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.     

Effect of Heat on the Fluorescence Properties of Tooth‐Colored Restorative Materials and Their Forensic Implications

During antemortem and postmortem comparison of dental records of carbonized victims, restorations may be part of such records. The objective of this study was to evaluate the effect of heat on the fluorescence behavior of contemporary tooth‐colored restorative materials and natural tooth structure when subjected to range of temperatures, using illumination with 405 nm wavelength light. A total of 132 human extracted teeth restored with tooth‐colored restorative materials were exposed to heat (200, 500, 900, 1200°C) in an oven for 30 min. Samples were imaged before and after heat treatment. All tooth‐colored restorative materials underwent changes in color and in fluorescence properties, at each of the temperatures used. Resin‐based restorative materials still fluoresced at 200°C, and at 500°C underwent major color changes due to volatilization of resin. Materials containing inorganic fluorophores still fluoresced at 900°C, while at 1200°C, none of the materials tested in this study showed any fluorescence.     

Resolving Reflection and Resolution in 3D Imaging of Fresh Bone

Structured light scanning is a noninvasive, accurate, and cost-effective 3D imaging technique, but due to reflection issues is yet to be utilized for tool mark analysis on fresh bone. During imaging, reflection from shiny surfaces, such as greasy bone, disrupts image formation. This study tested the David SLS-1 scanner's ability to image saw marks and explored six strategies to reduce reflection by [1] dulling the surface or [2] altering the projected light. The surface was dulled by freezing, talcum powder, dulling spray, or compressed air. The projected light was altered with a diffuser or limited to single pattern-coded. Results demonstrated that the resolution was insufficient for capturing minute details of striae. All six tests failed to reduce reflection sufficiently to produce complete images, but projecting vertical pattern-coded light showed the most promise. Future research is required concentrating on enhancing resolution and exploring the role of pattern-coded light in reducing reflection.     

Chemical trace XRF analysis to detect sharp force trauma in fresh and burned bone

Forensic anthropologists may not always be able to differentiate heat-induced fractures from fractures with other aetiologies, namely sharp force traumas, with clear nefarious impact on medico-legal conclusions. The objective of this study was to experimentally investigate if blade chemical traces are transferred to defleshed bone tissue and if they remain there after a burning event. This was accomplished by prompting sharp force traumas in 20 macerated fresh pig ribs with five different instruments, namely a stainless steel knife, an artisanal knife and a ceramic kitchen knife, a small axe and a large axe. Another pig rib was used as control, not being subjected to any trauma. All instruments were probed by X-ray fluorescence (XRF) to establish the composition of each blade. Bone samples, both pre-burned and post-burned (at 500 °C, 700 °C, 900 °C and 1100 °C), were then probed by XRF. All sharp force instruments left detectable chemical traces on pre-burned bone, although not in all samples. Furthermore, traces were still detected after experimental burning in most cases. Potentially, XRF can provide relevant information about the aetiology of fractures in burned and unburned bones, although the effect of soft tissues and diagenesis must still be investigated.     

Frozen human bone: a microscopic investigation

The taphonomic effects of prolonged extreme cold and freezing on human bone have received little research attention. Questions of specific interest include whether previously frozen bone can be identified and whether freezing alters the structural integrity enough to prevent histological aging. There is no evidence from previous studies that freezing damages the structural integrity, and to date no research investigating the freezing process on bone microstructure has been undertaken. This research attempts to distinguish histologically previously frozen bone from nonfrozen bone by identifying patterned defects. To determine the effects of freezing in bone microstructure using light and scanning electron microscopy (SEM), several human bone sections were subjected to prolonged freezing and allowed to thaw before thin sectioning. Light microscopy failed to demonstrate statistically significant differences between frozen and nonfrozen specimens. SEM analysis revealed fractures, although these lacked pattern and did not occur systematically throughout the section. Evidence of microstructural changes caused by liquid expansion, however, was remarkable but did not alter the structural integrity of the microstructure. The results of this study suggest that freezing does not alter the process of histomorphological analysis.     

Gunshot residues on dry bone after decomposition-a pilot study

Abstract: Very little literature exists concerning radiochemical and microscopic analyses of gunshot wounds in decomposed material, and even less concerning skeletonized samples; the most advanced technologies may provide useful indications for the diagnosis of suspect lesions, especially if gunshot wounds are no longer recognizable. However, we know very little of the survival of gunshot residues (GSR) in skeletonized samples. This study examined nine gunshot wounds produced on pig heads which then underwent skeletonization for 4 years, and four gunshot entries on human heads from judicial cases which were then macerated to the bone in water; the samples underwent scanning electron microscopy coupled with energy dispersive X‐ray (SEM‐EDX) analysis. Positive results for GSR were observed only in four of the nine animal samples and in all four human samples. Among the human samples, two lesions showed Pb and Sb, one lesion only Pb, and one Pb, Sb, and Ba. This pilot study showed the survival of GSR in skeletal material and therefore the crucial importance of SEM‐EDX analyses on skeletonized material. Further studies are needed in order to ascertain the role of environmental modifications of GSR.     

The Impact of Freezing on Bone Mineral Density: Implications for Forensic Research

It is common for researchers using animal or human remains for scientific study to freeze samples prior to use. However, effects of freezing on bone macro‐ or microstructure are relatively unknown. The research objective of this study was to determine whether freezing could potentially bias experimental results by analyzing changes in bone mineral density (BMD) with the freezing of remains over time. Eight fetal pigs were scanned to determine their initial BMD before freezing. Three piglets underwent a freeze‐thaw cycle to assess the effects of the freezing process. Four piglets were frozen and scanned weekly for 20 weeks to assess freezing over time. The overall average between the fresh initial scan and final frozen scan was significantly different (p < 0.001). Per contra, the final thawed BMD scans did not differ from the initial fresh scan (p = 0.418). Thus, completely thawed remains are recommended for experimental studies.     

The suitability of using domestic pigs (Sus spp.) as human proxies in the geophysical detection of clandestine graves

Research in many forensic science fields commonly uses domestic pigs (Sus spp.) as proxies for human remains, due to their physiological and anatomical similarities, as well as being more readily available. Unfortunately, previous research, especially that which compares the decompositional process, has shown that pigs are not appropriate proxies for humans. To date, there has not been any published research that specifically addresses whether domestic pigs are adequate human proxies for the geophysical detection of clandestine graves. As such, the aim of this paper was to compare the geophysical responses of pig cadavers and human donor graves, in order to determine if pigs can indeed be used as adequate human proxies. To accomplish this, ground penetrating radar (GPR) and electrical resistivity tomography (ERT) responses on single and multiple pig cadaver graves were compared to single and multiple human donor graves, all of which are in known locations within the same geological environment. The results showed that under field conditions, both GPR and ERT were successful at observing human and pig burials, with no obvious differences between the detected geophysical responses. The results also showed that there were no differences in the geophysical responses of those who were clothed and unclothed. The similarity of the responses may reflect that the geophysical techniques can detect graves despite what their contents are. The study implications suggest that experimental studies in other soil and climate conditions can be easily replicated, benefiting law enforcement with missing persons cases.     

Decomposing Human Blood: Canine Detection Odor Signature and Volatile Organic Compounds

The admissibility of human “odor mortis” discrimination in courts depends on the lack of comprehension of volatile organic compounds (VOCs) during the human decay process and of the lack in standardized procedures in training cadaver dogs. Blood was collected from four young people who died from traffic accidents and analyzed using HS‐SPME/GC‐MS at different decompositional stages. Two dogs, professionally trained, were tested to exactly locate blood samples, for each time point of the experiment. We found a long list of VOCs which varied from fresh to decomposed blood samples, showing differences in specific compounds. Dog performance showed a positive predictive value between 98.96% and 100% for DOG A, and between 99.47% and 100% for DOG B. Our findings demonstrated that decomposing human blood is a good source of VOCs and a good target for canine training.     

Analysis of pathological and non-pathological human skeletal remains by FT-IR spectroscopy

In this study, we report the chemical analyses of various non-pathological, tuberculosis and syphilis infected bone samples from different burial environments by Fourier transform infrared spectroscopy (FT-IR), in the framework of a general study of diagenesis. Dating human skeletal remains is one of the most important and yet unreliable aspects of forensic anthropology. In this paper, a new method has been suggested, using the crystallinity index and carbonate-phosphate index as a means of distinction between recent and archaeological, anthropological bone samples. Pathological bone samples were analyzed with the same method to see if changes in crystallinity interfere with the process of dating.     

Determining Volumetric Shrinkage Trends of Burnt Bone Using Micro-CT

Understanding the degree and pattern of shrinkage undergone by bone when subjected to heating is crucial to accurately deduce a biological profile from incinerated remains. X-ray microtomography (micro-CT) enables a nondestructive insight into hard tissue structural changes, while allowing for an accurate documentation of volumetric and trabecular shrinkage. Sheep ribs were experimentally burned at temperatures between 400 and 1000°C in 100°C increments and their volumetric shrinkage was calculated. Observed shrinkage ranged from 14.0% at 400°C to 45.5% at 1000°C. Bones burned at temperatures up to 600°C showed no significant difference, whereas the 700 and 800°C samples exhibited higher shrinkage. Bones burnt at 900 and 1000°C showed significantly higher shrinkage than the other temperature groups. Findings signify the potential of micro-CT in research on the effects of factors such as diagenesis or burning on the bone density, morphology and microarchitecture.     

An Examination of the Transition of Fracture Characteristics in Long Bones from Fresh to Dry in Central Florida: Evaluating the Timing of Injury

It is important to conduct timing of injury research analyzing fracture characteristics at known postmortem intervals (PMI) because bone can retain fresh characteristics throughout the PMI. Defleshed pig (Sus scrofa) long bones were fractured weekly in two environments (full sun and shade) over 14 weeks in Central Florida and fracture characteristics were categorized (N = 136) for analysis. Results of analysis of variance (ANOVA) using time in weeks (PMI) as a dependent variable indicate significant relationships between PMI and Fracture Angle (p < 0.001), Fracture Surface (p < 0.001), and Fracture Outline (p < 0.001). Fracture characteristics associated with perimortem trauma (smooth Fracture Surfaces and curved or V‐shaped Fracture Outlines) were commonly observed. Analysis of fracture characteristics for each environment demonstrated similar patterns. Overall, the loss of only fresh fracture characteristics for each bone was noted earlier in the PMI for the Central Florida region than previously reported.     

Effects of obesity on talar micro- and macro-morphology

The addition of information regarding obesity status to the forensic anthropological biological profile could significantly contribute to the identification of human skeletal remains since over 40% of the U.S. adult population is currently obese. This study examines the differences in talar shape and trabecular bone structure between obese and non-obese individuals. A sample of 20 obese and 20 non-obese divided evenly by sex was selected from the Texas State University Donated Skeletal Collection. Tali were imaged using x-ray computed tomography (voxel size: 28–38.7 μm). Image stacks were processed to produce binary images as well as trabecular thickness and spacing maps. Landmark-based geometric morphometric analyses were conducted to quantify shape variation. Shape coordinates were used to locate 100 geometrically homologous volumes of interest within each talus. Bone volume fraction, trabecular thickness, and trabecular spacing were extracted at each volume of interest. Within each sex, a one-way ANCOVA was used to determine if significant differences exist between obese and non-obese individuals in trabecular bone after controlling for age. The size of the talus as well as subtle aspects of shape were found to distinguish the sexes. The results further indicate that bone volume fraction significantly differs between obese and non-obese males. In females, bone volume fraction is correlated with age but does not differ between obese and non-obese. The study demonstrates that bone microstructure is a promising approach to estimating body mass or body mass index category but age effects diminish the potential for the talus to be used alone.     

An investigation of model forensic bone in soil environments studied using infrared spectroscopy

Infrared spectroscopy has been used to examine changes to bone chemistry as a result of soil burial. Pig carcasses were buried as part of a controlled field study, and pig bone was used in soil environments established in the laboratory. The variables of species type, bone pretreatment, soil type and pH, moisture content, temperature, and burial time were investigated. The crystallinity index (CI) and the organic and carbonate contents of the bones were monitored. The data revealed decreasing trends in the organic and carbonate contents and an increase in the CI of the bone with burial time. An acidic soil environment and soil type are the factors that have the most influence on bone chemistry as a result of burial. The study demonstrates the potential of infrared spectroscopy as a straightforward method of monitoring the changes associated with aging of bones in a variety of soil environments.     

Recovery of DNA from bone without demineralization

This study assessed the performance of five different DNA extraction methods for the recovery of DNA from bone: ChargeSwitch® gDNA Plant Kit, DNA IQ™ System Kit, DNeasy® Blood & Tissue Kit, PrepFiler® BTA Forensic DNA Extraction Kit and phenol-chloroform-isoamyl alcohol. DNA was extracted from pig rib and femur bones that was fresh, had undergone surface decomposition for three months, and had undergone surface decomposition for one year. Extracted DNA was analyzed using real-time PCR and amplification of an in-house PCR multiplex that assessed the quality and quantity of DNA and for the presence of inhibitors. The phenol-chloroform-based method consistently yielded the highest amounts of DNA and DNA IQ the lowest; however, all methods produced relatively high yields of DNA from both pig rib and femur samples that could be amplified without any detected inhibition. The data demonstrate that with reasonable quality bone samples any of the tested methods can isolate DNA that can be successfully analyzed. The effective use of internal PCR controls is also demonstrated.     

SEM and Stereomicroscopic Analysis of Cut Marks in Fresh and Burned Bone

This study documents the prevalence of cut mark characteristics in fresh and burned domestic pig ribs. Stab wounds from single edge serrated and smooth‐edged knives were inflicted in the vertebral and sternal regions of each fresh rib. Each rack of ribs was then divided into vertebral and sternal units. Vertebral units were defleshed and their associated cut marks were examined using a stereomicroscope. Sternal units were burned in an outdoor fire pit and examined with the addition of a scanning electron microscope (SEM). Linear cuts, V‐shaped cross‐sections, mounding, hinge fractures, and wastage were all observed on burned ribs. There was an overall decrease in the prevalence of all features (up to a 40% decrease), regardless of knife type, in burned ribs. Striations within cut marks were not observed in either fresh or burned ribs. Oblique faulting and bone lifts could only be observed using the SEM. Mounding and wastage were obliterated during the burning process. Therefore, cut marks in burned bone should ideally be examined for their characteristics utilizing an SEM.     

Sex Assessment from the Volume of the First Metatarsal Bone: A Comparison of Linear and Volume Measurements

Sexual dimorphism is a crucial characteristic of skeleton. In the last years, volumetric and surface 3D acquisition systems have enabled anthropologists to assess surfaces and volumes, whose potential still needs to be verified. This article aimed at assessing volume and linear parameters of the first metatarsal bone through 3D acquisition by laser scanning. Sixty‐eight skeletons underwent 3D scan through laser scanner: Seven linear measurements and volume from each bone were assessed. A cutoff value of 13,370 mm³ was found, with an accuracy of 80.8%. Linear measurements outperformed volume: metatarsal length and mediolateral width of base showed higher cross‐validated accuracies (respectively, 82.1% and 79.1%, raising at 83.6% when both of them were included). Further studies are needed to verify the real advantage for sex assessment provided by volume measurements.     

Characterization of the volatile organic compounds present in the headspace of decomposing animal remains, and compared with human remains

Human Remains Detection (HRD) dogs can be a useful tool to locate buried human remains because they rely on olfactory rather than visual cues. Trained specifically to locate deceased humans, it is widely believed that HRD dogs can differentiate animal remains from human remains. This study analyzed the volatile organic compounds (VOCs) present in the headspace above partially decomposed animal tissue samples and directly compared them with results published from human tissues using established solid-phase microextraction (SPME) and gas chromatography/mass spectrometry (GC/MS) methods. Volatile organic compounds present in the headspace of four different animal tissue samples (bone, muscle, fat and skin) from each of cow, pig and chicken were identified and compared to published results from human samples. Although there were compounds common to both animal and human remains, the VOC signatures of each of the animal remains differed from those of humans. Of particular interest was the difference between pigs and humans, because in some countries HRD dogs are trained on pig remains rather than human remains. Pig VOC signatures were not found to be a subset of human; in addition to sharing only seven of thirty human-specific compounds, an additional nine unique VOCs were recorded from pig samples which were not present in human samples. The VOC signatures from chicken and human samples were most similar sharing the most compounds of the animals studied. Identifying VOCs that are unique to humans may be useful to develop human-specific training aids for HRD canines, and may eventually lead to an instrument that can detect clandestine human burial sites.     

共聚焦激光扫描显微镜在法医病理学中的应用

共聚焦激光扫描显微镜(confocal laser scanning microscope,CLSM)是一种新兴的显微图像检测技术,能够进行光学切片和对检材进行三维重构,并且能够获得较之普通光学显微镜更高的空间分辨率.CLSM作为一种精密的观察工具,在法医病理学的显微图像检测中具有重要价值.本文综述了CLSM在心源性猝死、枪弹创分析以及神经系统损伤分析等相关领域的研究成果,探讨其在法医病理学研究以及法医检案中的应用价值.     

An experimental field protocol for investigating the postmortem interval using multidisciplinary indicators

This article proposes an experimental field protocol for investigating the postmortem interval using specially designed apparatus and human and pig cadavers. We further propose that this goal can only be achieved by a multidisciplinary group, comprised of forensic entomologists, pathologists, and anthropologists. The apparatus and collecting methods described by the authors establish the means by which data can be collected on several fronts simultaneously: the sequential arrival and variety of insects in the decay process, the character and manner of soft tissue decomposition, the sequence and nature of bone exposure and order of disarticulation of skeletal remains, and the influence of climate and season on decay rates and arthropod succession. A central feature of this protocol involves the construction and use of a dual-functioning insect trap that allows separate but simultaneous capture of arriving and emerging populations while successional and decompositional processes of the cadaver are left intact. Results of trap performance tests in an arid climate and preliminary arthropod data collected from field-exposed pig carcasses are presented. The use of this protocol could provide important and badly needed baseline data for both medical investigators and law enforcement personnel, information that is critical to understanding the causes, manner, and time of death, which the law requires to be ascertained.