Improving the strategy to identify historical military remains: a literature review and Y-STR meta-analysis.

The identification of historical military remains by Unrecovered War Casualties-Army (UWC-A) currently relies on Y-chromosome Short Tandem Repeat (Y-STR) testing when maternal relatives are not available, or when a mitochondrial DNA match does not provide sufficient certainty of identification. However, common Y-STR profiles (using Yfiler™) between sets of remains or families often prevent identification. To resolve these cases, an investigation of additional Y-DNA markers is needed for their potential inclusion into the DNA identification strategy. The number of genetic transmissions between missing soldiers and their living relatives needs to be considered to avoid false exclusions between paternal relatives. Analysis of 236 World War I/II (WWI/II) era pairs of relatives identified up to seven genetic transmissions between WWII soldiers and their living relatives, and nine for WWI. Previous Y-STR meta-analyses were published approximately 10 years ago when rapidly mutating markers were relatively new. This paper reports a contemporary literature review and meta-analysis of 35 studies (which includes 23 studies not previously used in meta-analysis) and 23 commonly used Y-STR's mutation rates to inform the inclusion of additional loci to UWC-A's DNA identification strategy. Meta-analysis found mutation data for a given Y-STR locus could be pooled between studies and that the mutation rates were significantly different between some loci (at P < 0.05). Based on this meta-analysis, we have identified two additional markers from PowerPlex® Y23 for potential inclusion in UWC-A's identification strategy. Further avenues for potential experimental exploration are discussed. Key points: From 236 UWC-A pairs of relatives, we observed up to nine genetic transmissions between WWI soldiers and their living relatives, and seven for WWII.MedCalc® software for meta-analysis utilizing the Freeman-Tukey transformation was run, which analysed 35 published studies and 23 commonly used loci. Previous Y-STR mutation rate meta-analyses are now 10 years old; this paper includes 23 studies that were not included in previous meta-analyses.Through meta-analysis, we identify two markers from PowerPlex® Y23 for potential inclusion in UWC-A's historical remains identification strategy (alongside Yfiler™). We discuss potential next steps for experimental exploration of additional Y-DNA markers.

Evaluating the suitability of current mitochondrial DNA interpretation guidelines for multigenerational whole mitochondrial genome comparisons.

Sanger sequencing of the mitochondrial DNA (mtDNA) control region was previously the only method available for forensic casework involving degraded samples from skeletal remains. The introduction of Next Generation Sequencing (NGS) has transformed genetic data generation and human identification using mtDNA. Whole mitochondrial genome (mtGenome) analysis is now being introduced into forensic laboratories around the world to analyze historical remains. Research into large pedigrees using the mtGenome is critical to evaluate currently available interpretation guidelines for mtDNA analysis, which were developed for comparisons using the control region. This study included mtGenomes from 225 individuals from the last four generations of the Norfolk Island (NI) genetic isolate pedigree consisting of 49 distinct maternal lineages. The data from these individuals were arranged into 2339 maternally related pairs separated by up to 18 meioses. Our results show that 97.3% of maternally related pairs were concordant at all nucleotide positions, resulting in the correct interpretation of "Cannot Exclude"; 2.7% of pairs produced an "Inconclusive" result, and there were no instances of false exclusion. While these results indicate that existing guidelines are suitable for multigenerational whole mtGenome analysis, we recommend caution be taken when classifying heteroplasmic changes as differences for human identification. Our data showed the classification of heteroplasmic changes as differences increases the prevalence of inconclusive identification by 6%, with false exclusions observed in 0.34% of pairs examined. Further studies of multigenerational pedigrees, however, are needed to validate mtGenome interpretation guidelines for historical case work to more fully utilize emerging advancements.

"Lest we forget": An overview of Australia's response to the recovery and identification of unrecovered historic military remains.

The Australian Defence Force (ADF) is responsible for the recovery and identification of its historic casualties. With over 30,000 still unrecovered from past conflicts including World War One (WW1) and World War Two (WWII), the Australian Army and Royal Australian Air Force have teams that research, recover, identify and oversee the burial (or reburial) of the remains of soldiers and airmen who continue to be found each year. The Royal Australian Navy is also responsible for its unrecovered casualties. Collectively the priorities of the various services within the ADF are the respectful recovery and treatment of the dead, thorough forensic identification efforts, resolution for families and honouring the ADF's proud history of service and sacrifice. What is unique about the approach of the ADF is that the respective services retain responsibility for their historic losses, while a joint approach is taken on policies and in the utilisation of the pool of forensic specialists. Section One describes the process undertaken by the Australian Army in the recovery, identification and burial or repatriation of soldiers through its specialised unit Unrecovered War Casualties - Army (UWC-A). Section Two describes the role of the Royal Australian Air Force in the recovery of aircraft and service personnel through their specialised unit Historic Unrecovered War Casualties - Air Force (HUWC-AF). An overview of the operations of each service and case studies is presented for each section.

Evaluation of rapid DNA using ANDE™ in a technical exploitation Level 2 laboratory workflow.

A trial of rapid DNA (rDNA), a fully automated DNA profiling system, within a technical exploitation (TE) workflow is an important endeavor. In the 2019 Ardent Defender (AD) exercise, the Deployable Technical Analysis Laboratory (DTAL), of the Canadian Department of National Defence (DND), evaluated the use of rDNA using ANDE™. Sixteen samples were processed during a pre-exercise "controlled" setting, 44 samples were from an "uncontrolled" environment during the exercise, and 22 samples were buccal swabs. The proportion of profiles suitable for upload to ANDE™ was 95.5% of buccal samples (21/22), 66.7% controlled samples, and 15.9% for uncontrolled samples. A considerable difference was observed in the proportions of complete DNA profiles obtained from all exploited items between the controlled (58.3%) and uncontrolled (15.9%) trials and in the proportions of samples where no DNA was detected (16.7% controlled trial vs. 56.8% uncontrolled trial). Overall, the trials highlighted the potential to gain identity intelligence using rDNA within a TE workflow and revealed the impact of operational constraints and the need to improve certain TE practices to gain the most benefit from rDNA. It also demonstrated the benefit of including an uncontrolled component for a more realistic indication of rDNA effectiveness in operational settings and highlighted operational practices impacting rDNA success. Mixture deconvolution was difficult as current guidelines do not consider some of the stochastic effects produced by the rDNA analysis; however, overall, the study demonstrated that rDNA using the ANDE™ instrument could be successfully incorporated into a TE workflow within a deployable laboratory.

Bloodstain pattern analysis: Does experience equate to expertise?

Bloodstain pattern analysis (BPA) has long been accepted by courts as an area of expertise; however, that position has recently been challenged. The discipline has been criticized for limited empirical research into practitioner determination error rates and whether determinations require specialized knowledge/expertise, including whether practitioner experience level influences accuracy. This study attempted to address these knowledge gaps as they relate to bloodstain pattern recognition. The aims were twofold: to establish whether practitioners would outperform lay non-practitioners, and whether practitioner experience influenced accuracy and error in determinations. Comparisons of practitioner responses under three scenarios (forced, casework, and definitive) were also made to assess conservatism/certainty in pattern recognition. Participants (both BPA practitioners and non-practitioners) analyzed photographs of bloodstain patterns and made determinations of the broad bloodstain category and specific bloodstain pattern type. When forced to provide only a single response, practitioners identified bloodstain categories and patterns significantly more accurately than non-practitioners (p = 0.0001, p < 0.00001, respectively). Practitioner accuracy in bloodstain pattern recognition was positively associated with experience level (p = 0.0429) and this was consistent regardless of response scenario. Although no significant difference in practitioner accuracy was observed across response scenarios, practitioner conservatism/certainty varied significantly among the broad bloodstain category and specific pattern types. Overall, these results support bloodstain pattern recognition as an area of expertise and that practitioner experience positively influences accuracy. Based on these results, a series of recommendations were proposed aimed at further improving practices within the discipline to maximize accuracy and reliability of BPA evidence.

A comparison of three shoe sole impression lifting methods at high substrate temperatures.

Footwear impressions are a common form of evidence found at crime scenes, and the accurate recovery and recording of such impressions is critical for shoe sole comparison and identification. The lifting of shoe sole impressions from hot surfaces (>30°C/86°F) and in hot environments has received little attention in the literature, particularly in relation to the recovery of class and randomly acquired characteristics (RACs) required for accurate comparisons. This study addressed this knowledge gap by comparing the performance of three common impression lifters (gelatin, adhesive, and vinyl static cling film) at recovering shoe sole impressions in dust from hot flooring substrates. Dry origin dust shoe sole impressions were made on ceramic tile, galvanized metal, and laminated wood flooring using a shoe that possessed two RACs and five class characteristics present on the sole. Substrates were left in direct full sun for five hours during a summer day prior to lifting. Performance was measured by the proportion of RACs and class characteristics visible in each lifted impression. Results demonstrated that the vinyl static cling film tested performed poorly across all substrates, particularly for metal (23.8% marks recovered), including notable shrinkage of the lifted impression. In contrast, adhesive (~96% marks recovered over all substrates), and to a lesser extent gelatin (~85%), lifts were highly successful on hot substrates. These data suggest that adhesive lifts can consistently and accurately recover shoe sole impressions from hot substrates. This study contributes critical information for crime scene examiners to improve and expand evidence recovery in hot environments.

Variation in decomposition stages and carrion insect succession in a dry tropical climate and its effect on estimating postmortem interval.

Insects have an important role in minimum postmortem interval (PMImin) estimation. An accurate PMImin estimation relies on a comprehensive study of the development and succession of local carrion insects. No published research on carrion insect succession exists for tropical north Queensland. To address this, we aimed to obtain preliminary observational data concerning the rate of decomposition and insect succession on pig carcasses in Townsville and compare these with other regions of Australia and overseas. Adult insects were collected daily from three pig carcasses for 30 d during summer and identified to family level. Observations of decomposition rate were made each day and progression through the stages of decomposition were recorded. Adult insects were identified to family and their presence/absence used as a proxy for arrival at/departure from the remains, respectively. These preliminary data highlight several interesting trends that may be informative for forensic PMImin estimation. Decomposition was rapid: all carcasses were at the dry/remains stage by Day 5, which was substantially quicker than all other regions in the comparison. Differences were also observed in the presence/absence of insect families and their arrival and departure times. Given the rapid progression through early decomposition, we argue that later-arriving coleopteran taxa may be more forensically informative in tropical Australia, in contrast with temperate regions where Diptera appear most useful. This research contributes preliminary observational data to understanding insect succession patterns in tropical Australia and demonstrates the critical need for comprehensive local succession data for each climatic region of Australia to enable accurate PMImin estimation. These data will inform future research targeted at gaining a more comprehensive understanding of insect succession in the Australian tropics.Key points:We obtained preliminary observational data concerning the rate of decomposition and insect succession on pig carcasses in tropical Australia.Decomposition was rapid: all carcasses were at the dry/remains stage by Day 5.Coleopteran taxa may be more forensically informative in tropical Australia than dipterans.

Detection of blood on clothing laundered with sodium percarbonate.

Laundering clothes with modern detergents containing sodium percarbonate can result in false negative results when certain presumptive and confirmatory tests are used to detect the presence of blood. This is problematic as blood evidence can be inadvertently overlooked and criminal activity concealed, simply by laundering bloodstained clothes in detergent. The aim of this research was to determine if the incidence of positive results using tetramethylbenzidine (TMB) reagent, luminol, Bluestar® Magnum, ABAcard® Hematrace® and RSID™-Blood was affected by treatment in hot and cold water, with and without the detergent, sodium percarbonate. This study identified that RSID-Blood consistently produced positive results irrespective of water temperature or the addition of sodium percarbonate. All other reagents returned positive results in the absence of sodium percarbonate, regardless of water temperature. The introduction of sodium percarbonate initiated negative results regardless of water temperature when testing with tetramethylbenzidine reagent, Bluestar® Magnum and ABAcard® Hematrace®. Luminol in the presence of sodium percarbonate responded differently to the temperature change of the water. Cold water returned positive results, however, hot water returned negative results. This research indicates that RSID™-Blood surpassed other blood screening tests identifying blood on sodium percarbonate treated cotton fabric. The results for luminol were varied depending on water temperature, however, luminol performed better as a presumptive test than TMB or Bluestar® Magnum.

A Comparison of Four Presumptive Tests for the Detection of Blood on Dark Materials.

Detection of blood on dark materials is difficult for crime scene examiners so presumptive tests are used to assist. This study compared the ability of luminol, leuko crystal violet, tetramethylbenzidine, and Combur Test®E to detect whole, diluted blood (1:100) and a key-shaped blood transfer stain (1:10), on dark cotton sheeting, tea towel, socks, synthetic carpet, and car mats. Powdered bleach was used to evaluate specificity of the blood detection tests. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and overall misclassification rate (OMR) assessed the quality of the blood tests. Luminol was the preferred test for diluted blood having the highest sensitivity (79%-96%), NPV (66%-93%), and the lowest OMR (3%-15%). Luminol was also found to be most efficient with a testing time on 25 items of 2 h 50 min compared with up to 8 h. Overall, luminol was the most effective method, also providing information on bloodstain patterns.

Tissue preservation in extreme temperatures for rapid response to military deaths.

Many deployable forensic capabilities, including those used by the Australian Defense Force (ADF), employ mobile battery-operated fridge/freezers for DNA sample preservation that are not suitable for rapid response application due to their size and weight. These fridge/freezers are expensive, require regular specialised maintenance, and have a set payload. A variety of transport media are successful preservatives for DNA samples, however, there is no research specifically targeted to their suitability for operational environments where temperatures exceed 50 °C. This research examined whether sodium chloride (NaCl), ethanol, and dimethyl sulfoxide (DMSO) could preserve muscle and bone samples (fresh and early decomposition) as effectively as refrigeration, when stored at 21 °C, 45 °C, 55 °C, and 65 °C for at least one week. A total of 78 muscle and 78 bone samples were collected from an unknown deceased individual. Half of each tissue type was stored at 30 °C for 48 h to induce early decomposition. Following this, samples were stored in the transport media for one week at the above temperatures, and a control set of samples were refrigerated (-4 °C) without any transport media. Preserved samples would need to provide DNA profiles comparable to the refrigerated samples for the transport media to be considered a successful replacement method. NaCl and 70% ethanol preserved muscle samples (fresh and decomposed) up to 65 °C, as well as 70% ethanol and 20% DMSO for fresh bone samples. These results were comparable with refrigeration and therefore, these preservatives could be used in rapid response operations by the military and for disaster victim identification. Conversely, under the conditions of this study, 20% DMSO and 70% ethanol failed to consistently produce full DNA profiles from decomposed bone, and NaCl performed poorly at preserving DNA from fresh and decomposed bone samples.

A new disaster victim identification management strategy targeting "near identification-threshold" cases: Experiences from the Boxing Day tsunami.

The international disaster victim identification (DVI) response to the Boxing Day tsunami, led by the Royal Thai Police in Phuket, Thailand, was one of the largest and most complex in DVI history. Referred to as the Thai Tsunami Victim Identification operation, the group comprised a multi-national, multi-agency, and multi-disciplinary team. The traditional DVI approach proved successful in identifying a large number of victims quickly. However, the team struggled to identify certain victims due to incomplete or poor quality ante-mortem and post-mortem data. In response to these challenges, a new 'near-threshold' DVI management strategy was implemented to target presumptive identifications and improve operational efficiency. The strategy was implemented by the DNA Team, therefore DNA kinship matches that just failed to reach the reporting threshold of 99.9% were prioritized, however the same approach could be taken by targeting, for example, cases with partial fingerprint matches. The presumptive DNA identifications were progressively filtered through the Investigation, Dental and Fingerprint Teams to add additional information necessary to either strengthen or conclusively exclude the identification. Over a five-month period 111 victims from ten countries were identified using this targeted approach. The new identifications comprised 87 adults, 24 children and included 97 Thai locals. New data from the Fingerprint Team established nearly 60% of the total near-threshold identifications and the combined DNA/Physical method was responsible for over 30%. Implementing the new strategy, targeting near-threshold cases, had positive management implications. The process initiated additional ante-mortem information collections, and established a much-needed, distinct "end-point" for unresolved cases.

The role of adenosine-related genes variants in susceptibility to essential hypertension.

OBJECTIVE: To test markers within adenosine-related genes: A1 and A2a receptors (ADORA1, ADORA2a) and adenosine deaminase (ADA) for potential involvement in essential hypertension (EH). DESIGN: Case-control association study investigating gene variants for the ADORA1, ADORA2a and ADA genes. PARTICIPANTS: The study used a cohort of 249 unrelated hypertensive individuals who were diagnosed with hypertension, and an age, sex and ethnically matched group of 249 normotensive controls. RESULTS: The association analysis indicated that both allele and genotype frequencies did not differ significantly between the case and control groups (P > 0.05) for any of the markers tested. CONCLUSION: The adenosine-related gene variants do not appear to alter susceptibility to the disease in this group of essential hypertensives. However, involvement of these genes and the adenosine system cannot be conclusively excluded from essential hypertension pathogenesis as other gene variants may still be involved.