA persistence under controlled conditions. The appearance of 70 bp amplicons in all samples collected from field and in most samples from controlled incubation experiments suggested that soft muscle tissues exposed to different environments can be used to perform SNP analysis. The full 4-plex multiplex amplification obtained from rabbit and pig preserved and dehydrated samples suggested that 96% ethanol, cell lysis solution (with and without 1% sodium azide) and dehydration can be used to preserve fresh and partially decomposed soft muscle tissues at room temperature for one year. The drop-out in amplification of larger amplicons in tissues preserved in 10% buffered formalin suggested that formalin was not suitable for long term storage. This system should therefore be considered as an additional method during Disaster victim identification (DVI) work to preserve fresh and partially decomposed samples. This study also suggested that the developed multiplex (4-plex) can be used to assess DNA persistence in human decomposing bodies and in experimental studies.

The forensic toxicologist faces challenges in the detection of drugs and poisons in biological samples due to transformations which occur both during life and after death. For example, changes can result from drug metabolism during life or from the use of formalin solution for post mortem embalming purposes. The former requires the identification of drug metabolites and the latter the identification of chemical reaction products in order to know which substances had been administered. The work described in this thesis was aimed at providing ways of tackling these challenges and was divided into two parts. Part 1 investigated the use of in vitro drug metabolism by human liver microsomes (HLM) to obtain information on drug metabolites and Part 2 investigated the chemical reactions of drugs and a carbamate pesticide with formalin solution and formalin-blood. The initial aim of part I was to develop an in vitro metabolism method using HLM, based on a literature review of previous studies of this type. MDMA was chosen as a model compound to develop the HLM method because its metabolism was known and standards of its metabolites were commercially available. In addition, a sensitive and selective method was developed for the identification and quantitation of hydrophilic phase I drug metabolites using LC/MS/MS with a conventional reverse-phase (C18) column. In order to obtain suitable retention factors for polar drug metabolites on this column, acetyl derivatives were evaluated for converting the metabolites to more lipophilic compounds and an optimal separation system was developed. Acetate derivatives were found to be stable in the HPLC mobile phase and to provide good chromatographic separation of the target analytes. In vitro metabolism of MDMA and, subsequently, of other drugs involved incubation of 4 µg drug substance in pH 7.4 buffer with an NADPH generating system (NGS) at 37oC for 90 min with addition of more NGS after 30 min. The reaction was stopped at 90 min by the addition of acetonitrile before extraction of the metabolites. Acetate derivatives of MDMA metabolites were identified by LC/MS/MS using multiple reaction monitoring (MRM). Three phase I metabolites (both major and minor metabolites) of MDMA were detected in HLM samples. 3,4-dihydroxy-methamphetamine and 4-hydroxy-3-methoxymethamphetamine were found to be major metabolites of MDMA whereas 3,4-methylenedioxyamphetamine was found to be a minor metabolite. Subsequently, ten MDMA positive urines were analysed to compare the metabolite patterns with those produced by HLM. An LC/MS method for MDMA and its metabolites in urine samples was developed and validated. The method demonstrated good linearity, accuracy and precision and insignificant matrix effects, with limits of quantitation of 0.025 µg/ml. Moreover, derivatives of MDMA and its metabolites were quantified in all 10 positive human urine samples. The urine metabolite pattern was found to be similar to that from HLM. The second aim of Part 1 was to use the HLM system to study the metabolism of some new psychoactive substances, whose misuse worldwide has necessitated the development of analytical methods for these drugs in biological specimens. Methylone and butylone were selected as representative cathinones and para-methoxyamphetamine (PMA) was chosen as a representative ring-substituted amphetamine, because of the involvement of these drugs in recent drug-related deaths, because of a relative lack of information on their metabolism, and because reference standards of their metabolites were not commercially available. An LC/MS/MS method for the analysis of methylone, butylone, PMA and their metabolites was developed. Three phase I metabolites of methylone and butylone were detected in HLM samples. Ketone reduction to β-OH metabolites and demethylenation to dihydroxy-metabolites were found to be major phase I metabolic pathways of butylone and methylone whereas N-demethylation to nor-methylone and nor-butylone were found to be minor pathways. Also, demethylation to para-hydroxyamphetamine was found to be a major phase I metabolic pathway of PMA whereas β-hydroxylation to β-OH-PMA was found to be a minor pathway. Formaldehyde is used for embalming, to reduce decomposition and preserve cadavers, especially in tropical countries such as Thailand. Drugs present in the body can be exposed to formaldehyde resulting in decreasing concentrations of the original compounds and production of new substances. The aim of part II of the study was to evaluate the in vitro reactions of formaldehyde with selected drug groups including amphetamines (amphetamine, methamphetamine and MDMA), benzodiazepines (alprazolam and diazepam), opiates (morphine, hydromorphone, codeine and hydrocodone) and with a carbamate insecticide (carbosulfan). The study would identify degradation products to serve as markers for the parent compounds when these were no longer detectable. Drugs standards were spiked in 10% formalin solution and 10% formalin blood. Water and whole blood without formalin were used for controls. Samples were analysed by LC/MS/MS at different times from the start, over periods of up to 30 days. Amphetamine, methamphetamine and MDMA were found to rapidly convert to methamphetamine, DMA and MDDMA respectively, in both formalin solution and formalin blood, confirming the Eschweiler-Clarke reaction between amine-containing compounds and formaldehyde. Alprazolam was found to be unstable whereas diazepam was found to be stable in both formalin solution and water. Both were found to hydrolyse in formalin solution and to give open-ring alprazolam and open-ring diazepam. Other alprazolam conversion products attached to paraformaldehyde were detected in both formalin solution and formalin blood. Morphine and codeine were found to be more stable than hydromorphone and hydrocodone in formalin solution. Conversion products of hydromorphone and hydrocodone attached to paraformaldehyde were tentatively identified in formalin solution. Moreover, hydrocodone and hydromorphone rapidly decreased within 24 h in formalin blood and could not be detected after 7 days. Carbosulfan was found to be unstable in formalin solution and was rapidly hydrolysed within 24 h, whereas in water it was stable up to 48 h. Carbofuran was the major degradation product, plus smaller amounts of other products, 3-ketocarbofuran and 3-hydrocarbofuran. By contrast, carbosulfan slowly hydrolysed in formalin-blood and was still detected after 15 days. It was concluded that HLM provide a useful tool for human drug metabolism studies when ethical considerations preclude their controlled administration to humans. The use of chemical derivatisation for hydrophilic compounds such as polar drug metabolites for analysis by LC/MS/MS with a conventional C18 column is effective and inexpensive, and suitable for routine use in the identification and quantitation of drugs and their metabolites. The detection of parent drugs and their metabolites or conversion and decomposition products is potentially very useful for the interpretation of cases in forensic toxicology, especially when the original compounds cannot be observed.

Cut mark analysis to date has been intermittently and superficially researched across a range of disciplines, despite its potential to significantly contribute to criminal investigation. The current study aims to elucidate cut mark analysis by proposing a novel classification system for the identification of knife cuts (kerfs) in bone. The system was devised, to record accurate and reliable information about cut marks and the criteria were tested for association with the knives that created them. Optical Microscopy was used to examine knife cuts on fleshed porcine bone. Incised cuts were made by a range of serrated, scalloped and fine-edged blades (n=9), by the author, and participants (n=23) were recruited to make marks on bone under the same force-measured conditions, using the Kistler force plate and a bespoke frame to control the level of height to which the knife can be raised above the bone prior to impact. Resultant kerfs were created by a single operator (n=86) and created by a range of individuals (n=186). The data suggests that consistent force was not achieved and the resultant marks on the bones made by the same knife had wide variation in their appearance and depth. The classification criteria tested did not provide discrete identification of knife blades from the assessment of kerf features; however, trends were identified from criteria including margin regularity, margin definition, floor width and wall gradient, which may form the basis for further investigation. Marks made by a single operator showed more significant associations (p<0.05) than group operators, and although kerfs from each share some trends, several significant relationships observed in marks made by a single operator are not shared by the participant group. Limitations of using optical microscopy included the inability to view all aspects of each mark, particularly when combined with variation in depth and angle produced by human operators. From the present results, it is suggested that the use of digital microscopy with a superior ability to build three dimensional images of indented marks would provide the necessary step forward to improve discrimination between knife classifications, based on the areas highlighted by the current research. This reinforces the need for further understanding of the mechanics of cut mark application in human individuals and their potential effects on kerf features.

Forensic science is defined as the application of scientific or technical practices to the recognition, collection, analysis, and interpretation of evidence for criminal and civil law or regulatory issues. A combination of computer science in the field of 3D reconstruction and molecular biology science and techniques were employed in this research aims to document and record a complete picture of the body decomposition process including the changes of the microbiome over the decomposition process. In this thesis, the possibility to reconstruct the crime scene and the decomposition process was investigated. In addition, a 3D model aiming to integrate the biological and thanatological information was generated. The possibility of utilising Autodesk 123D Catch software as a new tool for 3D reconstruction of a crime scene was thoroughly evaluated. First experiments demonstrated that the number of photos required to obtain the best result was specified to be from 20 to 30 photos as a minimum. In addition, significant experiments were performed in different conditions of sizes, locations, and different involved materials. The measurements were obtained from the models using the same software were compared with the real measurements of the tested objects. The result of the correlation between real and estimated measurements showed a very strong agreement ranging from 0.994 to 1.000. With reference to the documentation of the decomposition process, there are different factors, intrinsic and extrinsic, have been reported affecting the decomposition of a carrion/body. These factors mainly interact with the rates of the biological and chemical reaction happening after death. The biological reactions are mainly due to the activity of microorganism and insects. Pigs (Sus scrofa domesticus) were used as a model for human studies and the results obtained have been applied to other mammals without considering the effect of fur on the decomposition process and on the insect and microbial colonisation. In order to investigate this point, rabbits (Oryctolagus cuniculus) with and without fur were used in two sets of experiments at Huddersfield in summer 2014 and in spring 2015. The results obtained in this study showed a similarity of the decomposition stages between animals with and without fur. However, the decomposition process was faster during the summer due to the fast of insect colonisation and activity. In addition, the entomological data collected during the summer and spring experiments were demonstrated that the same taxa nearly were present in both seasons, except Hydrotaea (Diptera, Muscidae), which was presented only in the summer experiment, moreover, only one sample of Lucilia sericata (Calliphoridae) was detected in the spring season. Differences in colonisation time were observed only in spring experiment; animals without fur were colonised two days before animals with fur. The season could have affected the insect’s activity and the spread of the decomposition volatiles. The microbial communities during the decomposition process were investigated using BIOLOG EcoPlateTM and the hypervariable V1-3 region of 16S rRNA gene was used for their molecular identification based on pyrosequencing. Eurofins Genomic Operon using 454-GS Junior pyrosequencing platform (Roche) carried out these analyses. The functional diversity of the bacterial communities on all carcasses samples showed a considerable variability depending on the stage of the decomposition and the sampling region (Oral cavity, skin and interface-sand-carrion) in both seasons. Furthermore, over the molecular analyses of bacterial communities at the phylum level, four main phyla of bacteria were detected among analysed carrion during the decomposition process. These phyla were changed significantly during the stages of the decomposition and between sampling regions. While no difference was observed due to presence or absence of fur. On the other hand, the analysis at the family level was able to highlight differences at the temporal scale but as well as carrion with and without fur. The statistical analysis results showed a significant difference in the bacterial community family distribution among the presence of fur and among the decomposition stages, with significant differences among sampling regions and seasons.

Hollow-fibre liquid-phase microextraction (HF-LPME) was introduced in 1999 as a miniaturised version of liquid-liquid extraction (LLE) in order to reduce the consumption of organic solvents and offer an environmentally-friendly approach to extraction procedures. Since then, several studies have been published in the field of forensic and clinical toxicology applying the technique to a broad range of analytes; however more studies are necessary regarding its applicability to bioanalyses. The principle of HF-LPME is the extraction of analytes across a thin supported liquid membrane within the walls of a hollow fibre from a donor phase (DP) into an acceptor phase (AP). It is an extraction technique that encompasses several parameters that require optimisation for an efficient method; this is most effectively achieved by utilising a design of experiment (DoE) approach rather than the conventional one-factor-at-a-time (OFAT) approach. The main aim of this work is to further investigate the applicability of HF-LPME to the fields of forensic and clinical toxicology by developing and validating methods to extract various drugs from different biological matrices. Complex matrices, such as whole blood, are commonly used in forensic toxicology. Considering that not many studies have been performed on the application of HF-LPME to whole blood (only 10 up to the present day), this is an aspect that requires further investigation. For this, a fast, accurate and precise 3-phase HF-LPME method followed by LC-MS/MS analysis was developed and validated to simultaneously quantify 5 NBOMe drugs in human whole blood. NBOMe drugs are a group of substances part of the so-called “novel psychoactive substances” (NPS); drugs that have been emerging with increasing frequency over the last few years. NBOMes are associated to deaths as the causa mortis, and due to their high potency, these drugs are normally abused in micrograms. For that reason, the HF-LPME method developed had to present high sensitivity (LOD of pg/mL). The aim of the second part of this project was to challenge HF-LPME further by developing and validating methods to assess the potential application of HF-LPME in multi-drug analyses. Urine was selected as biological matrix, and the group of chosen analytes were 14 anti-hypertensive drugs and their metabolites with very different physical-chemical properties. HF-LPME has never been applied to such a broad spectrum of substances in previous bioanalytical studies. These drugs were divided into two groups (acidic and basic/neutral), and a total of four extraction methods (two for each group of analytes) were developed and optimised using chemometrics (DoE) then analysed by LC-MS/MS. Two of these methods were liquid-liquid extraction (LLE) methods that were developed and validated to be used as reference to which the two HF-LPME methods were compared. The LLE methods were sensitive, accurate, precise, and valid for application to real case samples. The HF-LPME methods presented some limitations due to the lack of isotopically-labelled analogues of each specific analyte as internal standards (IS); for non-exhaustive methods the use of these IS should be adopted as standard practise. Real urine samples from genuine patients were extracted using all 4 methods followed by LC-MS/MS analysis. By applying the methods to real case samples, it was possible to define that the HF-LPME methods were suitable for qualitative screening of urine to determine the level of compliance of patients under anti-hypertensive pharmacotherapy. However, for quantification of the drugs applying HF-LPME, further development is required to incorporate the use of isotopically labelled analogues. This study proved that HF-LPME is a potential asset not only for forensic but also for clinical toxicology. It can be a very powerful tool which, mainly due to its green-chemistry approach and pre-concentration capabilities, which allows direct injection into the analytical instrument, could potentially become a more used technique in the future. However, the analyst should be careful when developing HF-LPME methods, to bear in mind its limitations so that methods that are fit-for-purpose can be developed.