Browsing by Author "Heathfield, Laura"

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    A retrospective analysis of post-mortem procedures of sudden unexpected death in the young investigated at Salt River Forensic Pathology Services, Cape Town
    (2022) Hamadziripi, Dirk M; Heathfield, Laura; Mole, Calvin
    Sudden unexpected death in the young (SUDY) is the demise of a seemingly healthy individual aged between one and 40 years. The scope of SUDY investigation varies and there is little research regarding SUDY at Salt River Mortuary (SRM). Accordingly, the objectives of this study were to determine the number of SUDY cases admitted to SRM, document the scope of investigations, and identify candidates for retrospective molecular autopsies. A total of 1088 cases were admitted between 1 January 2010 and 31 December 2015, representing 3.3% (1088/32812) of the entire case load, 30 were excluded as the files were missing. Full autopsies (56.7%; 600/1058) were preferred to partial autopsies (5.6%; 59/1058) and external autopsies (37.7%; 399/1058). The most utilised ancillary tests were LODOX imaging (86.6%, 916/1058) and toxicology (34.8%; 368/1058). Specificity of cause of death was seen to be significantly associated with the extent of autopsy. A total of 35.7% (378/1058) of the cases were established as candidates for molecular autopsy on the criteria of being undetermined, having unspecific causes of death or having specific causes of death that are deemed hereditary. Findings of this study show that SUDY cases do not always undergo all ancillary tests coupled with retention of biological samples. These findings provide insight into the current gaps in the investigation of SUDY cases at SRM and highlight areas for improvement.
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    Assessment of 13 Forensic Molecular Markers for skin colour in South Africa
    (2018) Pharo, Gavin; Heathfield, Laura
    Molecular phenotyping is the use of informative genetic variation to estimate appearance. This concept can be applied in a forensic context to predict the appearance of suspects or decayed deceased individuals, which would otherwise remain unidentifiable. This concept has importance in a local context, as approximately 300 individuals remain unidentified, after conventional identification techniques, at Salt River Mortuary, every year. Ancestry Informative Markers (AIMs) are genetic variants with DNA which have been commonly associated with pigmentation phenotypes, and thus has value in predicting skin tone, hair colour and eye colour. This research study aimed to design and optimise an assay to genotype 13 AIMs associated with pigmentation, and then demonstrate the value of this assay by applying it to a case example and qualitatively predicting appearance. Primers were designed and PCR assays optimised to amplify each region, followed by Sanger sequencing on a case example. The case was that of an abandoned neonate, with unknown sex and ancestry. A comparison of the obtained genotypes to previous literature was performed to qualitatively estimate the skin tone, eye colour and hair colour of the decedent, which was not only in agreement with the forensic pathologist’s interpretation of sex and ethnicity, but provided richer detail with regards to ancestry, skin tone, eye colour and hair colour. The PCR assays were then further optimised into four multiplex assays with the intention of genotyping these AIMs by two SNaPshot® PCR assays (Applied Biosystems) in a larger control cohort to model the relationship between these AIMs and melanin index more objectively. Unfortunately, the scope of this research project did not allow for the completion of this additional aspect. Overall, these results indicate that these 13 AIMs have potential to predict pigmentation phenotypes of South African individuals. However, genotyping and modelling of the effects of these AIMs should be performed on a large cohort to further strengthen this conclusion.
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    The assessment of molecular markers for skin colour determination in the South African population
    (2017) Slabbert, Nandi; Heathfield, Laura
    Molecular markers associated with skin colour need to be investigated to determine viability and discriminatory power within the SA population. The use of phenotyping as a tool to aid forensic investigation is becoming incorporated internationally and its use within SA needs to be considered. This project proposes to examine the association between skin colour and two markers, which have been identified as potentially problematic within mixed ancestry populations. This will be done through designing and analysing a multiplex SNaPshot genotyping assay and independent pigmentation measurements.
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    Assessment of the suitability of blood samples collected for toxicological analysis for subsequent genetic analysis: A follow-up study one year later
    (2018) Musiyandaka, Fungisai Lorraine; Heathfield, Laura; Davies, Bronwen
    Drug usage, both of a recreational or pharmaceutical nature, is common, however the abuse of such substances is an international problem. In the Western cape, South Africa, the burden of drug-related fatalities is high compared to the rest of the country. The provincial Forensic Pathology Service may encounter cases where drug-related fatalities are unclear whether death was accidental or suicidal, or drug toxicity is inconsistent with the medical/social history. This may be due to genetic alterations with drug metabolism and it has been suggested that genetic analyses may be the next step in these cases. However, toxicology results from the National Forensic Chemistry Laboratory in the Western Cape may be delayed by months to years, meaning that upon interpretation of toxicology results, there is no chance to obtain another blood sample from the deceased individual for genetic analysis. It was therefore important to determine the suitability of blood samples collected and handled in toxicology environments for subsequent genetic tests. Previously, blood samples from 30 post-mortem cases were collected into two red-top (no additives), two grey-top (sodium fluoride/potassium oxalate) and one purple-top (EDTA) tubes. Samples from one red-top and one grey-top tube underwent toxicological analysis, followed by DNA analysis, while the remaining tubes (controls) underwent DNA analysis immediately. All samples were then stored for approximately one year, prior to this study. The DNA analysis was repeated on all blood samples (n = 150) and results were assessed in terms of storage time and tube type. DNA was not significantly degraded in any of the samples; however, DNA from red-top tubes had significantly lower concentrations compared to that from grey-top tubes (p < 0.001), regardless of whether the sample had undergone toxicological analysis. The very low yields of DNA from red-top tubes posed substantial challenges for PCR-based analysis, resulting in poor quality Sanger sequencing results. Some DNA from grey-top tubes, passed the quality assessments and hence further work is required to provide an informed decision on which tube type is better suited for genetic analyses.
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    Comparison of sars-cov-2 rapid tests and formal serological testing on deceased persons in Cape Town Metro
    (2022) Carlisle, Tayna; Heathfield, Laura; Martin, Lorna
    The COVID-19 disease was declared a global pandemic in 2020 and since, it is unclear how many people have truly been infected. Additionally, there is a paucity of research into post mortem antibody testing. An antibody screening tool that is suitable for use in the mortuary setting would go a long way to better document previous COVID-19 infections in deceased persons for surveillance purposes, which would add value to public health systems. This pilot study aimed to explore the use of the Sure Screen COVID- 19 IgG/IgM Rapid Test Cassette in a deceased population, and to compare it to the gold- standard antibody tests in South Africa, to determine the most suitable form of antibody testing for post-mortem samples. Thirty cases, with suspected COVID-19 infection in their lifetime, were recruited from Salt River and Tygerberg mortuaries following informed consent from next-of-kin. Positive COVID-19 PCR (PCP) test confirmation for SARS-CoV-2 was located for 19 of the participants. Blood was collected at autopsy into serum separator tubes which, were found to separate better when centrifuged immediately after sample collection. Sure Screen testing was carried out alongside Roche Diagnostics Elecsys Anti-SARS-CoV-2 and Abbott Architect SARS-CoV-2 IgG Assay. For the confirmed PCP cases, Elecsys' sensitivity was the highest at 94.74%, followed by Sure Screen IgG (78.95%). There was only one case with PCP confirmation with a negative Elecsys result and, in this instance, there was a longer interval between death and autopsy (8 days). No variables relating to time intervals between PCP, death and antibody testing were found to significantly influence the antibody test results. Overall Roche's Elecsys performed the best on our cohort of post-mortem serum samples, followed by Sure Screen, and lastly, Abbott's Architect assay. Based on these results alone, the Sure Screen test demonstrates potential as a screening tool in the mortuary setting, which should be followed up with Roche's Elecsys assay for diagnostic confirmation. However, it is recommended that the sample size be expanded to add weight to this preliminary conclusion.
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    DNA barcoding of forensically important blow flies (Diptera: Calliphoridae) within the Western Cape of South Africa
    (2019) Kulenkampff, Kyle Sieghard; Heathfield, Laura; Heyns, Marise
    In forensic entomology, determining species identity is a crucial step towards estimating post mortem interval. DNA barcoding can aid in the identification of unknown forensically relevant species, and this requires the comparison of DNA barcodes to reference data from known species. However, there is a lack of DNA barcode reference data of forensically relevant Calliphoridae species in the Western Cape (South Africa). DNA barcodes were generated for the COI and ITS2 markers for 41 forensically relevant Calliphoridae specimens, representing seven species from six localities in the Western Cape: Chrysomya albiceps (n = 3), Chrysomya chloropyga (n = 8), Chrysomya marginalis (n = 5), Chrysomya megacephala (n = 7), Hemipyrellia fernandica (n = 1), Lucilia cuprina (n = 8) and Lucilia sericata (n = 9). This data was combined with that from Cooke et al. (2018) (n = 40), and subjected to rigorous statistical and phylogenetic analyses. Phylogenetic analysis which combined data for both COI and ITS2 barcodes returned monophyletic clades for each species with increased support when compared to using each barcode individually. This combined dataset was able to discriminate between L. cuprina and L. sericata with full support (100% pP), which was not achieved previously. DNA barcodes were evaluated for intra- and inter-specific variance as well as haplotype patterning. No haplotype patterning was observed for either barcodes across sampled localities. Lastly, a single-blinded approach was used to assess the dataset, whereby DNA barcodes from ‘unknown’ specimens were correctly identified using this reference data. These identifications were more accurate than those using GenBank® or BOLD, highlighting the importance of using locally relevant reference data. This study has contributed new data pertaining to DNA barcodes for seven Calliphoridae species, which was previously scarce for the Western Cape, and this has directly contributed to an improvement in the accuracy of local species identification.
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    DNA barcoding of forensically important flies in the Western Cape
    (2016) Cooke, Tenielle Monique; Heyns, Marise; Heathfield, Laura
    One of the central applications of forensic entomology is the determination of the post mortem interval (PMI) from arthropod evidence associated with a corpse. Estimations of the PMI are based on succession and developmental patterns of specific species that visit the body. As first colonisers, Calliphoridae (blow flies) are often used by forensic entomologists to determine the PMI however, developmental rates of visiting fauna differ substantially which makes correct species identification vital. Traditional methods of identification which assign species based on keys that capitalise on morphological differences are insufficient for closely related species, especially during immature stages of the lifecycle or when the specimen is damaged. Molecular identification such as DNA barcoding has therefore become a popular method of identifying species. DNA barcoding characterises species by sequencing and analysing specific regions in the genome. This technique has been used to characterise species in various countries including parts of South Africa. Its application has also been demonstrated in a forensic setting but data for the Western Cape is minimal. This study therefore aimed to assess the utility of DNA barcoding for species level determination of four blow fly species common to the Western Cape of South Africa (Chrysomya chloropyga, Chrysomya albiceps, Chrysomya marginalis, and Lucilia sericata) as well as its ability to identify immature specimens. Ten adult specimens from each species were morphologically and molecularly identified using microscopy and DNA barcoding respectively. The standard DNA barcode, cytochrome c oxidase subunit I (COI) and a secondary marker, the second internal transcribed spacer (ITS2) were analysed. Phylogenetic analyses for both barcodes showed high interspecific divergence values which are desirable for species level differentiation by DNA barcoding. COI sequences from adult flies were also submitted and searched against BOLD for identification and only genus level identity could be achieved, indicating that, COI alone may be insufficient to discriminate between closely related species. DNA sequences from the adult specimens were then used as reference sequences for identification of seven unknown immature specimen using DNA barcoding of both COI and ITS2. Sequence similarity was assessed and identity was assigned based on >98% similarity scores, and all immatures were successfully identified. The use of more than one DNA marker to complement morphological data ensures higher confidence of species level identification. This method provides a reliable and consistent tool for entomologists to use for species identification which results in higher levels of accuracy in PMI estimations.
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    Evaluating the role of DNA evidence in sexual offence cases in Zambia between 2007 and 2014
    (2017) Makasa, Innocent; Heathfield, Laura
    Zambia has reported high incidences of sexual abuse against women and children in recent years. Zambian law categorises sexual offences into; rape, defilement, incest and others, with defilement constituting the majority of the cases (>89%). Between 2010 and 2012, only <39% of defilement cases were taken to court, and convictions were achieved in only 13% of the cases reported to the police. Literature was reviewed to determine factors which contributed towards the resolution of criminal cases, and it was found that DNA evidence was prominent in resolving crimes, specifically as an identification tool in sexual offences. Currently there is no empirical evidence describing how DNA evidence has been used in resolving sexual crimes in Zambia. The causes of low prosecution and conviction rates have also not been investigated. A retrospective study was therefore conducted to evaluate the role of DNA evidence in sexual offence cases in Zambia, reported to eight major police stations in Lusaka between 2007 to 2014 (n=1154). Sexual offence cases comprised rape (n=74, 6.4%), defilement of a child under the age of sixteen years (n=1028; 89.1%), incest (n=7; 0.6%) and others (n=45; 3.9%). Only 14 (0.1%) of the cases had forensic samples collected in the form of a vaginal swab for the sole purpose of determining the presence of semen. In all cases where a suspect was identified (60%), identification was based on the witness/victim testimonies, and in no case was forensic DNA evidence used to assist in identification or corroborate the testimonies. Overall, 28.1% cases were taken to court and the conviction rate was 12.4%. If no injuries were observed on a victim aged between 0 - 5 years, the case was not taken to court. It was also observed that the younger the victim, the more likely the accused was not identified (p < 0.001), victims did not know the date of occurrence (p < 0.001), and the case was closed due to insufficient evidence. These findings support the use of employing forensic DNA evidence in sexual offence cases to aid the identification of suspects, either in the absence of witness/victim testimonies or alongside as corroborative evidence, which is hypothesised to increase the number of cases prosecuted in Zambia. At the time of this study there was no standardised protocol for the forensic investigations of sexual offences in Zambia, which to some extent, led to numerous missing data. Development and use of the national protocol and use of a validated sexual assault evidence collection kit may help mitigate the deficiencies and inconsistencies witnessed during this study.
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    The evaluation of tests for the identification of semen
    (2016) Curry, Lyle; Heathfield, Laura
    The identification of bodily fluids for forensic purposes is typically classified as either presumptive or confirmatory. Presumptive tests (PT) are conducted first to screen for certain compounds which are relatively specific to particular fluids. Confirmatory tests are used to confirm the identity of a body fluid. Semen is one of the most common bodily fluids encountered in sexual assault cases and contains high concentrations of the acid phosphatase (AP) enzyme. The brentamine FB reagent reacts with the AP that is present in semen, and turns purple. If the colour change is observed within a specific time threshold, it is considered presumptively positive for semen. Cut-off time varies considerably between forensic laboratories, but in South Africa, the cut-off time is defined as 65 seconds. Additionally, semen may be considered to be from human origin if it reacts within 50 seconds. These cut off times have been arbitrarily defined, and there is little research in a local context to substantiate or inform the threshold time for the brentamine FB test for semen. Therefore this study assessed the sensitivity, specificity and kinetics of the brentamine FB test on semen from South African male volunteers (n=15), canines (n=2) and various fruit extracts and compared these results to purified human AP. Each semen sample was subjected to the PT in an indirect and direct method, and these tests were performed both on fresh and aged samples. The majority of fruit extracts yielded a distinctly different colour change compared to the purple that was produced from semen except for mushroom which also turned purple. Absorbance spectroscopy was used to determine the rate of the reaction at 525 nm. There were no significant differences between the rate of reaction for fresh and aged samples using both direct and indirect testing.
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    Exploring the Medico-legal death scene investigation of sudden unexpected death of infants admitted to Salt River mortuary, Cape Town, South Africa
    (2018) Bennett, Tracy; Heathfield, Laura; Martin, Lorna
    A death scene investigation (DSI) forms an integral part of the inquiry into death, particularly for sudden unexpected death of infants (SUDI). Global guidelines exist for DSI, however, it is unclear how many countries adhere to them, and to what extent they are followed. Therefore, a systematic literature review was undertaken to assess the scope of SUDI DSI performed internationally. It was found that national protocols have been established in some countries, and have shown value in guiding medico-legal examinations. Further, South Africa did not routinely perform DSI for SUDI cases, nor was there a protocol. This was largely attributed to the burden of SUDI cases as well as the lack of resources. Therefore, this study aimed to suggest realistic and feasible ways to improve DSI for local SUDI cases. This research study consisted of three phases: 1) A twoyear review of medico-legal case files from SUDI cases investigated at Salt River Mortuary; 2) The prospective observation of DSI for ten SUDI cases, using a semi-structured checklist; and 3) he distribution and analysis of a survey regarding SUDI DSI to all registered, qualified forensic pathologists in South Africa. The results showed that the SUDI death scenes were assessed in 59.2% of cases at Salt River Mortuary, with inconsistent levels of documentation or photography. Death scenes were never investigated in cases where the infant was pronounced dead on arrival at a medical facility. In both scene observations (n=10) and retrospective analysis (n=454) only one case incorporated a re-enactment, but the majority of infants were moved prior to DSI. The findings support the need for a standardised approach to DSI, coupled with specialised training for staff. Based on the available resources, this should focus on the establishment of guidelines pertaining to photography, handling medicine and scene reconstruction, as well as accurate use of relevant documentation.
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    Exploring the potential for biomarkers to aid forensic diagnosis of traumatic brain injury (TBI) – a systematic literature review and meta-analysis
    (2024) Velcich, Carly; Abrahams, Shameemah; Heathfield, Laura; Molefe, Itumeleng
    Background: Traumatic brain injury (TBI) is a prevalent condition worldwide. Understanding its pathophysiology is imperative for clinical diagnosis, treatment, and cause of death determination. Biomarkers could offer potential insight. Proteins involved in neuroinflammation, such as systemic inflammatory biomarkers interleukin (IL)-1β, IL-6, and IL-10 and astroglia-associated biomarkers S100 Calcium-Binding Protein B (S100β) and Glial Fibrillary Acidic Protein (GFAP), have been assessed as potential TBI biomarkers. The aim of this review was to evaluate recent articles that investigated these biomarkers in relation to TBI and relate this to a forensic diagnostic context. Methods: This review included 44 peer-reviewed articles from three major literature databases published from 2018 onwards, that investigated either IL-1β, IL-6, IL-10, GFAP, S100β, or a combination thereof, in relation to TBI. Studies conducted in a clinical or forensic setting were included. A meta-analysis was conducted on a subset of these studies. Results: Majority of the biomarkers were elevated in TBI versus control groups. The most promising biomarkers were GFAP and S100β, which in addition to being elevated also correlated with unfavourable outcomes and TBI severity. GFAP alone was increased in TBI patients with positive CT scans. The ILs had inconclusive results due to minimal studies and inconsistent study designs. A wide range of biomarker expression levels were noted across all articles (from 0.01 to 1.5 million pg/mL). The meta-analysis yielded a pooled effect size of 0.97. Discussion: Inconsistencies in results could potentially be explained by heterogenous TBI and control groups, various body specimens, and different immunoassays used. Thus, each biomarker should be investigated systematically whilst keeping other variables consistent to ensure definitive conclusions. Overall, none of the proteins could function as biomarkers of TBI alone. However, the meta-analysis did indicate a moderately significant association between biomarker levels and TBI occurrence. Future studies are needed to corroborate the findings.
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    Forensic genetic research on sudden unexpected death in an infant (SUDI) at Salt River Mortuary: experiences an perceptions of parents
    (2018) Louw, Susan; Heathfield, Laura; Wessels,Tina-Marié
    The unexpected and sudden death of an infant (SUDI) is a traumatic event. SUDI is defined as all deaths occurring suddenly and unexpectedly in infants under the age of one year. Molecular autopsy is used to determine the potential genetic contribution to SUDI and may lead to screening and interventions of at-risk family members. However, the potential of this may only be realised if the family members are willing to engage in the follow-up process. Next-of-kin experiences of participating in molecular autopsy research are unknown, and not previously done in South Africa. This study explored the experiences and perceptions of bereaved next-of-kin participating in forensic genetic research on SUDI at Salt River Mortuary, Cape Town. Methods Eleven participants, including the mothers and other family members for six SUDI cases participated in the study. These participants were recruited from a larger forensic molecular autopsy study conducted at the University of Cape Town. In order to explore the experiences and perceptions of next-of-kin, a qualitative approach was used and semi structured interviews were conducted. The interviews, transcribed verbatim, were analysed through thematic analysis. The perspective from the main researcher in the larger forensic molecular autopsy study was included to holistically explore the setting in which the genetics research took place. Results Four major themes were identified, namely (i) old wounds, (ii) my booboo, (iii) the sudden death and (iv) afterthought. Their main reasons for participating in the research were to find answers and to be of value in future cases of SUDI. Grief seemed to play a significant role in their understanding and engagement with regards to their research participation. Conclusion This study found that the grief and loss of at the time of obtaining consent may play a significant role in understanding and willingness for further engagement with molecular autopsy results. Understanding has previously been implicated in the willingness to engage with genetics results, however, it has not been explored in a mortuary setting. The understanding of genetics research is critical for further engagement that may have implications for the screening of other family members and future offspring. These findings may allow researchers to better engage with participants in genetics research on sensitive topics, including SUDI.
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    Forensic human identification: Generating Y-STR data for the South African population
    (2018) Reid, Kate Megan; Heathfield, Laura; Martin, Lorna
    Salt River Mortuary (SRM), Cape Town, investigates ~3500 cases of unnatural death annually, with an apparent burden of unclaimed bodies. A retrospective review was first undertaken to assess the number of these individuals who remained unidentified. Medicolegal records were examined (2010-2017), and ~9% of cases remained unidentified each year. DNA analysis was performed in 23.5% of cases. At the time of this study, unidentified bodies were in storage for up to two years, pending pauper burial. DNA profiling assists forensic human identification, and the analysis of markers on the Y-chromosome has particular importance in kinship analysis. To evaluate the statistical probability of DNA profiles matching between samples, reference data from the background population is required. Such data for the Y-chromosome is lacking for some populations groups in South Africa (SA). As such this study aimed to generate Y-chromosome data relevant to SA. Second to this, the obtainability of DNA profiles from unidentified decedents at SRM, prior to pauper burial, was investigated. Biological samples were obtained from 653 SA individuals (living: n=480; deceased: n=173) belonging to four major population groups. Following internal validation, samples were processed using the Promega PowerPlex® Y23 System. A cohort-representative subset of DNA profiles were also generated using the forensically validated Next Generation Sequencing (NGS) assay on the MiSeq FGx™ system, to assess concordance. Statistical analysis was performed using Arlequin and STATA packages. Full DNA profiles (i.e. haplotypes) were obtained from 626 samples (African: n=183; Coloured: n=170; Indian/Asian: n=111; White: n=162), with 599 haplotypes being unique to a single individual. Following optimisation, haplotypes were obtained from >99% and 85% of living and deceased individuals, respectively. Haplotypes were generated from numerous individuals stored for over one year, and DNA profile quality was not associated with time between death declaration and sample collection. NGS results confirmed the presence of one micro-variant and resolved allele-calling in five instances where the capillary electrophoresis assay was incorrect. Thus, concordance was observed in 98% of loci reviewed. Overall, haplotypes were successfully obtained for four different SA population groups, including refrigerated decedents, even 887 days after death declaration. This demonstrates that DNA profiling can be successful for decedents and efforts should be made to store DNA profiles for the possibility of familial searching and identification, even after burial. Identification of the multitude of unclaimed bodies at forensic facilities nationwide holds immense value for living family members, and provides closure for the acceptance of death and life thereafter.
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    Investigating the effect of NucleoSpin® Forensic Filters on DNA recovery from swabs
    (2021) Hitewa, Alina Ndahafa; Heathfield, Laura; Gibbon, Andrea; Mole, Calvin
    The burden of unresolved crime in South Africa highlights the need to improve methods of identifying perpetrators of crimes. One globally accepted method for human identification is forensic DNA profiling. Since trace evidence is often retrieved in small amounts, the optimal recovery of DNA from these samples is crucial. Methods for the recovery of touch DNA from swabs typically make use of a spin basket or filter, combined with a centrifugation step, to enhance the release of cells from the swab prior to DNA extraction. The NucleoSpin® Forensic Filter (Macherey-Nagel, Düren) is one such example, but it has not been thoroughly assessed on touch DNA samples. This study aimed to assess if the inclusion of the NucleoSpin® Forensic Filter significantly improved DNA recovery and DNA profiling success from cotton and flocked swabs used to collect touch DNA and buccal cells (control). Buccal cells and touch DNA samples were collected from 25 volunteers using each swab type (cotton and flocked) in duplicate. DNA was extracted from the samples using the NucleoSpin® DNA Forensic kit, one set with, and the other set without, NucleoSpin® Forensic Filters. DNA concentration was assessed using Qubit™ fluorometry and qPCR, and DNA profiling was done using the PowerPlex® ESX 16 system. The inclusion of the NucleoSpin® Forensic Filters significantly improved DNA concentration in buccal cells collected using flocked swabs (p = 0.035). However, no significant differences were noted for touch DNA samples, for either swab type. There was also no significant difference in DNA profiling success when NucleoSpin® Forensic Filters were used, regardless of swab and sample type. These results suggest that the NucleoSpin® Forensic Filters should not be included in the DNA extraction workflow, particularly for touch DNA samples. With only 16 % of touch DNA samples yielding full DNA profiles, there is the need to improve DNA recovery. Factors such as swab type and swab preservation buffers, should be investigated in future research.
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    Investigation into DNA recovered from human teeth for forensic applications
    (2020) Haikney, Tarryn; Heathfield, Laura; Gibbon Victoria
    In South Africa, there is a burden of unidentified deceased individuals in forensic mortuaries. When human remains are severely compromised, hard tissues may provide the only DNA source for identification. The QIAamp® DNA Investigator Kit is used in forensic laboratories worldwide, including in South Africa, to extract DNA for identification purposes. However, in local forensic casework, the DNA recovered from teeth is often of insufficient quantity and quality for generating a DNA profile. The phenol-chloroform DNA extraction method has demonstrated improved, yet inconsistent results, when used on hard tissues. Therefore, this study assessed DNA recovery from 52 human control teeth from three deceased individuals, using an optimised phenol-chloroform method. This method involved an overnight demineralisation, two additions of phenol/chloroform/isoamyl alcohol (25:24:1) and an ethanol precipitation, as used by the Australian Federal Police. Quantitative PCR (Quantifiler™ Trio DNA Quantification Kit) and DNA profiling (PowerPlex® ESI 16 System) were then used to assess DNA quantity and quality. Results were compared to those obtained from the same teeth but extracted using the QIAamp® DNA Investigator Kit. The phenol-chloroform method recovered DNA with significantly higher yields (p = 0.0454) and significantly less degradation (p < 0.0001). Despite this improvement, there was no significant difference in DNA profiling success. This study also did a preliminary analysis of other factors affecting results and suggested that premolars might be the best tooth type with regards to DNA quantity, quality and profiling. Furthermore, dental disease and jawbone had a significant impact on results from teeth. Lastly, the phenol-chloroform method was applied to six teeth from a marine decomposition case to assess its performance in a local forensic setting. DNA metrics were particularly poor in this casework example, highlighting how different forensic and control environments are and the need for further optimisation. Overall, this study supports the use of the phenol-chloroform method and has provided a preliminary suggestion of the best tooth type, jawbone and tooth condition for DNA analysis for forensic human identification.
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    Molecular Forensic Investigations into Animal Sexual Abuse
    (2020) Natha, Khilona; Heathfield, Laura; Mole, Calvin
    Animal sexual abuse (ASA) involves the sexual molestation of animals by humans. The identification of semen provides a legally-accepted indicator that sexual activity occurred, while forensic DNA analysis provides a lead to a potential suspect. After conducting a systematic literature review, no previous research investigating semen and/or DNA recovery from animals over time was found. Therefore, this pilot study aimed to assess the recovery of human semen and DNA from animal fur over a two-week period to establish baseline data pertaining to evidence retention in the ASA context. This pioneer study also attempted to contribute towards the development of a suitable animal fur model on which to perform experiments. Daily swabbing and testing of semen from three fur models (unpreserved baboon fur, preserved nyala hides and faux fur) showed that semen could still be detected at 14 days using standard presumptive and confirmatory tests. Although DNA degradation showed a statistically significant increase over time, forensically usable DNA profiles (≥ 12 fully typed short tandem repeat loci) were consistently obtained. There was significantly higher DNA degradation in samples from the baboon fur compared to the others, while DNA concentrations were significantly different between each fur model. These differences highlight that future research must consider the choice of fur model to best represent the animal of interest; e.g. dissected fur from a recently deceased animal would best mimic a fatal ASA case. The insight regarding the choice of animal model hopes to be of benefit for future research, which should focus on the influence of more realistic variables (e.g. movement and body heat) on semen and DNA retention on animal fur. Overall, this study successfully generated baseline data, and provides a foundation for additional research, which hopes to eventually assist in the interpretation of forensic evidence in the global burden of ASA.
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    Optimisation of DNA extraction from teeth submerged in sea water in False Bay, South Africa
    (2018) Longden-Thurgood, Chandra; Gibbon,Victoria; Heathfield, Laura
    Extracting forensically useable DNA from human remains recovered from an open marine environment is problematic, and in some cases, impossible. The reason is unclear given the lack of research on marine decomposition, DNA survival in seawater, and possible methods to optimise the DNA extraction workflow. Compounding this problem is the fact that South Africa experiences a high number of unidentified human bodies entering its mortuaries each year, and these individuals often remain unidentified. The aim of the study was to extract forensically useable DNA from pig (Sus scrofa) teeth submerged in-situ in an open marine environment, by a process of optimisation and implementation. Detailed environmental information was available for this study. A DNA extraction technique was developed and optimised on “fresh” control pig teeth (n = 13). The developed methods for decontamination, tooth sampling, and the optimised DNA extraction protocol were successfully performed on these, with forensically useable DNA obtained. However, this was not the case for the subsample of experimental pig teeth (n = 6) tested. Implementation of the developed method on a larger sample of experimental teeth (n = 28) was warranted to assess the recovery of nDNA and mtDNA. Amplification of nDNA by qPCR was successful in 60% (17/28) of samples for a 96 bp fragment, and in 46% (13/28) for 200 bp. By comparison, mtDNA showed a detection rate of 57% (16/28) for a 486 bp fragment via PCR amplification. In seven samples mtDNA was detected where nDNA was not, demonstrating improved survivability in seawater. Colder and more stable seawater temperatures is hypothesised to have preserved molecular elements. DNA hydrolysis and the possibility of DNAase activity from marine bacteria, may have contributed to poor DNA preservation in the other samples. Recovery of DNA from teeth submerged in an open marine environment is complex and requires further investigation in human samples to improve the identification process for individuals who have died at sea.
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    Optimisation of sample preparation for DNA extraction from formalin fixed paraffin embedded tissues of unresolved sudden unexpected death cases
    (2020) Viljoen, Rabia; Heathfield, Laura; Mole, Calvin
    A retrospective case review revealed an increase in sudden unexpected death (SUD) admittance at Salt River Mortuary (SRM) between 2014 and 2018, and that 40 % of SUD occurred in young individuals between the ages of 1 and 40 years old (SUDY). Despite extensive investigations, the cause of death remained undetermined in 26 % of SUDY cases. These dormant cases may benefit from retrospective post-mortem molecular autopsies for investigation into genetic causes of death. Often, formalin fixed paraffin embedded tissues (FFPETs) are the only archival sources of DNA available for retrospective analyses. This study aimed to optimise DNA recovery from FFPETs for potential use in molecular autopsies of unresolved SUDY cases. To this end, DNA was extracted from FFPET sections using the QIAamp® DNA FFPE tissue kit; the thickness and number of sections were varied. DNA was assessed using spectrophotometry, real-time PCR and digital capillary electrophoresis. Results showed that finer sectioning (1-µm thick as compared to 3-µm and 5-µm thick), improved DNA concentrations, purities and DNA fragment lengths. Increasing the number of 1-µm thick sections from 30 to 100, significantly improved DNA yield. DNA was not significantly more degraded for FFPETs stored for up to three years, which holds promise in the effectiveness of the technique for aged samples. The DNA extraction method developed in this study yielded a median of 320 ng (287 ng - 698 ng) of DNA with 55 % of DNA fragments being at least 400 bp in size. These results are especially informative for downstream molecular analyses, indicating that genotyping or sequencing assays need to be designed to target amplicons less than 400 bp in size. The degraded nature of the FFPET samples also suggests that massively parallel sequencing might be suited for downstream molecular analysis for determining cause of death in unresolved SUDY cases.
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    Post-mortem Molecular Investigation: exploring genetic variation in CYP2D6 in deceased individuals at Salt River Mortuary
    (2018) Vincent, Devin Michael; Heathfield, Laura; Davies, Bronwen
    Drug use is a major burden in Cape Town, South Africa, and at times may be fatal. Individuals suspected to have demised from drug intoxication are referred for medico-legal investigation, in order for cause of death to be determined. Sometimes, it remains ambiguous as to whether the drug intoxication was suicidal or accidental, even after a full post-mortem examination. Literature has shown that molecular analysis of genetic variants in genes encoding for drug metabolising enzymes may provide insight into the manner of death. At Cape Town’s Salt River Mortuary, numerous toxicological-related cases yield ambiguous results, which may potentially be resolved with molecular analyses. However, no optimised molecular assay to sequence drug metabolising enzymes currently exists in a local context. The aim of this project was to design and optimise a molecular-based assay to sequence the drug metabolising enzyme, CYP2D6. Subsequent to primer design, exons in CYP2D6 were amplified and sequenced. The optimised assay was then applied to DNA from two decedents suspected to have demised from drug intoxication. Following a toxicological drug screen, certain drugs metabolised by CYP2D6 were reported. The assay revealed genetic variants within CYP2D6; both individuals were heterozygous for 138insT, rendering one allele in each individual defective. While one decedent also exhibited variants with normal and unknown haplotypes, the other decedent was homozygous for *17 (decreased functionality), overall making the former an intermediate (altered) or extensive (normal) metaboliser and the latter, an intermediate metaboliser of specific drugs. Quantitative toxicological results were unavailable; consequently, the contribution of the metabolism phenotype on death in these cases could not be established. However, the genetic variants, combined with the presence of these drugs in each case, suggests altered drug metabolism, which should be investigated further and interpreted within each case context. These findings would also be beneficial to the decedents’ living relatives, who may also carry these variants. Overall, this study demonstrates the value of molecular analyses in forensic investigations of toxicological-related fatalities, and lays the foundation for additional future research, particularly since the molecular assay has now been successfully optimised.
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    Post-mortem toxicogenetics: determining the suitable of blood samples collected for routine toxicological analyses for use in subsequent genetic analyses
    (2018) Vuko, Loyiso Abongile Marvin; Davies, Bronwen; Heathfield, Laura; Auckloo, Kathrina
    South Africa has one of the highest prevalences of drug misuse and abuse in Africa. Salt River Mortuary (Cape Town, South Africa), along with other national Forensic Pathology Service providers, receives many cases of suspected drug-related deaths. In some cases, the traditional autopsy – when viewed together with the decedent's history – is not able to indicate whether a drug-related death is accidental or suicidal in relation to altered drug metabolism. Literature has shown that this can be investigated by sequencing gene(s) encoding the implicated metabolising enzyme(s) in a postmortem genetic analysis. However, as such an analysis would normally be performed following the obtainment of postmortem toxicological results, it is imperative to investigate whether blood samples retrieved back from a toxicology laboratory would be sufficient for the said genetic analysis, despite the handling involved in the process of toxicological investigation. To this end, blood samples from 30 deceased individuals in which drug use/abuse may have contributed to death, were collected into two red-top tubes (plain), two grey-top tubes (containing sodium fluoride and potassium oxalate) and one EDTAcontaining purple-top tube (control). DNA was immediately extracted from one of each colour tube, while the duplicate red-top and grey-top tubes first underwent a process of toxicological analyses, and then underwent DNA extraction. The concentration, degradation, purity, contamination, and quality of DNA were assessed using real-time PCR, spectrophotometry, forensic DNA profiling, and Sanger sequencing. In contrast to the grey-top tubes, the results showed that the red-top tubes were most suitable for the aforementioned genetic analysis. Overall, the study not only demonstrated that postmortem genetic analysis using samples retrieved from a toxicology laboratory is possible in the local context, but also provided guidelines around the pre-analytical phase of the analysis. These results illustrate the opportunity to investigate these toxicogenetic avenues further, particularly in future expansion of services currently provided at Salt River Mortuary, which may provide families more information about circumstances of their relative’s death.