Understanding the burden of unidentified bodies: a systematic review.

While human identification is a crucial aspect of medico-legal investigations, many individuals remain unidentified each year across the world. The burden of unidentified bodies is often referred to when motivating for improved methods of identification, and anatomical teaching, yet the actual burden is somewhat unclear. A systematic literature review was undertaken to identify articles that empirically investigate the number of unidentified bodies experienced. Despite the large number of articles returned, an alarmingly low number (24 articles) provided specific and empirical details on the number of unidentified bodies, demographics and trends thereof. It is possible that this lack of data is due to the variable definition of 'unidentified' bodies and the use of alternative terminology such as 'homelessness' or 'unclaimed' bodies. Nevertheless, the 24 articles provided data for 15 forensic facilities across ten countries of both developed and developing statuses. On average, developing countries experienced more than double (9.56%) the number of unidentified bodies when compared to developed nations (4.40%). While facilities were mandated under different legislations and infrastructures available varied greatly, the most common issue faced is the lack of standardised procedures for forensic human identification. Further to this, the need for investigative databases was highlighted. Through addressing the standardisation of identification procedures and terminology, alongside the appropriate utilisation of existing infrastructure and database creation, the number of unidentified bodies could be significantly reduced globally.

Towards molecular autopsies: Development of a FFPE tissue DNA extraction workflow.

Sudden unexpected death (SUD) is a devastating event and forms a substantial proportion of the cases investigated at forensic mortuaries each year. Despite post-mortem investigations, the cause of death may remain undetermined. There is potential for these unresolved cases to benefit from retrospective molecular autopsies for investigation into genetic mutations which may have contributed towards death. Often, formalin fixed paraffin embedded tissues (FFPET) are the only archival sources of DNA available for retrospective analyses. However, extracting usable DNA from FFPET is challenging as current methods yield poor quality and quantity DNA. Thus, this study aimed to optimise DNA recovery from FFPET by investigating several variables within the DNA extraction workflow, including the selection of tissue type, number and thickness of tissue sections, deparaffinisation method, and DNA extraction kit. The quantity and quality of DNA recovered were assessed using spectrophotometry, real time PCR, digital capillary electrophoresis and DNA profiling. This study was the first to implement a nuclei quantification using microscopy to guide the selection of the best tissue type to use for DNA analysis. The use of a greater number of thinner tissue sections (100 sections, each 1 µm) significantly improved DNA concentration, purity and fragment length. Additionally, the combination of Deparaffinization Solution with the QIAamp® DNA FFPE Tissue Kit proved most favourable with a median DNA yield of 320 ng and 55% of DNA fragments greater than 400 bp. Isolated DNA was of single source, indicating no contamination in the workflow, and FFPET blocks that were stored for up to 3.5 years did not significantly affect DNA degradation (p = 0.1764). These results are especially informative for designing library preparation and sequencing workflows for determining cause of death in unresolved SUD cases.

Evaluation of direct PCR for routine DNA profiling of non-decomposed deceased individuals.

Forensic DNA profiling is a standard method used in the attempt to identify deceased individuals. In routine investigations, and if available, the preferred sample type is usually blood. However, this requires the invasive re-opening of the body, days or weeks after the autopsy, which is undesirable in resource-constrained mortuary settings. Motivated by the ease of sampling as well as reduced health and safety risks, this study aimed to establish the success rate of generating a full DNA profile on first attempt from buccal swab lysates using a direct PCR approach. Buccal swab samples were collected from 100 unidentified deceased males, and were subjected to direct DNA profiling with use of the Promega PowerPlex® Y23 Kit. At the time of sample collection, these individuals had been stored for between 1 and 887 days. This study shows that full DNA profiles were initially obtained from 73% of samples, which constitutes the first empirical data pertaining to first time success rates of direct PCR from post-mortem buccal lysates. Further investigation of partial and failed DNA profiles using real-time PCR showed that samples did not contain PCR inhibitors, DNA was not degraded, but DNA concentration was particularly low. Repeating DNA profiling with increased lysate input and extra PCR cycles yielded an additional six full DNA profiles, resulting in an overall success rate of 79%. Overall, DNA profile success rate was not associated with the duration of storage (p = 0.387). Lastly, massively parallel sequencing with the ForenSeq™ Signature DNA Prep kit provided more informative profiles for three additional samples. These results indicate that blood should therefore remain the sample of choice in a post-mortem setting, yet buccal lysates hold potential to be optimised further, which may ease the human identification workflow.

A first for forensic genetics in Africa: successful identification of skeletal remains from the marine environment using massively parallel sequencing.

In unrelated circumstances, two young adult males allegedly went missing off the coast of Cape Town, South Africa, within two months of each other. Weeks after the second disappearance, a decomposed human lower limb was recovered from a beach in Cape Town, followed by a washed-up decomposed hand three days later. An item of female clothing was found with the remains, and preliminary analysis of the skeleton indicated a female, leading to confusion regarding the possible identity of the decedent. Consequently, DNA analyses were requested to determine the biological sex of the remains, and whether the two sets of remains originated from the same individual. Various samples were collected, including bone, nails and swabs of soft tissue. DNA quantity and quality varied between sample types, with better results obtained from metacarpal bone and swab lysates. DNA profiling revealed a male sex, which suggests cognitive bias may have played a role in initial sex estimations. In addition, massively parallel sequencing confidently matched the two sets of remains (random match probability: 1 in 2.70 x 1031). These results were a first for Africa where massively parallel sequencing was successfully used and assisted in the identification of human remains, thus, affording closure to the next-of-kin. Moreover, this constitutes the first global report where soft tissue lysates from a marine decomposition case yielded full DNA profiles with a massively parallel sequencing approach.

A review of the optimisation of the use of formalin fixed paraffin embedded tissue for molecular analysis in a forensic post-mortem setting.

Molecular analyses in a post-mortem setting are becoming increasingly common, particularly in cases of sudden unexplained death, with the aim of identifying genetic mutations which may be responsible for causing death. In retrospective investigations, the access to suitable autopsy biological samples may be limited, and often formalin fixed paraffin embedded (FFPE) tissue is the only sample available. The preservation of tissue in formalin is known to damage DNA through crosslinking activity. This results in the extraction of severely fragmented DNA of variable yields, which subsequently reduces the ability to perform downstream molecular analyses. Numerous studies have investigated possible improvements to various aspects of the DNA extraction and amplification procedures from FFPE tissue and this review aims to collate these optimization steps in a cohesive manner. A systematic review was performed of three major databases, which identified 111 articles meeting the inclusion criteria. Five main areas for optimization and improvements were identified in the workflow: (1) tissue type, (2) fixation process, (3) post-fixation, (4) DNA extraction procedure and (5) amplification. It was found that some factors identified, for example tissue type and fixation process, could not be controlled by the researcher when conducting retrospective analyses. For this reason, optimization should be performed in other areas, within the financial means of the laboratories, and in accordance with the purposes of the investigation. Implementation of one or more of the optimization measures described here is anticipated to assist in the extraction of higher quality DNA. Despite the challenges posed by FFPE tissue, it remains a valuable source of DNA in retrospective molecular forensic investigations.