Luminol and the postmortem interval estimation - influence of taphonomic factors.

The estimation of the postmortem interval (PMI) is one of the key challenges for forensic anthropologists. Although there are several methods referenced for this purpose, none is sufficiently effective. One of the main reasons justifying the complexity of this task is the influence of several taphonomic factors.The study of the Luminol technique has stood out as a promising method for estimating PMI, complementing the existing methods, since it is an economic, easy and reproducible method that operates as a presumptive test. However, it is not known which taphonomic factors can influence the results obtained by this technique.The aim of this study is to test the influence of taphonomic factors, such as temperature, humidity, soil type and pH, on the estimation of the PMI by the Luminol technique.In order to test the influence of the referred factors, a sample consisting of 30 clavicles, with known and similar PMI, collected from autopsies, was distributed as evenly as possible by six vases and buried with different decomposition conditions for a period of 12 months. After the exhumation and sample preparation, the Luminol technique was applied.It was possible to clearly observe differences in the results. Thus, according to our research, it is possible to conclude that the results obtained by the application of Luminol are influenced by taphonomic factors. Therefore, the context in which a body is found should always be considered for applying this technique.

All that glitters is not gold: X-ray fluorescence analysis of a fixed dental prosthesis from Colecção de Esqueletos Identificados Século XXI, Portugal (CEI/XXI).

Access to better health care anticipates that more medical devices can be found alongside skeletal remains. Those employed in oral rehabilitation, with available brands or batch/series, can prove useful in the identification process. A previous study in the Colecção de Esqueletos Identificados Século XXI described macroscopically the dental prostheses. An unusual case of a dental device with chromatic alterations demonstrated to require a more detailed analysis. The individual, a 53-year-old male, exhibited, at both arches, a fixed tooth-supported rehabilitation, with gold colouring classified initially as a gold-palladium alloy. Simultaneously, a green pigmentation deposit was observable in bone and prosthesis. This investigation aimed to verify the elemental composition of the dental prosthesis alloy. Elemental analysis was performed by X-ray fluorescence in two regions (labial surface of the prosthetic crown and the root surface of the lower right lateral incisor). Both the spectra and the qualitative results found higher levels of copper and aluminium, followed by nickel, iron, zinc, and manganese. No gold or palladium was detected. The most probable assumption is that a copper-aluminium alloy was used, as its elemental concentration corresponds to those measured in similar devices. Dental prostheses of copper-aluminium alloys have been made popular since the 1980s, particularly in the USA, Japan, and Eastern Europe. Apart from the biographical information, it was also known that the individual's place of birth was an Eastern European country, which highlighted the usefulness of this type of information when dealing with missing people cases.

Age assessment in unaccompanied minors: assessing uniformity of protocols across Europe.

Age assessment of migrants is crucial, particularly for unaccompanied foreign minors, a population facing legal, social, and humanitarian challenges. Despite existing guidelines, there is no unified protocol in Europe for age assessment.The Forensic Anthropology Society of Europe (FASE) conducted a comprehensive questionnaire to understand age estimation practices in Europe. The questionnaire had sections focusing on the professional background of respondents, annual assessment numbers, requesting parties and reasons, types of examinations conducted (e.g., physical, radiological), followed protocols, age estimation methods, and questions on how age estimates are reported.The questionnaire's findings reveal extensive engagement of the forensic community in age assessment in the living, emphasizing multidisciplinary approaches. However, there seems to be an incomplete appreciation of AGFAD guidelines. Commonalities exist in examination methodologies and imaging tests. However, discrepancies emerged among respondents regarding sexual maturity assessment and reporting assessment results. Given the increasing importance of age assessment, especially for migrant child protection, the study stresses the need for a unified protocol across European countries. This can only be achieved if EU Member States wholeheartedly embrace the fundamental principles outlined in EU Directives and conduct medical age assessments aligned with recognized standards such as the AGFAD guidelines.

In-Situ Anaerobic Heating of Human Bones Probed by Neutron Diffraction.

The first neutron diffraction study of in-situ anaerobic burning of human bones is reported, aiming at an interpretation of heat-induced changes in bone, which were previously detected by vibrational spectroscopy, including inelastic neutron scattering techniques. Structural and crystallinity variations were monitored in samples of the human femur and tibia, as well as a reference hydroxyapatite, upon heating under anaerobic conditions. Information on the structural reorganization of the bone matrix as a function of temperature, from room temperature to 1000 °C, was achieved. Noticeable crystallographic and domain size variations, together with O-H bond lengths and background variations, were detected. Above 700 °C, the inorganic bone matrix became highly symmetric, devoid of carbonates and organic constituents, while for the lower temperature range (<700 °C), a considerably lower crystallinity was observed. The present pilot study is expected to contribute to a better understanding of the heat-prompted changes in bone, which can be taken as biomarkers of the burning temperature. This information is paramount for bone analysis in forensic science as well as in archeology and may also have useful applications in other biomaterial studies.

DXAGE 2.0 - adult age at death estimation using bone loss in the proximal femur and the second metacarpal.

The accurate age at death assessment of unidentified adult skeletal individuals is a critical research task in forensic anthropology, being a key feature for the determination of biological profiles of individual skeletal remains. We have previously shown that the age-related decrease of bone mineral density (BMD) in the proximal femur could be used to assess age at death in women (Navega et al., J Forensic Sci 63:497-503, 2018). The present study aims to generate models for age estimation in both sexes through bone densitometry of the femur and radiogrammetry of the second metacarpal. The training sample comprised 224 adults (120 females, 104 males) from the "Coimbra Identified Skeletal Collection," and different models were generated through least squares regression and general regression neural networks (GRNN). The models were operationalized in a user-friendly online interface at https://osteomics.com/DXAGE2/ . The mean absolute difference between the known and estimated age at death ranges from 9.39 to 13.18 years among women and from 10.33 to 15.76 among men with the least squares regression models. For the GRNN models, the mean absolute difference between documented and projected age ranges from 8.44 to 12.58 years in women and from 10.56 to 16.18 years in men. DXAGE 2.0 enables age estimation in incomplete and/or fragmentary skeletal remains, using alternative skeletal regions, with reliable results.

The dental prosthesis (removable and fixed) from the Colecção de Esqueletos Identificados Século XXI (CEI/XXI).

For identification of the unknown, the analysis of individualizing characteristics is a paramount procedure, which allows the reconciliation of antemortem with postmortem data. Worldwide, populations are commonly affected by tooth loss, leading to procedures of oral rehabilitation, such as dental prostheses. Although the potential of these devices for human identification is well known, the lack of marks or serial numbers as well as scarce systematic register on databases may difficult its use. The objective of this paper is to present and describe the cases with dentures and bridges of an identified osteological collection, the Colecção de Esqueletos Identificados Século XXI. All the individuals were macroscopically observed, and the cases presenting dental prostheses were registered. A total of thirty-one individuals (20 females; 11 males), ages 38 to 91 years, presented 49 dental prosthesis (removable or fixed), of various materials. The removable acrylic dentures (n = 42) were the most frequent. No identifying marks or production labels were found. This research reinforces two focal points: the value of studying and recognizing medical devices and their application for identification, and the relevance of identified skeletal collections for the advance of forensic science in the current multidisciplinary approach.

New data about the 21st Century Identified Skeletal Collection (University of Coimbra, Portugal).

After the publication of the first article in 2014, 21st Century Identified Skeletal Collection, housed in the Laboratory of Forensic Anthropology, Department of Life Sciences at the University of Coimbra, Portugal, has been growing. Currently, the collection is composed of 302 complete adult skeletons of both sexes, which means that in 5 years it has doubled. The collection consists mostly of elderly individuals, with only 12.25% of the individuals aged less than 61 years old. All individuals are Portuguese nationals who died between 1982 and 2012. Ninety individuals exhibit prostheses, other medical devices and signs of surgical procedures. Moreover, a sub-collection of experimentally burned skeletons is under development, and currently includes 56 individuals (18.54% of the collection). The 21st Century Identified Skeletal Collection constitutes a fundamental tool for forensic anthropology research, including the development and validation studies of methods that focus on elderly individuals, as can be ascertained by the numerous scientific publications and academic scholarship that have been produced in previous years.

Chemosteometric regression models of heat exposed human bones to determine their pre-burnt metric dimensions.

OBJECTIVES: Heat exposure can lead to apparently random osteometric changes that hinder the application of metric methods used for biological profiling. The impracticality of using objective and burn-specific osteometric methods reduces the chances of establishing the biological profiles of unknown individuals based on their skeletal remains. We investigated the potential of chemometry analysis based on infrared spectroscopy to predict the amount of heat-induced osteometric changes and how this reflected into sex estimation. MATERIAL AND METHODS: Bones from 41 identified adult skeletons (24 females and 17 males with ages between 62 and 90 years old) were experimentally burnt to maximum temperatures ranging from 450°C to 1,100°C (attained after 65 to 240 min). Measurements were taken both before and after each experiment and powder samples were analyzed through FTIR-ATR. Correlations among heat-induced metric changes and chemometric indices (crystallinity index; B-type carbonates; carbonate [A + B] to carbonate B ratio; hydroxyl to phosphate ratio; 630 cm-1 , 1450 cm-1 , 3572 cm-1 , and 3642 cm-1 ) were tested. Significant variables were used to build regression models to predict heat-induced metric change which were then tested on an independent set of samples. Agreement in sex estimation between the pre- and post-burnt samples was also evaluated. RESULTS: All indices were significantly correlated to heat-induced metric changes (α = .01) and the highest correlations were obtained for the 630 cm-1 , 3572 cm-1 , and crystallinity index. We confirmed that regression models based on chemometrics obtained from infrared spectra through FTIR-ATR are better at estimating heat-induced metric changes affecting bone and at sexing remains than other osteometric methods such as those based on correction factors or on metric references specific to calcined bones. DISCUSSION: Regression models avoid the subjectivity associated with the application of other methods. While the latter can be applied only to calcined bones, which is difficult to assess sometimes, regression models can be applied to all bones regardless of their condition. Also, regression models have the advantage of allowing to infer about heat-induced metric change on a case-by-case basis.

A validation study of the Stoyanova et al. method (2017) for age-at-death estimation quantifying the 3D pubic symphyseal surface of adult males of European populations.

The age-at-death estimation thresholds have recently been shifted towards a more objective assessment of the aging process. Such a non-subjective approach offers quantitative methods of age estimation; for instance, the method relating to the surfaces of pubic symphyses of males published by Stoyanova et al. (J Forensic Sci 62:1434-1444, 2017). A validation study was conducted to test the method performance in European samples. The sample consisted of 96 meshes of pubic symphyses of male individuals (known sex and age) that came from four different samples (two Portuguese collections, one Swiss, and one Crete). Stoyanova's method based on five regression models (three univariate and two multivariate models) performed worse in our sample, but only when the whole sample (without age limitation) was included. A sample limited to individuals under 40 years of age achieved better results in our study. The best results were reached through the thin plate spline algorithm (TPS/BE) with a root mean square error of 5.93 years and inaccuracy of 4.47 years. Generally, the multivariate regression models did not contribute to better age estimation. In our sample in all age categories, age was systematically underestimated. The quantitative method tested in this study works best for individuals under 40 years of age and provides a suitable basis for further research.

Age at death estimation by cementochronology: Too precise to be true or too precise to be accurate?

OBJECTIVES: Cementochronology based on annual deposition of acellular cementum is acknowledged for its superior performance for estimating age-at-death but is also disregarded because of its suspicious effectiveness. This article aims to provide a standardized framework for the validation of the technique and to define and test cementochronology's performance and limitations. MATERIALS AND METHODS: To determine the boundaries of the cementum aging technique, we applied a certified protocol on a sample of 200 healthy canines from individuals of known age, sex, postmortem, and inhumation intervals from anthropological and anatomical collections. We scored readability and preservation of cementum and measured the agreement between estimates, i.e., the precision, and assessed the quality of the accordance between estimates and chronological age, i.e., the accuracy. To investigate the applicability on ancient material, 200 additional canines extracted from archeological assemblages were included. Accuracy and precision were analyzed for each age group in considering postmortem intervals and taphonomical conditions. RESULTS: A strong correlation was found between chronological age and estimates (r = .927; p = .000) but results reveal an association between readability of incremental lines and chronological age (p < .05) and a notable difference in both precision and accuracy between individuals under and over 50 years. Results also demonstrate that taphonomy can be a serious obstacle increasing imprecision by a factor of three. DISCUSSION: Improperly adopted, cementochronology can lead to precise but inaccurate estimations. If methodological, physiological, and taphonomical factors are taken into account, then, and only then, cementochronology will serve as a versatile and powerful tool for age-at-death 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.

Surviving a transfixing gunshot wound to the head 70 years ago.

Surviving a gunshot wound to the head is a rare event, particularly in the past when medical treatment was much less advanced than it is today. Moreover, the finding of such a case as an identified specimen within a museum collection is even more uncommon. This led us to report on this unique case in this paper as it poses a challenge to forensic anthropology and provides a unique educational oppourtunity. The skull from the Collection at the Cranium Museum in the Department of Morphology and Genetic at the Federal University of São Paulo (UNIFESP) dates back to 1946. For trauma registration the bone location, severity, trauma aetiology, trauma classification, description, callus formation, periosteal reaction, degree and success of repair, and an estimate of the time elapsed since the trauma, were all assessed. To explore the case radiologically a CT scan of the skull was performed. Considering the survival of the patient and the morphology of the wound it is likely that the injury was caused by a small calibre, low-velocity gunshot. The bullet path shows an almost vertical direction on the right side of the individual's splanchno and neurocranium. The path of the projectile is consistent with a suicide attempt, although the possibility of a homicide simulating a suicide cannot be discarded. This case highlights how informative such cases can be to the practice of forensic anthropology.

Potential of Bioapatite Hydroxyls for Research on Archeological Burned Bone.

The estimation of the maximum temperature affecting skeletal remains was previously attempted via infrared techniques. However, fossilization may cause changes in the composition of bones that replicate those from burned bones. We presently investigated the potential of three OH/P indices (intensity ratios of characteristic infrared bands for OH and phosphate groups, respectively) to identify bones burned at high temperatures (>800 °C) and to discriminate between fossil and burned archeological bones, using vibrational spectroscopy: combined inelastic neutron scattering (INS) and FTIR-ATR. The INS analyses were performed on two unburned samples and 14 burned samples of human femur and humerus. FTIR-ATR focused on three different samples: (i) modern bones comprising 638 unburned and 623 experimentally burned (400-1000 °C) samples; (ii) archeological cremated human skeletal remains from the Bronze and Iron Ages comprising 25 samples; and (iii) fossil remains of the Reptilia class from the Middle Triassic to the Eocene. The OH/P indices investigated were 630 cm-1/603 cm-1, 3572 cm-1/603 cm-1, and 3572 cm-1/1035 cm-1. The OH signals became visible in the spectra of recent and archeological bones burned between 600 and 700 °C. Although they have episodically been reported in previous works, no such peaks were observed in our fossil samples thus suggesting that this may be a somewhat rare event. While high crystallinity index values should always correspond to clearly visible hydroxyl signals in burned bone samples, this is not always the case in fossils which may be used as a criterion to exclude burning as the agent responsible for high crystallinity ratios.

A test and analysis of Calce (2012) method for skeletal age-at-death estimation using the acetabulum in a modern skeletal sample.

In forensic anthropology, the age-at-death of an adult individual is one of the most complex parameters of the biological profile to estimate. The present study aims to evaluate the reliability of the Calce (2012) method for the estimation of age-at-death through acetabulum changes in a sample of Portuguese origin. This method consists of the global analysis of acetabular age-related morphology with focus on three specific traits, namely the acetabular groove, the osteophyte development of the acetabular rim, and the apex growth. This method was tested in 120 individuals sampled from the Twenty-first Century Identified Skeletal Collection (University of Coimbra, Portugal). The test sample is composed of 60 males and 60 females, aged between 25 and 99 years, with well-preserved os coxae. The results showed that only 60% of the individuals were correctly attributed to the age group defined by the technique. The comparison with previous studies in other populations shows significant inter-population differences in the relationship between the acetabulum variables used by Calce and age-at-death. The obtained results advise caution in the use of the Calce (2012) method to estimate the age-at-death of unidentified skeletons.

The costal remains of the El Sidrón Neanderthal site (Asturias, northern Spain) and their importance for understanding Neanderthal thorax morphology.

The study of the Neanderthal thorax has attracted the attention of the scientific community for more than a century. It is agreed that Neanderthals have a more capacious thorax than modern humans, but whether this was caused by a medio-lateral or an antero-posterior expansion of the thorax is still debated, and is key to understanding breathing biomechanics and body shape in Neanderthals. The fragile nature of ribs, the metameric structure of the thorax and difficulties in quantifying thorax morphology all contribute to uncertainty regarding precise aspects of Neanderthal thoracic shape. The El Sidrón site has yielded costal remains from the upper to the lower thorax, as well as several proximal rib ends (frequently missing in the Neanderthal record), which help to shed light on Neanderthal thorax shape. We compared the El Sidrón costal elements with ribs from recent modern humans as well as with fossil modern humans and other Neanderthals through traditional morphometric methods and 3D geometric morphometrics, combined with missing data estimation and virtual reconstruction (at the 1st, 5th and 11th costal levels). Our results show that Neanderthals have larger rib heads and articular tubercles than their modern human counterparts. Neanderthal 1st ribs are smaller than in modern humans, whereas 5th and 11th ribs are considerably larger. When we articulated mean ribs (size and shape) with their corresponding vertebral elements, we observed that compared to modern humans the Neanderthal thorax is medio-laterally expanded at every level, especially at T5 and T11. Therefore, in the light of evidence from the El Sidrón costal remains, we hypothesize that the volumetric expansion of the Neanderthal thorax proposed by previous authors would mainly be produced by a medio-lateral expansion of the thorax.

Luminol chemiluminescence: contribution to postmortem interval determination of skeletonized remains in Portuguese forensic context.

Postmortem interval (PMI) determination is one of the main challenges of forensic anthropology, and there are several referenced methods that consider intrinsic and extrinsic factors to the skeletonized remains. Therefore, there is an important need to develop a precise, economic, easy and reproducible technique, which will operate as a presumptive test. The chemical reaction that occurs in the presence of luminol, chemiluminescence, is used as a work tool to evaluate the PMI. This is given by luminol high affinity for haemoglobin, which is secured in bone tissue by its high mineral content, after the blood supply ceases postmortem. We evaluated a luminol-based technique, in which we measured the chemiluminescence of skeletonized remains as an indicator of the PMI. We assessed the usage of this technique for PMI estimation and for deciding whether remains have forensic interest, particularly in the Portuguese medicolegal context. Additionally, we evaluate the reproducibility of this technique. The test sample consists of 50 skeletonized individuals of both sexes, adults, between 20 and 98 years, with a known time since death and without bone pathologies. The results of Kappa statistic, with values between 0.79 and 0.97, showed high interobserver and intraobserver agreement when applying the luminol test as a presumption test. We concluded that this technique alone is not precise and of limited value to determine forensic relevance in the Portuguese context. However, it can be used as a presumptive test, reducing time and costs in criminal investigation.

The circles of life: age at death estimation in burnt teeth through tooth cementum annulations.

Age at death estimation in burnt human remains is problematic due to the severe heat-induced modifications that may affect the skeleton after a burning event. The objective of this paper was to assess if cementochronology, which focuses on the cementum incremental lines, is a reliable method of age estimation in burnt remains. Besides the classical approach based on the counting of incremental lines, another approach based on the extrapolation of incremental lines taking into account the cement layer thickness and the incremental line thickness was investigated. A comparison of the performance of the two techniques was carried out on a sample of 60 identified monoradicular teeth that were recently extracted at dentist offices and then experimentally burnt at two maximum temperatures (400 and 900 °C). Micrographs of cross-sections of the roots were taken via an optical microscope with magnification of ×100, ×200 and ×400. Incremental line counting and measurements were carried out with the ImageJ software. Age estimation based on incremental line counting in burnt teeth had no significant correlation with chronological age (p = 0.244 to 0.914) and led to large absolute mean errors (19 to 21 years). In contrast, age estimation based on the extrapolation approach showed a significant correlation with known age (p = 0.449 to 0.484). In addition, the mean absolute error of the latter was much smaller (10 to 14 years). The reason behind this discrepancy is the heat-induced dimensional changes of incremental lines that affect their visibility and individualization thus complicating line counting. Our results indicated that incremental lines extrapolation is successful at solving this problem and that the resulting age estimation is much more reliable.

Validation and reliability of the sex estimation of the human os coxae using freely available DSP2 software for bioarchaeology and forensic anthropology.

OBJECTIVES: A new tool for skeletal sex estimation based on measurements of the human os coxae is presented using skeletons from a metapopulation of identified adult individuals from twelve independent population samples. For reliable sex estimation, a posterior probability greater than 0.95 was considered to be the classification threshold: below this value, estimates are considered indeterminate. By providing free software, we aim to develop an even more disseminated method for sex estimation. MATERIALS AND METHODS: Ten metric variables collected from 2,040 ossa coxa of adult subjects of known sex were recorded between 1986 and 2002 (reference sample). To test both the validity and reliability, a target sample consisting of two series of adult ossa coxa of known sex (n = 623) was used. The DSP2 software (Diagnose Sexuelle Probabiliste v2) is based on Linear Discriminant Analysis, and the posterior probabilities are calculated using an R script. RESULTS: For the reference sample, any combination of four dimensions provides a correct sex estimate in at least 99% of cases. The percentage of individuals for whom sex can be estimated depends on the number of dimensions; for all ten variables it is higher than 90%. Those results are confirmed in the target sample. DISCUSSION: Our posterior probability threshold of 0.95 for sex estimate corresponds to the traditional sectioning point used in osteological studies. DSP2 software is replacing the former version that should not be used anymore. DSP2 is a robust and reliable technique for sexing adult os coxae, and is also user friendly.

A method for estimating gestational age of fetal remains based on long bone lengths.

The estimation of gestational age (GA) in fetal human remains is important in forensic settings, particularly to assess fetal viability, in addition to often being the only biological profile parameter that can be assessed with some accuracy for non-adults. The length of long bone diaphysis is one of the most frequently used methods for fetal age estimation. The main objective of this study was to present a simple and objective method for estimating GA based on the measurements of the diaphysis of the femur, tibia, fibula, humerus, ulna, and radius. Conventional least squares regression equations (classical and inverse calibration approaches) and quick reference tables were generated. A supplementary objective was to compare the performance of the new formulae against previously published models. The sample comprised 257 fetuses (136 females and 121 males) with known GA (between 12 and 40 weeks) and was selected based on clinical and pathological information. All measurements were performed on radiographic images acquired in anonymous clinical autopsy records from spontaneous and therapeutic abortions in two Portuguese hospitals. The proposed technique is straightforward and reproducible. The models for the GA estimation are exceedingly accurate and unbiased. Comparisons between inverse and classical calibration show that both perform exceptionally well, with high accuracy and low bias. Also, the newly developed equations generally outperform earlier methods of GA estimation in forensic contexts. Quick reference tables for each long bone are now available. The obtained models for the estimation of gestational age are of great applicability in forensic contexts.

Rather yield than break: assessing the influence of human bone collagen content on heat-induced warping through vibrational spectroscopy.

Warping has been used to determine the pre-burning condition of human skeletal remains. In the literature, this modification has been associated more often with the burning of fleshed and green bones, but it also arises during the burning of dry bones. The objective of this paper was to assess if bone collagen content has a significant effect on the occurrence of warping in a sample of experimentally burned human bones. The presence of collagen was analyzed in two different samples through a vibrational spectroscopy technology-FTIR. One of them was composed of 40 archeological bones from the seventeenth to twentieth centuries AD. The other one was composed of bones from 14 skeletons belonging to the 21st century identified skeletal collection. The results confirmed that the amide I band assigned to the collagen was much more intense in bones presenting heat-induced warping. Nonetheless, although significant (p = 0.040), the collagen content was not as useful as other variables to the regression model we proposed for explaining warping. Factors such as the maximum temperature (p < 0.001) and burning time (p = 0.001) contributed more significantly. Results demonstrated that the mere preservation of collagen is not enough to explain warping. Burning dynamics seem to have an important role as well although we failed to clearly document its specificities. Other factors such as the asymmetric distribution of collagen and other components within bone, the gravity force, the shape of the bone, and the position in which it is burned may also play an important role on heat-induced changes and require further analysis.