Validity of postmortem computed tomography for use in forensic odontology identification casework.

Forensic Odontology (FO) identification compares antemortem (AM) and postmortem (PM) dental datasets and is widely accepted as a primary identifier. Traditionally, a PM dental examination is undertaken in the same manner as a dental examination conducted for a living patient. Recently, the increased forensic application of computed tomography (CT) offers an alternative source of PM data. While charting from PMCT is widely accepted as less accurate, the impact on reconciliation is unknown. This study aims to determine if reconciliation outcome differs when PM dental data is collected from PMCT, compared with conventional PM examination. PMCT data was reviewed for 21 cases previously completed using conventional PM dental examination. Operators blinded to original identification outcomes charted from CT images before comparing to AM data to form an opinion regarding identity. Opinions formed were compared with original identification outcomes. Differences in PM dental charting between the two methods and the evidentiary value of AM and PM datasets were assessed to determine driving factors of differences in identification outcome. Compared to conventional PM dental examination, PMCT examination resulted in similar or less certain identification outcomes. Discrepancies in outcome were driven by the quality of AM and PM datasets rather than inaccuracies in charting from PMCT. Based on the results of this study, both conventional and PMCT methods of PM dental examination can reach similar identification outcomes. However, operators remained more certain in establishing identity when conducting conventional PM dental examinations especially when AM data was lacking.

From clean spaces to crime scenes: Exploring trace DNA recovery from titania-coated self-cleaning substrates.

Titanium dioxide (titania, TiO2) is frequently used as a coating for a variety of self-cleaning products, such as antifogging vehicle mirrors, ceramic tiles, and glass windows because of its distinct physiochemical features. When exposed to light TiO2 causes photocatalytic decomposition of organic contaminants, potentially compromising DNA integrity. The impact of TiO2-coated commercial glasses, Bioclean® and SaniTise™, on trace DNA persistence, recovery, and profiling was investigated. DNA in saliva and touch samples deposited on self-cleaning glass slides exposed to indoor fluorescent light for up to seven days was more degraded than control samples indicating some degree of fluorescent light-induced photocatalytic activity of the self-cleaning surfaces. When exposed to sunlight, DNA yields from saliva and touch samples deposited on the titania-coated substrates decreased rapidly, with a corresponding increase in DNA degradation. After three days no DNA samples applied to self-cleaning glass and exposed to natural sunlight yielded STR profiles. These results suggest that the photocatalytic activation of TiO2 is the likely mechanism of action underlying the extreme DNA degradation on the Bioclean® and SaniTise™ glasses. Consequently, rapid sample collection and use may be warranted in casework scenarios involving TiO2-coated materials.

Metal-DNA interactions: Exploring the impact of metal ions on key stages of forensic DNA analysis.

Forensic DNA analysis continues to be hampered by the complex interactions between metals and DNA. Metal ions may cause direct DNA damage, inhibit DNA extraction and polymerase chain reaction (PCR) amplification or both. This study evaluated the impact of metal ions on DNA extraction, quantitation, and short tandem repeat profiling using cell-free and cellular (saliva) DNA. Of the 11 metals assessed, brass exhibited the strongest PCR inhibitory effects, for both custom and Quantifiler Trio quantitation assays. Metal ion inhibition varied across the two quantitative PCR assays and the amount of DNA template used. The Quantifiler Trio internal PCR control (IPC) only revealed evidence of PCR inhibition at higher metal ion concentrations, limiting the applicability of IPC as an indicator of the presence of metal inhibitor in a sample. Notably, ferrous ions were found to significantly decrease the extraction efficiency of the DNA-IQ DNA extraction system. The amount of DNA degradation and inhibition in saliva samples caused by metal ions increased with a dilution of the sample, suggesting that the saliva matrix provides protection from metal ion effects.

Quantitative PCR overestimation of DNA in samples contaminated with tin.

Metals can pose challenges while conducting forensic DNA analysis. The presence of metal ions in evidence-related DNA extracts can degrade DNA or inhibit PCR as applied to DNA quantification (real-time PCR or qPCR) and/or STR amplification, leading to low success in STR profiling. Different metal ions were spiked into 0.2 and 0.5 ng of human genomic DNA in an "inhibition study" and the impact was evaluated by qPCR using the Quantifiler™ Trio DNA Quantification Kit (Thermo Fisher Scientific) and an in-house SYBR Green assay. This study reports on a contradictory finding specific to tin (Sn) ions, which caused at least a 38,000-fold overestimation of DNA concentration when utilizing Quantifiler Trio. This was explained by the raw and multicomponent spectral plots, which indicated that Sn suppresses the Quantifiler Trio passive reference dye (Mustang Purple™, MP) at ion concentrations above 0.1 mM. This effect was not observed when DNA was quantified using SYBR Green with ROX™ as the passive reference, nor when DNA was extracted and purified prior to Quantifiler Trio. The results show that metal contaminants can interfere with qPCR-based DNA quantification in unexpected ways and may be assay dependent. The results also highlight the importance of qPCR as a quality check to determine steps for sample cleanup prior to STR amplification that may be similarly impacted by metal ions. Forensic workflows should recognize the risk of inaccurate DNA quantification of samples that are collected from substrates containing tin.

A custom hybridisation enrichment forensic intelligence panel to infer biogeographic ancestry, hair and eye colour, and Y chromosome lineage.

Massively parallel sequencing can provide genetic data for hundreds to thousands of loci in a single assay for various types of forensic testing. However, available commercial kits require an initial PCR amplification of short-to-medium sized targets which limits their application for highly degraded DNA. Development and optimisation of large PCR multiplexes also prevents creation of custom panels that target different suites of markers for identity, biogeographic ancestry, phenotype, and lineage markers (Y-chromosome and mtDNA). Hybridisation enrichment, an alternative approach for target enrichment prior to sequencing, uses biotinylated probes to bind to target DNA and has proven successful on degraded and ancient DNA. We developed a customisable hybridisation capture method, that uses individually mixed baits to allow tailored and targeted enrichment to specific forensic questions of interest. To allow collection of forensic intelligence data, we assembled and tested a custom panel of hybridisation baits to infer biogeographic ancestry, hair and eye colour, and paternal lineage (and sex) on modern male and female samples with a range of self-declared ancestries and hair/eye colour combinations. The panel correctly estimated biogeographic ancestry in 9/12 samples (75%) but detected European admixture in three individuals from regions with admixed demographic history. Hair and eye colour were predicted correctly in 83% and 92% of samples respectively, where intermediate eye colour and blond hair were problematic to predict. Analysis of Y-chromosome SNPs correctly assigned sex and paternal haplogroups, the latter complementing and supporting biogeographic ancestry predictions. Overall, we demonstrate the utility of this hybridisation enrichment approach to forensic intelligence testing using a combined suite of biogeographic ancestry, phenotype, and Y-chromosome SNPs for comprehensive biological profiling.

"Identified", "probable", "possible" or "exclude": The influence of task-irrelevant information on forensic odontology identification opinion.

In a mass disaster situation, identification of the deceased utilising comparison of dental features is frequently heavily relied upon to facilitate rapid and accurate outcomes. The method consists of the comparison of clinical and radiographic records depicting oral structures and dentition to allow an opinion to be produced on a presumed identity. Current forensic odontology identification opinions are expressed as categories of levels of identification. Categories such as "Identified", "Probable", "Possible" and "Exclude" are used in various forensic odontology identification scales. The boundaries between the levels of the scales are not fixed; hence, category selection is highly subjective. It is uncertain how extrinsic factors such as exposure to contextual task-irrelevant information or operator experience influence category selection. In this study, forensic odontologist and dentist participants read task-irrelevant context case information containing either strong or weak identification or non-identification suggestions before evaluating and comparing pairs of true matching and non-matching dental radiographs. They were then asked to form an opinion regarding identification using one of four categories from the INTERPOL scale. Context information was found to influence categorical decisions. The magnitude and direction of influence depended on the type of participant, the true match status of the radiographs, and the strength and direction of bias of the context. The results of this study demonstrate the contextual effect and fluidity of the boundaries between the categories on the identification scale and highlight the need for stringent protocols to be developed regarding the use of these categorical scales to enable decision making to be more objective.

Ethics reporting in forensic science research publications - A review.

An essential element of compliance with ethical standards in scientific research is the reporting of a verifiable declaration of ethical approval and, when human subjects are involved - informed consent, in published works. The level of reporting of ethical permission for research published in forensic and investigative sciences journals has not been explored to date. Hence, we examined the reporting of ethical approval and informed consent in original research utilising human or animal subjects published in six forensic science journals from 2010 to 2019. We identified 10,192 articles and retained 3010 that satisfied the inclusion criteria of utilising human (91.2%), or animal (7.0%) or both (1.8%) subjects or tissues in experiments. Just over a third (1079/3010) of all studies declared obtaining ethical approval, with 927 (85.9%) of those indicating the name of the ethical committee, but only 392 (36%) provided an approval code. Furthermore, while consent was said to have been sought in 527 (17.5%) of studies, only 155 of those reported that written informed consent was obtained, eleven stated oral (verbal) consent, while the remaining 357 studies (67.7%) did not report the process used to gain consent. Ethical approval reporting rates differed between different research types, availability of financial support and whether authors were affiliated to academia or industry. The results demonstrate a low level of declaration of ethical approval and informed consent in forensic science research and publication, requiring urgent rectification. We support the adoption of the model proposed by Forensic Science International: Genetics as baseline recommendations to facilitate consistent nomenclature, transparency, and standard of ethical reporting in forensic science.

Unveiling forensically relevant biogeographic, phenotype and Y-chromosome SNP variation in Pakistani ethnic groups using a customized hybridisation enrichment forensic intelligence panel.

Massively parallel sequencing following hybridisation enrichment provides new opportunities to obtain genetic data for various types of forensic testing and has proven successful on modern as well as degraded and ancient DNA. A customisable forensic intelligence panel that targeted 124 SNP markers (67 ancestry informative markers, 23 phenotype markers from the HIrisplex panel, and 35 Y-chromosome SNPs) was used to examine biogeographic ancestry, phenotype and sex and Y-lineage in samples from different ethnic populations of Pakistan including Pothwari, Gilgit, Baloach, Pathan, Kashmiri and Siraiki. Targeted sequencing and computational data analysis pipeline allowed filtering of variants across the targeted loci. Study samples showed an admixture between East Asian and European ancestry. Eye colour was predicted accurately based on the highest p-value giving overall prediction accuracy of 92.8%. Predictions were consistent with reported hair colour for all samples, using the combined highest p-value approach and step-wise model incorporating probability thresholds for light or dark shade. Y-SNPs were successfully recovered only from male samples which indicates the ability of this method to identify biological sex and allow inference of Y-haplogroup. Our results demonstrate practicality of using hybridisation enrichment and MPS to aid in human intelligence gathering and will open many insights into forensic research in South Asia.

Comparison of Isohelix™ and Rayon swabbing systems for touch DNA recovery from metal surfaces.

A previous study evaluating two swabbing systems found that DNA was best recovered from sterile metal substrates using an Isohelix™ swab wetted with isopropyl alcohol rather than a Rayon swab with water as the wetting agent. We tested the same swabbing systems on metal (aluminum, brass, and stainless steel) and plastic substrates in a regularly touched environment to simulate the non-deliberate transfer of touch evidence likely seen in a casework scenario, to ascertain the performance of these swabs in an uncontrolled situation. Higher amounts of touch DNA were recovered with Isohelix™ swabs (0.5 - 3.3 ng) compared to Rayon swabs (0.13 - 1.2 ng). The Isohelix™ swabbing system was found to significantly recover more touch DNA (p = 0.04) from the metal substrates than the Rayon swabbing system, consistent with the findings of our previous work. The results contribute to our understanding of the impact of sample collection techniques on touch DNA recovery from problematic metal surfaces and suggest that supplemental cleaning of substrates as a precautionary step against the spread of infections may affect touch DNA persistence and the recovery efficiency of swabs.

The biasing impact of irrelevant contextual information on forensic odontology radiograph matching decisions.

The potential biasing effect of irrelevant context information on the forensic odontology method of radiograph-based identification has never been empirically investigated despite being a recognized problem in other forensic science disciplines. This study examines the effect of irrelevant context information on the probability judgment of match (JOM) of practicing forensic odontologist and dentist participants who were asked to match pairs of dental radiographs supplemented with irrelevant case information. The irrelevant case information contained domain task-irrelevant context information which varied in strength (strong or weak). It suggested either supportive or contradictory bias relative to the actual match status of the radiograph pairs. The dental radiographs consisted of verified match and non-match radiographs pairs sampled and de-identified from actual forensic cases. Changes in accuracy and JOM between supportive and contradictory contexts conditions revealed a contextual bias. Mixed model analysis showed that strong supportive context increased the odds ratio of correct decisions by a factor of 2.4 [1.23, 4.46]; p = 0.0097. Consistent with the biasing effect, the JOM score differences between strong supportive and contradictory irrelevant context information were 1.03 and 0.43 respectively for the non-match and match decisions. The direction of context suggestion (p = 0.0067), the radiograph match status (p = 0.014), and their interactions (p = 0.0061), were all found to impact the participants' decision. The weak context information was not strong enough to have a significant effect on accuracy or JOM scores. This study demonstrates that radiograph match judgment is affected and can be biased by strong irrelevant contextual information.

A review of the current understanding of burned bone as a source of DNA for human identification.

Identification of incinerated human remains may rely on genetic analysis of burned bone which can prove far more challenging than fresh tissues. Severe thermal insult results in the destruction or denaturation of DNA in soft tissues, however genetic material may be preserved in the skeletal tissues. Considerations for DNA retrieval from these samples include low levels of exogenous DNA, the dense, mineralised nature of bone, and the presence of contamination, and qPCR inhibitors. This review collates current knowledge in three areas relating to optimising DNA recovery from burned bone: 1) impact of burning on bone and subsequent effects on sample collection, 2) difficulties of preparing burned samples for DNA extraction, and 3) protocols for bone decalcification and DNA extraction. Bone decalcification and various DNA extraction protocols have been tested and optimised for ancient bone, suggesting that prolonged EDTA (Ethylenediaminetetraacetic acid) demineralisation followed by solid-phased silica-based extraction techniques provide the greatest DNA yield. However, there is significantly less literature exploring the optimal protocol for incinerated bones. Although burned bone, like ancient and diagenetic bone, can be considered "low-copy", the taphonomic processes occurring are likely different. As techniques developed for ancient samples are tailored to deal with bone that has been altered in a particular way, it is important to understand if burned bone undergoes similar or different changes. Currently the effects of burning on bone and the DNA within it is not fully understood. Future research should focus on increasing our understanding of the effects of heat on bone and on comparing the outcome of various DNA extraction protocols for these tissues.

Interpretation, confidence and application of the standardised terms: Identified, Probable, Possible, Exclude and Insufficient in forensic odontology identification.

Forensic odontology identification scales are used to express certainty of identifications of deceased persons. These standardized scales are assumed to convey unambiguous expert opinions and facilitate communication between forensic odontologists and end users. However, to date no studies have investigated how the experts interpret and use these scales. Forensic odontology identification scales are used to express certainty of identifications of deceased persons. These standardized scales are assumed to convey unambiguous expert opinions and facilitate communication between forensic odontologists and end users. However, to date no studies have investigated how the experts interpret and use these scales. This paper aims to examine the interpretation of the DVISYS forensic identification scale and choices of the levels in the scale subsequent to, and derived from, comparison of pairs of dental radiographs by extending the analysis of the data collected in the study by Page and Lain et. al. 2017. The studied variables: self-reported confidence, forced binary decision of match and non-match, choice of level in the DVISYS scale (Identified, Probable, Possible, Insufficient and Exclude) were further analysed in this study using mixed models for relationships between the choices of level in the identification scale and the fundamental beliefs of likelihood of identification. The results of this further analysis showed that the reported confidence of the decisions was correlated to the difficulty of cases, and as confidence decreased the use of less definitive terms ('Probable', 'Possible' and 'Insufficient') increased. 'Probable' and 'Possible' were used mainly in underlying beliefs below that of 'Identified' whereas 'Insufficient' was used mainly to convey a sublevel of 'Exclude'. The use of 'Insufficient' in this study was not consistent with the prescribed definition of the term. The participants of the original study were not aware of the difficulty grading of the cases nor were required to grade them, however the reported confidence was systematically correlated to difficulty. Furthermore, indicated confidence level was correlated with choice of level on the scale in general, but the interpretation of the definition and application of the terms varied. The findings reported here contribute to the foundational knowledge of factors governing the interpretation and application of the DVISYS forensic odontology identification scale and suggest that this scale may need to be modified.

The use of gelling agents to preserve burnt teeth within the dental alveoli for dental human identification - a study utilising sheep mandibles.

Dental comparison is one of the primary methods of scientific identification of severely incinerated human remains. However, due to the fragile nature of the remains dental structures may be lost or damaged during recovery and transportation, limiting the amount of evidence available for examination. In addition to protecting the head, stabilization of the oral structures with an adhesive substance that will not interfere with the dental examination is ideal. A number of materials have been described in previous studies, however, no optimal method has yet to be indicated. Many of these materials contain petrochemicals, which have been shown to be a contamination risk. Wheatpaste solution has been demonstrated to be a viable alternative but has demonstrated handling issues and is not optimal in some environments. This study explores the stabilization of burnt teeth utilizing gelatin and agar solutions as alternatives to wheatpaste. Like wheatpaste solution, these materials are inexpensive, simple to use and are free from petrochemicals. Anterior sections of sheep mandibles were incinerated and subsequently solutions of agar, gelatin or wheatpaste were applied. The jaw fragments were then subjected to vibration and the number of teeth retained within the bone was recorded and compared to untreated incinerated jaw fragments. Although agar solution demonstrated serious handling issues, gelatin solution provided stabilization equivalent to that of wheatpaste. Gelatin also performed well at lower temperature conditions under which wheatpaste has been shown to perform poorly.

Comparison of bone demineralisation procedures for DNA recovery from burned remains.

Recovering DNA from modern incinerated bones can be challenging and may require alteration of routine DNA extraction protocols. It has been postulated that incinerated bones share some similarities with ancient bones, including fragmented DNA, surface contamination and highly mineralised structure, all of which can inhibit the successful recovery of genetic material. For this reason, ancient DNA extraction protocols are often used for incinerated modern samples; however, their effectiveness is still somewhat unclear. Much of this uncertainty exists around the demineralisation step of extraction, specifically the length of incubation and retention or removal of supernatant. As obtaining human samples for forensic research can be challenging, porcine models (Sus scrofa domesticus) are often used as substitutes. This study developed real time PCR assays for porcine nuclear DNA in order to investigate the effects of modified demineralisation protocols on DNA yield from femurs exposed to either short (60 min) or prolonged (120 min) burning. Gradient PCR results indicated 56 °C was the ideal amplification temperature for targeted amplicons, with melt curve analysis showing short and long amplicons corresponded to 80.3 °C and 83 °C peaks respectively. Results of altered extraction protocol showed a trend towards higher DNA yields from longer demineralisation periods however this was not significant. By comparison, retaining supernatant post-demineralisation resulted in significantly greater DNA yields compared to discarding it (P < 0.009). Although DNA content yield decreased with burn duration, the demineralisation treatment variations appeared to have the same effect for all burn lengths. These results suggest that for incinerated modern bone retaining the supernatant following demineralisation can dramatically increase DNA yield.

Evaluation of the efficiency of Isohelix™ and Rayon swabs for recovery of DNA from metal surfaces.

PURPOSE: We investigated the recovery and extraction efficiency of DNA from three metal surfaces (brass, copper, steel) relevant to forensic casework, and plastic (control) using two different swabbing systems; Rayon and Isohelix™ swabs, with sterile water and isopropyl alcohol respectively, as the wetting solutions. METHODS: Twenty nanograms of human genomic DNA were applied directly to Isohelix™ and Rayon swabs; and to the metal and plastic substrates. All substrates were left to dry for 24 h, followed by single wet swabbing and extraction with the DNA IQ™ System. DNA extracts were quantified using real time quantitative PCR assays with SYBR green chemistry. RESULTS: DNA was extracted from directly seeded Isohelix™ swabs with a high efficiency of 98%, indicating effective DNA-release from the swab into the extraction buffer. In contrast, only 58% of input DNA was recovered from seeded Rayon swabs, indicating higher DNA retention by these swabs. Isohelix™ swabs recovered 32 - 53% of DNA from metal surfaces, whilst the Rayon swabs recovered 11-29%. DNA recovery was lowest from copper and highest from brass. Interestingly, Rayon swabs appeared to collect more DNA from the plastic surface than Isohelix™ swabs, however, due to the lower release of DNA from Rayon swabs they returned less DNA overall following extraction than Isohelix™ swabs. CONCLUSION: These results demonstrate that DNA samples deposited on metal surfaces can be more efficiently recovered using Isohelix™ swabs wetted with isopropyl alcohol than Rayon swabs wetted with sterile water, although recovery is affected by the substrate type.

A comparison of crystal structure in fresh, burned and archaic bone - Implications for forensic sampling.

Standard protocols for extracting DNA from bone are variable and are largely dependent on the state of preservation. In archaic samples, endogenous DNA is believed to be tightly bound to crystal aggregates in the Hydroxyapatite (HAp) matrix requiring prolonged demineralisation to allow its release. By comparison, fresh bone contains abundant cellular material, discounting the need for demineralisation. Recommendations for incinerated bone, specifically how viable sampling sites should be selected and the ideal techniques for DNA recovery are unclear, and the protocol used is often selected based on macroscopic sample appearance. It has been postulated that like archaic bone, burned bone is 'highly degraded' and therefore aDNA techniques may present better results for DNA recovery than using fresh protocols. However, little research has been undertaken comparing the crystal structure of burnt, fresh and archaic bone. This study uses a combination of XRPD and SEM analysis to compare the crystalline profile and microscopic appearance of burned bone subjected to temperatures ranging from 100-1000°C, with archaic and fresh samples. Although macroscopically visually different, fresh samples and samples heated up to 500°C showed no microscopic differences or significant changes in crystallinity. By comparison, samples heated above 500°C became significantly more crystalline, with HAp crystal size increasing dramatically. Archaic samples were different again, more closely resembling the amorphous fresh samples than the highly crystalline incinerated samples. These results suggests that, potentially, samples burned at 500°C or lower can be treated as fresh samples, whilst samples exposed to higher temperatures may require adapted protocols. Whether or not these highly burned samples require demineralisation needs to be investigated.

Forensic touch DNA recovery from metal surfaces - A review.

Trace evidence such as touch (also known as contact) DNA has probative value as a vital forensic investigative tool that can lead to the identification and apprehension of a criminal. While the volume of touch DNA evidence items submitted to forensic laboratories has significantly increased, recovery and amplification of DNA from these items, especially from metal surfaces, remains challenging. Currently little is understood with regards to the underlying mechanisms of metal-DNA interactions in the context of forensic science and how this may impact on DNA recovery. An increased understanding of these mechanisms would allow optimisation of methods to improve outcomes when sampling these materials. This paper reviews the basis of DNA binding to metal substrates, the merits and limitations of current methods and future perspectives of improving recovery and amplification of touch DNA from metal surfaces of forensic interest.

A mini-multiplex SNaPshot assay for the triage of degraded human DNA.

Short Tandem Repeat (STR) genotyping is currently the primary DNA-based method for human identification, however it can have limited success when applied to degraded human remains. Massively parallel sequencing (MPS) provides new opportunities to obtain genetic data for hundreds of loci in a single assay with higher success from degraded samples. However, due to the extra requirement for specialised equipment, expertise and resources, routine use of MPS may not be feasible or necessary for many forensic cases. Here we describe the development of a mini-multiplex SNaPshot screening tool (Miniplex) for human samples which allows the qualitative comparison of short mitochondrial and nuclear DNA targets, as well as the interrogation of biogeographic ancestry, lineage, and phenotype single nucleotide polymorphisms (SNPs). This tool is useful to triage samples based on sample quality prior to downstream identification workflows and provides broad biological profile data for intelligence purposes.