The Potential of X‐Ray Diffraction in the Analysis of Burned Remains from Forensic Contexts*

Abstract: In view of the difficulties in extracting quantitative information from burned bone, we suggest a new and accurate method of determining the temperature and duration of burning of human remains in forensic contexts. Application of the powder X‐ray diffraction approach to a sample of human bone and teeth allowed their microstructural behavior, as a function of temperature (200–1000°C) and duration of burning (0, 18, 36, and 60 min), to be predicted. The experimental results from the 57 human bone sections and 12 molar teeth determined that the growth of hydroxylapatite crystallites is a direct and predictable function of the applied temperature, which follows a nonlinear logistic relationship. This will allow the forensic investigator to acquire useful information about the equilibrium temperature brought about by the burning process and to suggest a reasonable duration of fire exposure.     

Bone Mineral Change During Experimental Calcination: An X‐ray Diffraction Study

The effects of calcination (400–1200°C) on pig bones have been studied using powder X‐ray diffraction (XRD) and secondary modifications, such as color change and weight loss. The characterisation by powder XRD confirmed the presence of the crystalline phase of hydroxyapatite, and comparison of the results obtained at different temperatures suggested that at 650°C, all the organic components and carbonate substitutions were completely removed. Accordingly, these samples were white. In addition, the crystallinity degree and the crystallite size progressively increased with the calcination temperature until 650°C, remaining stable until 1200°C. Below 650°C, bone samples presented organic compounds, resulting in background noise in the diffractogram and gray or black color. In addition, impurities in the lattice correspond to low crystallite sizes.     

Scanning Electron Microscopy–Energy‐Dispersive X‐Ray (SEM/EDX): A Rapid Diagnostic Tool to Aid the Identification of Burnt Bone and Contested Cremains

This study investigates the use of Scanning electron microscopy–energy‐dispersive X‐ray (SEM‐EDX) as a diagnostic tool for the determination of the osseous origin of samples subjected to different temperatures. Sheep (Ovis aries) ribs of two experimental groups (fleshed and defleshed) were burned at temperatures of between 100°C and 1100°C in 100°C increments and subsequently analyzed with the SEM‐EDX to determine the atomic percentage of present elements. Three‐factor ANOVA analysis showed that neither the exposure temperature, nor whether the burning occurred with or without soft tissue present had any significant influence on the bone's overall elemental makeup (p > 0.05). The Ca/P ratio remained in the osseous typical range of between 1.6 and 2.58 in all analyzed samples. This demonstrates that even faced with high temperatures, the overall gross elemental content and atomic percentage of elements in bone remain stable, creating a unique “fingerprint” for osseous material, even after exposure to extreme conditions.     

Using Paint to Investigate Fires: An ATR‐IR Study of the Degradation of Paint Samples Upon Heating

Fire investigation is a challenging area for the forensic investigator. The aim of this work was to use spectral changes to paint samples to estimate the temperatures to which a paint has been heated. Five paint samples (one clay paint, two car paints, one metallic paint, and one matt emulsion) have been fully characterized by a combination of attenuated total reflectance Fourier transform infrared (ATR‐IR), Raman, X‐ray fluorescence spectroscopy and powder X‐ray diffraction. The thermal decomposition of these paints has been investigated by means of ATR‐IR and thermal gravimetric analysis. Clear temperature markers are observed in the ATR‐IR spectra namely: loss of ν(C = O) band, >300°C; appearance of water bands on cooling, >500°C; alterations to ν(Si–O) bands due to dehydration of silicate clays, >700°C; diminution of ν(CO₃) and δ(CO₃) modes of CaCO₃, >950°C. We suggest the possible use of portable ATR‐IR for nondestructive, in situ analysis of paints.     

Preliminary Validation of Handheld X‐Ray Fluorescence Spectrometry: Distinguishing Osseous and Dental Tissue from Nonbone Material of Similar Chemical Composition

One of the tasks of a forensic anthropologist is to sort human bone fragments from other materials, which can be difficult when dealing with highly fragmented and taphonomically modified material. The purpose of this research is to develop a method using handheld X‐ray fluorescence (HHXRF) spectrometry to distinguish human and nonhuman bone/teeth from nonbone materials of similar chemical composition using multivariate statistical analyses. The sample materials were derived primarily from previous studies: human bone and teeth, nonhuman bone, nonbiological materials, nonbone biological materials, and taphonomically modified materials. The testing included two phases, testing both the reliability of the instrument and the accuracy of the technique. The results indicate that osseous and dental tissue can be distinguished from nonbone material of similar chemical composition with a high degree of accuracy (94%). While it was not possible to discriminate rock apatite and synthetic hydroxyapatite from bone/teeth, this technique successfully discriminated ivory and octocoral.     

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.     

Forensic Comparison of Automotive Aluminum Wheel Fragments Using Synchrotron Radiation X‐ray Fluorescence with 18‐ and 116‐keV Excitation X‐rays

The performance of synchrotron radiation X‐ray fluorescence (SR‐XRF) spectrometry for nondestructive discrimination of small fragments of automotive aluminum wheels was studied. Fragments (< 500 × 500 μm²) of 45 kinds of wheels were first analyzed by scanning electron microscopy–energy dispersive spectroscopy (SEM‐EDS) and then by SR‐XRF. Despite the Mg/Al intensity ratio being a useful identification index, SEM‐EDS was not efficient enough because of the absence of other meaningful indicators of comparison. Conversely, pairwise comparison was conducted and a 92.9% identification was achieved via SR‐XRF using 18‐keV X‐rays. Trace heavy elements in the high‐energy region were detected by SR‐XRF using 116‐keV X‐rays, and an 82.9% identification was obtained. Combined use of 18‐ and 116‐keV X‐rays allowed 98.2% identification. Hence, SR‐XRF is a powerful tool for nondestructive discrimination of automotive aluminum wheels with high precision using trace elements in a wide energy region.     

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.     

Practical Considerations in Trace Element Analysis of Bone by Portable X‐ray Fluorescence

Forensic anthropologists are more often turning to nondestructive methods to assist with skeletal analyses, specifically for trace elemental analyses. Portable XRF (pXRF) instruments are versatile and are able to be used in diverse settings or for specimens of a shape and size that cannot be accommodated by laboratory‐based instruments. Use of XRF requires knowledge of analysis parameters such as X‐ray penetration and exit depth. Analysis depth was determined by examining pure elements through known thicknesses of equine bone slices. Correlation between the element's X‐ray emission energy and the depth of reading was observed. Bone surfaces from a small unknown historic cemetery were analyzed before and after sanding of the periosteal surface to observe possible changes in XRF readings based on potential diagenesis. Results validate the pXRF device as a powerful and convenient instrument for nondestructive analysis, while highlighting limitations and considerations for the analysis of osseous materials.     

Detection of methamphetamine in mouse femurs exposed to high temperature

Bone samples are valuable for examining the cause of death and circumstance leading up to death when body fluids are not available for forensic toxicological analysis. Examined were heat-induced changes in methamphetamine and amphetamine concentrations in femurs removed from methamphetamine-injected mice to determine if the burned bones could be used for toxicology testing. The femurs were heated at 100°C, 300°C, or 500°C for 10 or 30 min. The tissue structure of the heated femurs was preserved at 100°C for 30 min but was destructed at higher temperatures. Methamphetamine and amphetamine were detected in femurs heated at 100°C for 10 min, 100°C for 30 min, and 300°C for 10 min (with methamphetamine and amphetamine concentrations ranging from 0.36 to 35 μg/g and 0.54 to 47 μg/g, respectively). Methamphetamine and amphetamine were detectable when heated above their decomposition temperature as a result of limited heat transfer do to protection provide by the femoral muscle. Thus, the bone could be a useful analytical sample in cases of burn-related deaths, where it is difficult to collect body fluids.     

Thermal Modifications of Root Transparency and Implications for Aging: A Pilot Study

Root transparency has proven to be related to age and has been considered by different odontological methods for age estimation. Very little is known concerning possible variations of root transparency with heat, although the applicability of the method to burnt remains depends on the possible modifications of this specific variable. This pilot study presents the results of an experiment performed on 105 teeth obtained from dental patients and autopsy material, heated in an industrial oven at 50°C, 100°C, 150°C and 200°C. Root transparency was measured before and after the charring experiment. The heating process proved to radically modify root transparency, which decreased in 20% of samples at 50°C, in 34.6% at 100°C, in 50% at 150°C, in 77% at 200°C. The overall correlation index (CI) between decrease in root transparency and increase in temperature amounted to 0.96. These results show that heat may modify root transparency and suggest caution in using methods based on root transparency for age estimation.     

Testing a Novel 3D Printed Radiographic Imaging Device for Use in Forensic Odontology

There are specific challenges related to forensic dental radiology and difficulties in aligning X‐ray equipment to teeth of interest. Researchers used 3D printing to create a new device, the combined holding and aiming device (CHAD), to address the positioning limitations of current dental X‐ray devices. Participants (N = 24) used the CHAD, soft dental wax, and a modified external aiming device (MEAD) to determine device preference, radiographer's efficiency, and technique errors. Each participant exposed six X‐rays per device for a total of 432 X‐rays scored. A significant difference was found at the 0.05 level between the three devices (p = 0.0015), with the MEAD having the least amount of total errors and soft dental wax taking the least amount of time. Total errors were highest when participants used soft dental wax—both the MEAD and the CHAD performed best overall. Further research in forensic dental radiology and use of holding devices is needed.     

A Case of Contested Cremains Analyzed Through Metric and Chemical Comparison

Since the 1980s, cremation has become the fastest growing area of the U.S. funeral industry. At the same time, the number of litigations against funeral homes and cremation facilities has increased. Forensic anthropologists are often asked to determine whether the contents of an urn are actually cremated bone, and to address questions regarding the identity of the remains. This study uses both metric and chemical analyses for resolving a case of contested cremains. A cremains weight of 2021.8 g was predicted based on the decedent's reported stature and weight. However, the urn contents weighed 4173.5 g. The urn contents also contained material inconsistent with cremains (e.g., moist sediment, stones, ferrous metal). Analysis using XRD and SEM demonstrated that the urn contained thermally altered bone as well as inorganic material consistent with glass fiber cement. Although forensically challenging, cremains cases such as this one can be resolved using a multidisciplinary approach.     

The Optimal Combination of Cartilage Source and Apparatus for Long‐Term In Vitro Chondrocyte Viability Analysis

Abstract: Most studies of long‐term chondrocytes survival were for tissue banks. They showed a gradual reduction in the viable chondrocytes percentage as a function of time and ambient temperature, but the samples were harvested under optimal conditions. The aim of our study was to determine the most reliable combination of cartilage source and assay for the in vitro postmortem chondrocyte viability analysis in the conditions that imitate a dead body. Osteochondral cylinders were procured from femoral condyles and talar trochleas of three male donors and stored in the cell culture media at 4 ± 2°C and 23 ± 2°C. The samples were analyzed by a cell viability analyzer and a confocal laser scanning microscope (CLSM) initially 24–36 h after death and then in 4‐week intervals. The results reconfirmed the significant influence of time (p = 0.0002), but not of the temperature (p = 0.237). The largest reproducibility was presented for the knee joint and the CLSM.     

Multimethod Resolution of a Small‐Scale Case of Commingling

This study highlights the use of multiple methods for resolving a case of commingled human remains. Skeletal remains were located in a marijuana field in rural northern California by law enforcement. Although initially buried in shallow graves, the remains of two decedents were disturbed by large carnivores and scattered over a wide area. The remains were submitted by law enforcement for forensic anthropological analysis and resolution of commingling. To segregate the remains of the two individuals, a number of methods were employed, including: (i) physical matching of fragmented remains; (ii) articulation to evaluate joint congruence; (iii) visual pair‐matching of bilateral elements; (iv) osteometric pair‐matching; (v) evaluation of taphonomic patterns; (vi) DNA analysis; and (vii) portable X‐ray spectrometry (pXRF). This multimethod approach resulted in accurate resolution of the commingling and facilitated reconstruction of the biological profiles, taphonomic patterns, and trauma analysis for each individual.     

Orthodontic Treatment: Real Risk for Dental Age Estimation in Adults?

Dental age estimation becomes a challenge once the root formation is concluded. In living adults, one dental age indicator is the formation of secondary dentine, also associated with orthodontic treatment as well as root shortening. The aim of this study was to establish whether these secondary effects of orthodontic treatment could generate a statistically significant difference in dental age estimations when using Kvaal's method. The study sample included 34 pairs of pre‐ and postorthodontic panoramic radiographs, from different individuals with exactly the same age and sex distribution. Females 65%, median age 17.5 years, and males 35%, median age 22.5 years, were included. After data collection, dental age was estimated per tooth using formulae previously published. The risk of obtaining over‐estimation of age was calculated. (RR = 1.007). The changes caused by orthodontic treatment do not have any significant effect on age estimation when Kvaal et al.'s method is applied on panoramic radiographs.     

Age Estimation Based on Pulp Cavity to Tooth Volume Ratio Using Postmortem Computed Tomography Images

Pulp cavity size is known to decrease with age and can therefore serve as an indicator for age estimation. Here, we evaluated whether reconstructed images of multidetector‐row computed tomography (MDCT) acquired before forensic autopsy are useful for estimating age at death. Images of 136 mandibular first premolars obtained from bodies of known age at death were analyzed, and the volume of the regions corresponding to pulp cavity and that of the whole tooth were determined using a voxel counting function. The pulp cavity was clearly distinguishable from dental hard tissue on the reconstructed images when using a cutoff value of 1400 Hounsfield units. Regression analysis adjusted for sex showed that estimated age correlated significantly with the pulp cavity to tooth volume ratio (r = 0.76). MDCT is gaining more widespread use in forensic medicine, and analyzing dental images to obtain parameters for age prediction is a practical approach for postmortem identification.     

The Survival of Gunshot Residues in Cremated Bone: An Inductively Coupled Plasma Optical Emission Spectrometry Study

Gunshot residue (GSR) has been sought and demonstrated on many types of material and with many techniques. Inductively coupled plasma optical emission spectrometry (ICP‐OES) could be a useful method on difficult substrates, but a systematic study on burnt material has never been performed. Hence, this study aims at evaluating the usefulness and reliability of this method on burnt samples. Sixteen adult bovine ribs (eight with soft tissues, eight totally skeletonized) were shot using two kinds of projectile (both 9 mm full metal‐jacketed or unjacketed). Then, every sample was led to complete calcination in an electric oven. The area of the gunshot entrance wound was swabbed and analyzed by ICP‐OES; the results were also correlated with a previously published parallel study by scanning electron microscopy (SEM) equipped with an SEM–energy dispersive X‐ray analyzer. ICP‐OES proved to be very sensitive and reliable even on degraded material and can be an appropriate nondestructive method for detecting residues on difficult and delicate substrates such as burnt bone.