Estimating the Temperature of Heat‐exposed Bone via Machine Learning Analysis of SCI Color Values: A Pilot Study

Determining maximum heating temperatures of burnt bones is a long‐standing problem in forensic science and archaeology. In this pilot study, controlled experiments were used to heat 14 fleshed and defleshed pig vertebrae (wet bones) and archaeological human vertebrae (dry bones) to temperatures of 400, 600, 800, and 1000°C. Specular component included (SCI) color values were recorded from the bone surfaces with a Konica‐Minolta cm‐2600d spectrophotometer. These color values were regressed onto heating temperature, using both a traditional linear model and the k‐nearest neighbor (k‐NN) machine‐learning algorithm. Mean absolute errors (MAE) were computed for 1000 rounds of temperature prediction. With the k‐NN approach, the median MAE prediction errors were 41.6°C for the entire sample, and 20.9°C for the subsample of wet bones. These results indicate that spectrophotometric color measurements combined with machine learning methods can be a viable tool for estimating bone heating temperature.     

X‐ray Scattering Evaluation of Ultrastructural Changes in Human Dental Tissues with Thermal Treatment

Micro‐ and ultrastructural analysis of burned skeletal remains is crucial for obtaining a reliable estimation of cremation temperature. Earlier studies mainly focused on heat‐induced changes in bone tissue, while this study extends this research to human dental tissues using a novel quantitative analytical approach. Twelve tooth sections were burned at 400–900°C (30‐min exposure, increments of 100°C). Subsequent combined small‐ and wide‐angle X‐ray scattering (SAXS/WAXS) experiments were performed at the Diamond Light Source synchrotron facility, where 28 scattering patterns were collected within each tooth section. In comparison with the control sample, an increase in mean crystal thickness was found in burned dentine (2.8‐fold) and enamel (1.4‐fold), however at a smaller rate than reported earlier for bone tissue (5–10.7‐fold). The results provide a structural reference for traditional X‐ray scattering methods and emphasize the need to investigate bone and dental tissues separately to obtain a reliable estimation of cremation temperature.     

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.     

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.     

The Effect of Soft Tissue on Temperature Estimation from Burnt Bone Using Fourier Transform Infrared Spectroscopy

This study investigated the effect of soft tissue and different exposure times on the prediction of burning temperatures of bone when using Fourier transform infrared spectroscopy (FTIR). Ovis aries rib bones were burnt at different temperatures and for varying time intervals. Results of a linear regression analysis indicated that burn temperatures can be predicted with a standard error of ±70°C from defleshed bone spectra. Exposure time does not have a significant impact on prediction accuracy. The presence of soft tissue has a significant impact on heat‐induced changes of the bone matrix in low (<300°C) as well as high temperatures (>800°C), slowing down combustion in the former and accelerating it in the latter (p < 0.05). At medium temperatures, no significant difference was noted. These results provide forensic investigators a new perspective with which to interpret the results of crystallinity measures derived from burnt bone.     

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.     

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.     

The Stability of Prostate-Specific Antigen in Semen Under Various Temperatures

Prostate-specific antigen (PSA) is most commonly used for identifying semen, especially in the absence of sperm. However, PSA concentration varies according to storage temperature and duration, and little is known about its stability in semen. This study was therefore aimed to determine the stability under five different temperatures: −80, −20, 4, 25, and 37°C; and nine different durations: 1, 2, 3, 5, 7, 14, 30, 90, and 180 days. All samples were stored at −80°C after being secreted from the volunteers' body until analyzed. Results showed that the PSA concentration declined significantly over time under all temperatures studied except −80°C. At −20 and 4°C, PSA was still detectable on Day 180 with 50% and 70% decrease from its original concentration, respectively. At 25 and 37°C, PSA was detected up to Day 7 and 3, respectively. This information might assist forensic scientists understand more about PSA nature and integrate it into their works.     

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.     

The Effect of Temperature and Exposure Time on Stability of Cholesterol and Squalene in Latent Fingermarks Deposited on PVDF Membrane

Cholesterol and squalene are fatty materials of latent fingermarks that can be utilized for dating methodologies and visualization techniques. Previous studies have suggested these compounds undergo degradation in fingermarks as a function of time (days) and light at ambient temperature. However, studies assessing how their composition changes at low and high temperatures over short periods of time (hours) have not been published previously. Here, we performed quantitative analysis of cholesterol and squalene in natural fingermark residue using PVDF membrane, after exposure to a range of temperatures (−20 to 100°C) for 4 and 8 h. We found that levels of both fatty materials remained constant at −20 to 60°C, but both showed significant reduction at 100°C, over short exposure times. These results indicate that cholesterol and squalene are detectable at −20 to 60°C, whereas at 100°C or higher, both are lost due to rapid thermal degradation.     

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.     

Preliminary results of an investigation on postmortem variations in human skeletal mass of buried bones

Extreme fragmentation can complicate the inventory of human skeletal remains. In such cases, skeletal mass can provide information regarding skeleton completeness and the minimum number of individuals. For that purpose, several references for skeletal mass can be used to establish comparisons and draw inferences regarding those parameters. However, little is known about the feasibility of establishing comparisons between inherently different materials, as is the case of curated reference skeletal collections and human remains recovered from forensic and archaeological settings. The objective of this paper was to investigate the effect of inhumation, weather and heat exposure on the skeletal mass of two different bone types. This was investigated on a sample of 30 human bone fragments (14 trabecular bones and 16 compact bones) that was experimentally buried for two years after being submitted to one of four different heat treatments (left unburned; 500?°C; 900?°C; 1000?°C). Bones were exhumed periodically to assess time-related mass variation. Skeletal mass varied substantially, decreasing and increasing in accordance to the interchanging dry and wet seasons. However, trends were not the same for the two bone types and the four temperature thresholds. The reason for this appears to be related to water absorption and to the differential heat-induced changes in bone microporosity, volume, and composition. Our results suggest that mass comparisons against published references should be performed only after the skeletal remains have been preemptively dried from exogenous water.     

Quantifying the Temperature of Maggot Masses and its Relationship to Decomposition

Numerous Calliphoridae species have been observed to form larval aggregations during the feeding stage of development, resulting in localized increases in temperature. This study investigates the relationship between maggot numbers in a mass and heat generation. Single‐species aggregations (Lucilia sericata) of various sizes (50–2500 individuals) were reared in the laboratory at a constant ambient temperature of 22°C. Internal mass temperatures were recorded every 5 min throughout the feeding stage of development. Results showed that mass temperatures increased with mass numbers (p‐value < 0.001), ranging from 2.5 to 14°C above ambient. A minimum mass size of 1200 produced overall temperatures that were significantly warmer than ambient, diverging away from 22°C after c. 26 h. These results indicate that the microclimate of a mass has the potential to differ significantly from ambient, which may be influencing larval development rates and should therefore be factored into mPMI estimates to increase accuracy.     

Variation in Developmental Time for Geographically Distinct Populations of the Common Green Bottle Fly,Lucilia sericata (Meigen)*

Abstract: Time between death and discovery of remains, or postmortem interval (PMI), can be assessed using blow fly maggot age. Forensic entomologists rely on published, often nonlocal, species‐specific developmental tables to determine maggot age. In a series of common garden experiments, we investigated the developmental rate variation between populations of Lucilia sericata collected from Sacramento, CA, San Diego, CA, and Easton, MA at 16°C, 26°C, and 36°C. For the 16°C trial the time measurement started at egg hatch, while for the higher temperatures the experiment began at oviposition; the wandering stage signified the endpoint for all experiments. The distribution of developmental times differed significantly (ANOVA, p < 0.001) between the three populations within each temperature treatment. We discovered that regional variation of developmental times within a blow fly species exists. This study demonstrates the importance of assembling local population‐specific developmental tables when estimating larval age to determine PMI.     

Effects of Different Temperatures on the Development of Dermestes Frischii and Dermestes Undulatus (Coleoptera,Dermestidae): Comparison Between Species

Dermestidae could be useful in forensic investigations to assess the PMI as adults and larvae colonize dried remains. We reared two species of Dermestidae (Dermestes frischii and Dermestes undulatus) to understand the effects of different temperatures on the length of their whole life cycle and on their immature stages. Both species were reared at 23°C ± 0.5, RH 75% and at 26°C ± 0.5, 75% RH. Our result shows that the temperature is the main factor that influences the development of those species; in fact, increasing temperature leads to a shorter development cycle (59.8 ± 0.5 and 38.1 ± 0.2 for D. frischii; 50.6 ± 0.6 and 36.2 ± 0.2 for D. undulatus). Furthermore, we found that the number of the molts before the pupa decreases from 5–7 to 5–6 for D. frischii and from 4–6 to 4–5 for D. undulatus, respectively, at 23°C and 26°C.     

Temperature‐dependent Development of Parasarcophaga similis (Meade 1876) and its Significance in Estimating Postmortem Interval

Flesh flies are commonly found insects on decaying corpses that appears slightly later than blowflies, and their development patterns are significant indicators for minimum postmortem interval (PMI_min) estimation. In this study, the flesh fly Parasarcophaga similis (Meade 1876) was reared at nine constant temperatures ranging from 15°C to 35°C to examine indicators for estimating their age. We generated three development models, including isomorphen diagram, isomegalen diagram, and thermal summation model. Larval body length at different rearing temperatures was fit into an L = a + bT + cT² + dT³ equation with which the relationship between the larval body length (L) and the time after larviposition (T) was confirmed. The pupal stage was categorized into 13 substages according to intrapuparial morphological changes, and a detailed table was generated of the pupal developmental stages at five rearing temperatures, 15°C, 20°C, 25°C, 30°C, and 35°C. This study provides fundamental data in supporting P. similis as an indicator for PMI_min estimation.     

Fluorescence and Structural Degradation in Composite Resins as a Function of Temperature*

Abstract: Detecting composite resin upon postmortem examination can be difficult. Ultraviolet illumination has been suggested to ease location of this material; however, this may not be advisable in incineration situations. Understanding of the chemical and physical properties of resin as a function of temperature is an important parameter in identification of this material in incineration circumstances. Twenty-seven discs of resin, Quixx (Dentsply), Filtek Supreme (3 M), and Tetric Ceram (Ivoclar) were prepared and exposed to increasing heating conditions of 200°C–900°C in 100°C increments for 30 min. Analysis was performed with Fourier transform infrared spectroscopy, ultraviolet-visible light spectrophotometry, scanning electron microscopy/energy dispersive X-ray spectroscopy, optical microscopy, and UV illumination. Characterization of the material occurred at each temperature range. The organic components and the fluorescence properties were lost at temperatures above 300°C. The inorganic component remained through 900°C. This information can aid in detection of resin in high temperature circumstances.     

Heat Induced Changes to Dental Resin Composites: A Reference in Forensic Investigations?*

The objective was to investigate color change and surface damage in dental resin composites exposed to high temperatures over different time intervals for comparative purposes. Samples were prepared using two resins - Z100(R) (R1) and Charisma (R2), heated at the following temperatures: 200 degrees C, 400 degrees C, 600 degrees C, 1000 degrees C, for 15, 30 and 45 min (n = 104 for each resin sample). Color (DeltaE) and brightness (DeltaL) changes were analyzed by spectrophotometry using the CIE Lab system and surface changes by Scanning Electron Microscopy (SEM). R1 showed more intense color changes after heat exposure than R2. DeltaL values were found to be the best parameter for evaluation of light and color change. A biphasic pattern after thermal exposure was detected, from dark brown to light white. SEM showed more intense alterations in R2 than in R1. These results indicate that the parameters observed in both resins are useful as a guide in forensic analyses.     

Tracking Movement and Temperature Selection of Larvae of Two Forensically Important Blow Fly Species Within a “Maggot Mass”

The current study responds to the lack of understanding about the temperatures experienced by individual blow fly larvae within “maggot masses.” The temperature selection of both aggregating (in a mass) and nonaggregating larvae was compared and their pattern of movement assessed. Infrared imaging determined the temperatures within a mass and in the vicinity of the constituent individual larvae, whose movements were tracked by dyeing their tissues red. Individual Chrysomya rufifacies larvae selected temperatures above 27°C, significantly higher than the temperature selected by Calliphora vicina larvae (24.5°C). However, this same difference was not seen within a mass, with both species selecting temperatures around 28°C. Larval movement in a mass was nonrandom, indicating that larvae actively select their position in a mass. Furthermore, larvae have a strong tendency to select the hottest part of a mass; therefore, maximum mass temperatures might provide a reliable proxy for the actual temperatures experienced by larvae.     

Effect of Temperature on Six Different Developmental Landmarks within the Pupal Stage of the Forensically Important Blowfly Calliphora vicina (Robineau‐Desvoidy) (Diptera: Calliphoridae)

This study investigates the pupal development times of the blow fly Calliphora vicina, which were studied in the laboratory at six different constant temperatures (15, 20, 23, 25, 28, and 30°C each ± 1°C). Lower thresholds (t_L) for development were estimated from the linear regression of the developmental rates on each temperature. These data have made it possible to calculate the accumulated degree days (ADD) necessary for C. vicina to complete the larval stage and to achieve adult emergence. The minimal duration of development from oviposition to adult emergence was found to be inversely related to temperature. Additionally, six landmarks in pupal development are showed, and for each of the landmarks, the ADD value was calculated for every rearing temperature involved. These data assist in calculating the duration of the pupal stage based on morphological characteristics and would be of great value for future forensic entomological casework.