Human blood, saliva, semen and vaginal secretions are the main body fluids encountered at crime scenes. In the “Live-Time” era of forensic science it has become evident that the current challenge in the examination of body fluids is that non-destructive screening methods of greater specificity are required for body fluid identification compared to the presumptive tests currently utilised. Further to this, a method suitable for routine application is strongly sought after to determine the age of body fluid stains as it could enable police forces to make informed decisions regarding the relevance of forensic biological evidence recovered from crime scenes. The focus of this research was to investigate the use of analytical techniques (ATR-FTIR spectroscopy and protein analyses; SDS-PAGE and the Bradford assay) in the application of robust confirmatory body fluid identification and age determination. The findings of this research demonstrated that human blood, saliva, semen and vaginal secretions could successfully be detected and differentiated from one another when analysed with ATR-FTIR spectroscopy, based on the unique spectral pattern and combination of peaks corresponding to macromolecule groups, and SDS-PAGE, based on separation patterns of various proteins within each of the body fluids. Direct ATR-FTIR spectroscopic examination of blood and vaginal secretion stains enabled successful detection and identification in stains aged up to 18 months and 6 months, respectively. In contrast, stains of saliva and semen aged up to 18 months and 9 months, respectively, could not be detected when directly analysed. However, when the stains were extracted with a simple water-based method, all four body fluids could be detected. Age determination analysis with ATR FTIR spectroscopy demonstrated that peak intensities and ratios were not appropriate variables to discriminate between body fluids stains and extracts. Successful detection of extracted blood, semen and vaginal secretion stains aged up to 7 days was also achieved with SDS-PAGE, although saliva stains were not detected when extracted. The age of extracted samples appeared to have no impact on the detection of the proteins. Furthermore, comparison of average total protein yield obtained with the Bradford assay from aged extracted body fluid stains demonstrated no correlation with protein concentration and sample age for any of the body fluids examined. Overall, this research has demonstrated the successful application of both ATR-FTIR spectroscopy and SDS-PAGE for the identification of human body fluids. ATR-FTIR spectroscopy in particular has reproducibly demonstrated detection and identification of body fluids, which has great potential to be utilised in the routine screening of biological evidence due to its quick and robust application within forensic science.

Single-cell DNA analysis is not routinely carried out in a forensic setting as it is considered unreliable due to challenges associated with DNA amplification, contamination and profile interpretation. In light of the development of increasingly sensitive techniques, the question of the reliability of single-cell DNA analysis in terms of both processing and interpretation is addressed in the first part of this thesis. Optimising all stages of the DNA analysis process has provided a sensitive method which facilitates the successful outcome of a useable profile from single-cells. Although no consensus profile can be generated for this sample type, interpretation guidelines have been set to enable the robust analysis of single cells. It has been concluded that single-cells can be reliably amplified and profiled for forensic purposes. Both DNA and textile fibres have a proven track record in forensic casework yet their analysis is rarely combined. As an application of the aforementioned single-cell DNA analysis, this project explores the possibility that when fibres are transferred from one surface to another, they could also be acting as a vector for the wearer’s own DNA, through cells that have adhered to the fibre surfaces. Fluorescent staining and microscopy is used to detect the cells in situ on the fibre surface, which are then recovered and processed for DNA using the previously optimised single-cell analysis methods, along with a newly developed DNA assay designed for the amplification of low DNA template samples. The results of this study have demonstrated that cells can be visualised in situ on the fibre surface and that there is potential for cell transfer to occur. It has been concluded however, that from a casework point of view, targeting transferred fibres for cells may not be the best approach as it is time consuming and has not been shown to be effective in this study. The final part of this thesis is focused on the efficacy of massively parallel sequencing (MPS) technology for samples that are expected to be severely degraded due to age or exposure to a hostile environment. The ability of both the recently launched Illumina ForenSeq™ DNA Signature Prep Kit for nuclear DNA markers and an in-house method for the sequencing of degraded mitochondrial DNA, have been tested to determine if MPS offers a more comprehensive evaluation of degraded material than the traditional PCR-CE methods. The results of the ForenSeq kit have demonstrated the effectiveness of its low molecular weight STR and SNP markers for amplifying low template, degraded DNA samples, with alleles amplified using less than 20 pg total DNA input. This kit has also therefore shown application in the field of bioarchaeology, as it can provide the biological sex of the sample, biogeographic ancestry information and also aids detection of sample/control contamination. The in-house mitochondrial DNA assay resulted in the successful amplification and sequencing of samples for which no nuclear DNA was amplified. The high depth of read coverage in these samples, average of 18,000, allowed for the identification of even low level variants.

The aim of this work was to establish an analytical method for the simultaneous detection of five major opium alkaloids in poppy seeds by liquid chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS). Once opium alkaloids were detected in poppy seeds, toxicological studies were carried out to establish if these compounds were detected in oral fluid (OF) of participants who ingested muffins containing poppy seeds. It is known that the ingestion of poppy seeds has caused positive opiate drug test results and much work has been reported in the scientific literature in the last 20 years. Researchers in the field have investigated alternatives to differentiate between heroin administration and that of other opiate drugs versus poppy seed ingestion. Most of the work which has been carried out relates to establishing illicit heroin use by examining biological matrices for the presence of acetylcodeine, thebaine, papaverine, noscapine and their associated metabolites. The research methodology consisted of establishing an LC-ESI-MS method for the simultaneous detection of five major opium alkaloids (morphine, codeine, thebaine, papaverine and noscapine). A deuterated internal standard (morphine-d3) was used for the quantitation of alkaloids in harvested poppy seeds and oral fluid samples. Due to technical difficulties, 3 LC-MS instruments were employed in this work. Electrospray ionisation was employed in all mass spectrometers but the analysers included an ion trap with octopole, a triple quadrupole and a hybrid quadrupole Orbitrap. Suitable extraction procedures were determined and harvested seeds purchased from a number of supermarkets were analysed for the presence of five alkaloid compounds using the LC-MS method. A small scale pilot study with 6 participants was carried out to establish if it was possible to fail an OF drug test for opiates after consuming poppy seed muffins. OF samples were collected post ingestion using Quantisal™ kits and the level of each of the opiates was monitored. The findings were that an LC-ESI-MS method was established for the simultaneous detection and quantitation of five major alkaloids. However, the method development process involved finding a solution to co-elution of morphine and codeine. The process also included resolving the issue of thebaine producing two peaks with identical mass spectra and separated by a difference of 6 minutes in retention time. Varying levels of alkaloids were identified in harvested poppy seeds: levels of these compounds differed considerably within and between batches of poppy seeds. These findings could be attributed to a number of factors, for example, where and how the plants were grown and methods of harvesting. Two poppy seed muffins were consumed as part of a toxicology study. Morphine was detected in the 5 minute sample in 5 out of the 6 participants with concentrations in OF of 0.5-0.8 ng mL-1; codeine was detected in 2 of the 6 participants at 1.5 and 2.6 ng mL-1. Thebaine, noscapine and papaverine were also detected in OF of a number of participants, which has not been previously reported in the literature. However, it should be noted that the values calculated are only estimated since the peak area ratios obtained were found to be less than the lowest concentration (10 ng mL-1) in the linear calibration range. In conclusion, an LC-ESI-MS method for the simultaneous detection and quantitation of five major opium alkaloids has been established and has been used to detect alkaloids in harvested poppy seeds and oral fluid samples. From a small pilot toxicology study, oral fluid results indicate that levels of morphine and codeine do not exceed the SAMSHA 40 ng mL-1 cut-off after ingestion of a realistic amount of poppy seeds contained within bakery products.