Human brains preserve in diverse environments for at least 12 000 years.

The brain is thought to be among the first human organs to decompose after death. The discovery of brains preserved in the archaeological record is therefore regarded as unusual. Although mechanisms such as dehydration, freezing, saponification, and tanning are known to allow for the preservation of the brain on short time scales in association with other soft tissues (≲4000 years), discoveries of older brains, especially in the absence of other soft tissues, are rare. Here, we collated an archive of more than 4400 human brains preserved in the archaeological record across approximately 12 000 years, more than 1300 of which constitute the only soft tissue preserved amongst otherwise skeletonized remains. We found that brains of this type persist on time scales exceeding those preserved by other means, which suggests an unknown mechanism may be responsible for preservation particular to the central nervous system. The untapped archive of preserved ancient brains represents an opportunity for bioarchaeological studies of human evolution, health and disease.

Assessment of the efficiency of DNA isolation and profiling applying a temperature-driven method in human remains.

The identification of human remains is of utmost importance in a variety of scenarios. One of the primary identification methods is DNA. DNA extraction from human remains could be difficult, particularly in situations where the remains have been exposed to environmental conditions and other insults. Several studies tried to improve extraction by applying different approaches. ForensicGEM Universal (MicroGem) is a single-tube approach to DNA extraction and a temperature-driven method that could have some advantages with respect to previous techniques, among them, reducing the risk of contamination, not requiring specialized equipment, or several steps to perform. The aim of this study was to assess, for the first time, the efficiency of DNA extraction and quality of STR profiles applying the MicroGem protocol and modifications of this protocol from tooth samples in comparison with automatic extraction (AE). Our results indicated that AE and MicroGem performed similar, though with variability depending on the MicroGem modifications, increasing the DNA yield and STR profile quality when DNA is concentrated with Microcon. These findings demonstrated the efficiency of this methodology for DNA extraction from human remains while also providing a simple and quick technique suitable to apply in a variety of forensic scenarios.

Technical note: Temperature estimation accuracy based on colourimetry of embalmed human and fresh non-human burned bone.

Estimation of the exposure temperature of skeletal remains can be done by means of colourimetry and a previously published decision model for the colourimetric data, resulting in clusters that represent a range of exposure temperature. The method was based on thermally altered freshly burned human skeletal remains. However, in practice the origin or pre-burning condition of (possibly fragmentary) burned remains can be unknown. Further, in order to use the colourimetric analysis, and accompanying decision model, it is important to have a reference or test set. Fresh human material is not available for this purpose in all countries. Hence, the classification accuracy of the decision model was tested for, in some countries more readily available substitutes for fresh bone; embalmed human bone and non-human bone. The model yielded high accuracies for these sample materials, making it possible to create a reference or test set from fresh non-human and embalmed human bone as substitute for human, and also to use the decision model for these deviating samples in practice.

The influence of age on greater sciatic notch morphology: testing the Walker method in an Australian population.

Sex estimation is an integral aspect of a forensic biological profile. The pelvis, being the most dimorphic part of the skeleton, has been studied in considerable detail relative to morphological and metric variation. However, empirical data on the effect of age on pelvic morphology relative to sex-specific morphological variation is limited, especially in regard to the estimation of skeletal sex. This study assesses whether there are age-related differences in the distribution of the Walker (2005) morphological scores for the greater sciatic notch (GSN) in an Australian population. Three-dimensional volumetric reconstructions derived from multi-detector computed tomography (MDCT) scans of 567 pelves of 258 females and 309 males aged 18 to 96 years were scored following Walker (2005). Differences in score distributions and means by sex and age group were tested using Pearson's chi-squared test and ANOVA, respectively. The accuracy of sex estimates derived from logistic regression equations was explored using leave-one-out cross-validation. Significant differences were found in score distribution and means among age groups in females, but not in males. There was a tendency toward higher scores in older females. The overall sex estimation accuracy was 87.5%. When comparing age groups 18-49 and 70 + years, estimation accuracy decreased in females (99% vs. 91%), while the opposite was found for males (79% vs. 87%). These findings suggest that age affects GSN morphology. Higher mean scores in older females imply that, on average, the GSN becomes narrower with increasing age. It is thus recommended due consideration of estimated age when assessing sex based on the GSN in unidentified human remains.

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.

A comparison of maceration methods for the preparation of infant skeletal remains for forensic anthropological analysis.

Very little literature currently exists prescribing which maceration method to use when preparing infant human remains, resulting in bone quality that is suitable for forensic anthropological analysis. The aim of the study was to test five maceration methods to determine which is most suitable for infant remains for forensic anthropological analysis. The sample included five neonate pig carcasses (Sus scrofa domesticus), ranging between one to three days old. Five maceration methods were tested on the pig carcasses (one pig per maceration method) to determine their effectiveness. The methods included invertebrate maceration by meal worms, chemical maceration by bleach, chemical maceration by borax solution, enzymatic maceration by laundry detergent and sodium carbonate solution, and chemical maceration by sodium hypochlorite. A scoring method was created to assess the effectiveness of each maceration method. Invertebrate maceration and chemical maceration using bleach were the least successful methods of maceration (total maceration score = 8 respectively). Chemical maceration using borax and chemical maceration using sodium hypochlorite achieved complete maceration of the skeletal remains; however, they both resulted in artifacts that are unsuitable for forensic analysis (total maceration score = 14 respectively). Enzymatic maceration using laundry detergent and sodium carbonate was the most successful method (total maceration score = 17). The detergent technique subsequently successfully macerated all five sets of infant human remains. This study has validated that the enzymatic maceration technique using laundry detergent and sodium carbonate can be used to effectively macerate the remains of infant skeletal remains for forensic anthropological analysis.

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.

Sex estimation from coxal bones using deep learning in a population balanced by sex and age.

In the field of forensic anthropology, researchers aim to identify anonymous human remains and determine the cause and circumstances of death from skeletonized human remains. Sex determination is a fundamental step of this procedure because it influences the estimation of other traits, such as age and stature. Pelvic bones are especially dimorphic, and are thus the most useful bones for sex identification. Sex estimation methods are usually based on morphologic traits, measurements, or landmarks on the bones. However, these methods are time-consuming and can be subject to inter- or intra-observer bias. Sex determination can be done using dry bones or CT scans. Recently, artificial neural networks (ANN) have attracted attention in forensic anthropology. Here we tested a fully automated and data-driven machine learning method for sex estimation using CT-scan reconstructions of coxal bones. We studied 580 CT scans of living individuals. Sex was predicted by two networks trained on an independent sample: a disentangled variational auto-encoder (DVAE) alone, and the same DVAE associated with another classifier (Crecon). The DVAE alone exhibited an accuracy of 97.9%, and the DVAE + Crecon showed an accuracy of 99.8%. Sensibility and precision were also high for both sexes. These results are better than those reported from previous studies. These data-driven algorithms are easy to implement, since the pre-processing step is also entirely automatic. Fully automated methods save time, as it only takes a few minutes to pre-process the images and predict sex, and does not require strong experience in forensic anthropology.

Sex estimation by CT image analysis of the rib cage in a Mediterranean population.

Sexual dimorphism in the human species is key to the development of sex estimation techniques in the human skeleton. This dimorphism is manifested, as in other regions of the skeleton, in the bones that constitute the thoracic cage, according to the existing bibliography. In this aspect, the study of the human skeleton through 3D images has also proved to be useful for the development and validation of sex estimation methodologies for the reconstruction of the osteobiological profile.For this purpose, a sample of 240 thoracic CT scans of adult individuals was selected from a collection of 3D images belonging to the University of Granada, provided by the Castilla-La Mancha Health Service (SESCAM). Different measurements of the thoracic bones (ribs R2 to R5 width, sternum length and width, and clavicles width) have been taken with OsiriX software, with the aim of developing discriminant functions for sex estimation.The obtained results are positive, allowing sex estimation through 3D images of the thorax with up to 89.6% accuracy through discriminant functions, which shows the usefulness of image analysis for the reconstruction of the osteobiological profile.

Postmortem interval estimation of human skeletonized remains through luminol chemiluminescence.

Postmortem interval (PMI) estimation represents a significant challenge in the forensic sciences, particularly when dealing with human skeletal remains. A screening protocol for distinguishing possible remains of forensic interest is a crucial tool for judicial purposes. In this context, luminol chemiluminescence emerges as a promising method, with low overall costs and required time. This method is primarily used as a presumptive test, based on the understanding that the intensity of the chemiluminescence reactions decreases with an increase in the postmortem interval, thus underlining its practical implications.This research aims to expand previous research on the potential of luminol chemiluminescence, evaluating its usefulness in estimating PMI. Our sample comprised 239 human clavicles, with known PMI. The luminol solution was sprayed on each powder bone sample in a dark room, observed by the naked eye and photographed. The intensity of the chemiluminescence reaction was measured using a binary and a 5-level scale.The present results reveal that this method is a suitable tool for PMI estimation as a presumptive test, reducing time and costs in criminal investigations. The findings underscore the high sensitivity of luminol chemiluminescence for detecting recent PMI but also highlight a notable incidence of false positives. Thus, our results confirm luminol chemiluminescence as a powerful tool for dating time of death, particularly for identifying forensic relevant remains. Still, the relatively low specificity indicates that it should be complemented with additional tests for further confirmation and scientific validation of the remains' forensic relevance.

Sex estimation from patellar measurements in a contemporary Italian population: a machine learning approach.

Biological sex estimation in forensic anthropology is a crucial topic, and the patella has shown promise in this regard due to its sexual dimorphism. This study uses 12 machine learning models for sex estimation based on three patellar measurements (maximum height, breadth, and thickness). Data was collected from 180 skeletons of a contemporary Italian population (83 males and 97 females) as well as from an independent sample of 21 forensic cases (13 males and 8 females). Statistical analyses indicated that each of the variables exhibited significant sexual dimorphism. To predict biological sex, the classifiers were built using 70% of a reference sample, then tested on the remaining 30% of the original sample and then tested again on the independent sample. The different classifiers generated accuracies varied between 0.85 and 0.91 on the reference sample and between 0.71 and 0.95 for the validation sample. SVM classifier stood out with the highest accuracy and seemed the best model for our study.This study contributes to the growing application of machine learning in forensic anthropology by being the first to apply such techniques to patellar measurements in an Italian population. It aims to enhance the accuracy and efficiency of biological sex estimation from the patella, building on promising results observed with other skeletal elements.

Ballistic long bone fracture pattern: an experimental study.

When dealing with badly preserved cadavers or skeletal human remains, the assessment of death circumstances remains challenging. When forensic evidence cannot be taken from the skin and soft tissue, the information may only be deduced from more resistant elements such as bone. Compared to cranial gunshot injuries, reliable data on ballistic long bone trauma remains scarce. This study aims to define ballistic fracture characteristics in human long bones. The shaft of 16 femurs and 13 humeri from body donors was perpendicularly shot with a 9-mm Luger full metal jacket bullet at an impact velocity of 360 m/s from a distance of 2 m. Some bones were embedded in Clear Ballistics Gel®, and some were shot without soft tissue simulant in order to better visualise the fracture propagation on the high-speed camera. The fractures were examined macroscopically and compared between the sample groups. We consistently found comminuted fractures with a stellate pattern. Fracture details were classified into entrance, exit and general characteristics. For some traits, we detected different occurrence values in the group comparison. The results indicate that some of the traits depend on bone properties such as shaft diameter, bone length and cortical thickness. The presence of ballistic gel also influenced some fracture traits, emphasising the relevance of soft tissue simulant in osseous gunshot experiments. This study revealed new insights in the detailed fracture pattern of human long bones. These may serve as guidelines for the identification and reconstruction of gunshot trauma in human long bones.

The influence of biological relatedness on sexual dimorphism and sex classification based on external morphology of the frontal bone.

The most significant sexual differences in the human skull are located in the upper third of the face (the frontal bone), which is a useful research object, mainly in combination with virtual anthropology methods. However, the influence of biological relatedness on sexual dimorphism and frontal bone variability remains unknown. This study was directed at sexual difference description and sex classification using the form and shape of the external surface of the frontal bones from a genealogically documented Central European osteological sample (nineteenth to twentieth centuries). The study sample consisted of 47 cranial CT images of the adult members of several branches of one family group over 4 generations. Three-dimensional virtual models of the frontal bones were analyzed using geometric morphometrics and multidimensional statistics. Almost the entire external frontal surface was significantly different between males and females, especially in form. Significant differences were also found between this related sample and an unrelated one. Sex estimation of the biologically related individuals was performed using the classification models developed on a sample of unrelated individuals from the recent Czech population (Cechová et al. in Int J Legal Med 133: 1285 1294, 2019), with a result of 74.46% and 63.83% in form and shape, respectively. Failure of this classifier was caused by the existence of typical traits found in the biologically related sample different from the usual manifestation of sexual dimorphism. This can be explained as due to the increased degree of similarity and the reduction of variability in biologically related individuals. The results show the importance of testing previously published methods on genealogical data.

Comparison of the effectiveness and efficiency of the grid and link search methods to recover scattered skeletal remains.

The grid and link search methods are used to recover scattered skeletal remains. Neither have not been compared robustly and clear guidelines for the link method have not been sufficiently developed. The study aimed to compare the effectiveness and efficiency of both methods and propose guidelines for the link method. The scattering patterns of two scavengers of forensic relevance-slender mongooses (Galerella sanguinea) and black-backed jackals (Canis mesomelas)-were recreated using four pig skeletons (Sus scrofa domesticus). Two groups (n = 6 each) were assigned a different method to recover the scattered remains. The length of the search and when each bone was located for each scatter pattern was recorded for each group and scatter pattern. A Likert scale questionnaire assessed participants' perceptions of their assigned method. A paired t-test (p = 0.005) compared the efficiency of each method and the questionnaire answers. Both methods were effective, recovering 100% of all remains. The link method was more efficient for both scatter patterns, despite there being no statistical significance (jackal: p = 0.089; mongoose: p = 0.464). Participants indicated favorable views for both methods; however, the link method scored significantly more favorably (p = 0.01) for efficiency. Specific guidelines were developed for the use of the link method. The link method is suggested for the recovery of scattered remains in forensic contexts, especially when the scavenger, its behavior, and scattering pattern is known or suspected.

Exploring cranial macromorphoscopic variation and classification accuracy in a South African sample.

To date South African forensic anthropologists are only able to successfully apply a metric approach to estimate population affinity when constructing a biological profile from skeletal remains. While a non-metric, or macromorphoscopic approach exists, limited research has been conducted to explore its use in a South African population. This study aimed to explore 17 cranial macromorphoscopic traits to develop improved methodology for the estimation of population affinity among black, white and coloured South Africans and for the method to be compliant with standards of best practice. The trait frequency distributions revealed substantial group variation and overlap, and not a single trait can be considered characteristic of any one population group. Kruskal-Wallis and Dunn's tests demonstrated significant population differences for 13 of the 17 traits. Random forest modelling was used to develop classification models to assess the reliability and accuracy of the traits in identifying population affinity. Overall, the model including all traits obtained a classification accuracy of 79% when assessing population affinity, which is comparable to current craniometric methods. The variable importance indicates that all the traits contributed some information to the model, with the inferior nasal margin, nasal bone contour, and nasal aperture shape ranked the most useful for classification. Thus, this study validates the use of macromorphoscopic traits in a South African sample, and the population-specific data from this study can potentially be incorporated into forensic casework and skeletal analyses in South Africa to improve population affinity estimates.

Spina bifida, the normal, the pathological and the in-between: first evidence from a forensic osteological collection.

Spina bifida (SB), a rare congenital disorder, is often mentioned as an individualizing factor in Forensic Anthropology. A lack of empirical data regarding SB is noticed in the scientific literature. Moreover, within the scope of anthropological research on SB disparities in terminology, classification systems, and methodological approaches result in incomparable results. The wide range (1,2%-50%) of "spina bifida occulta" reported prevalences is a good example. This research aims to analyze and debate the standard diagnostic criteria of SB on human skeletal remains, and attempts to elaborate on an universal system, premised on the distinction between SB as a pathology, and cleft neural arch (CNA) as an anatomical variant, according to Barnes (1994, p. 360 [1). A study-base of 209 individuals (88 males; 121 females; 44-99 years old) from the 21st Century Identified Skeletal Collection from the University of Coimbra (CEI/XXI) was macroscopically analyzed, focusing on the sacrum and remaining vertebrae. Four individuals presented complete posterior opening of the sacral canal (2,6%[4/156]). The observed bone changes, combined with the analysis of the entire skeleton, indicate that CNA, rather than SB linked to a neural tube defect, is the most reliable explanation for these cases. Overall, CNA was observed on 11 skeletons (7.05% of 156). The viability and applicability of the developed methodology for the identification of SB/CNA in forensic and/or osteological contexts are discussed, as well as the possibility of a lower prevalence of SB occulta, in the general population, than speculated before. HIGHLIGHTS: • Spina bifida has been studied so far under different methodologies, classification systems and nomenclature, leading to unstandardized and incomparable data. • Spina bifida as a pathological manifestation of a neural tube defect, as opposed to cleft neural arch as a simple form of skeletal variation. • Both spina bifida and complete sacral cleft fit the criteria of an individualizing trait in Forensic Anthropology.

Stereomicroscopic analysis of cut marks inflicted by a knife with blade damage.

Skeletal cut mark analysis provides relevant information on the general class characteristics of suspect knife. However, there is a lack of research on the influence of blade damage on cut mark analysis. This study aimed to thoroughly investigate the effects of damaged knife blades on cut marks' morphological and morphometrical characteristics. Fifteen undamaged, non-serrated knives were used to cut human ribs to make control cut marks. The knife blades were then damaged by a series of cuts on the bones. A comparison was made between the control cuts and 3 groups of cut marks inflicted by blades that had been damaged to different degrees. The results showed that the damaged blades created wider cut marks than undamaged one. Kerf morphology was likely to imitate the cuts made by the serrated blades such as an elliptical shape, a V-shaped cross-section and the presence of coarse striations. Wear-related features can affect cut mark analysis as the marks left behind by blades damaged to different degrees showed dissimilar dimensions and morphologies. The findings of this study can be applied to a forensic investigation when cut marks were caused by a knife with a damaged blade.

Awareness of forensic anthropology in Switzerland: a survey among forensic practitioners, police, and prosecutors.

Forensic anthropology (FA) as a specialized discipline has been practised in multi-lingual Switzerland for over a decade. A variety of expertise regarding osteological assessments as well as facial image comparison (FIC) is provided by different centres. Nevertheless, information is lacking about the awareness of FA and its benefits for forensic investigations among forensic stakeholders. Therefore, a survey was sent to Swiss anthropologists (AN) and related professions (police officers, prosecutors, and forensic pathologists) to assess three main aspects: (1) the experience of working (biological/forensic) anthropologists within FA; (2) how FA is perceived by other professions within the legal system; and (3) identify gaps (if any) in understanding of FA with the aim to suggest avenues for improvement if necessary. The results show that awareness of FA varies by occupation and cantonal regions. In areas where close collaborations between forensic anthropologists (FAs) and other stakeholders have been formally established, be it with focus on osteological analyses or FIC, the awareness of FA competencies was superior to areas where this was not the case. An overwhelming majority of forensic actors expressed interest in continuing education related to the role of FA. These findings indicate that facilitation of communication and collaboration leads to improvement in the awareness of the competencies of FAs and their contribution to forensic investigations.

Machine learning and deep learning enabled age estimation on medial clavicle CT images.

The medial clavicle epiphysis is a crucial indicator for bone age estimation (BAE) after hand maturation. This study aimed to develop machine learning (ML) and deep learning (DL) models for BAE based on medial clavicle CT images and evaluate the performance on normal and variant clavicles. This study retrospectively collected 1049 patients (mean± SD: 22.50±4.34 years) and split them into normal training and test sets, and variant training and test sets. An additional 53 variant clavicles were incorporated into the variant test set. The development stages of normal MCE were used to build a linear model and support vector machine (SVM) for BAE. The CT slices of MCE were automatically segmented and used to train DL models for automated BAE. Comparisons were performed by linear versus ML versus DL, and normal versus variant clavicles. Mean absolute error (MAE) and classification accuracy was the primary parameter of comparison. For BAE, the SVM had the best MAE of 1.73 years, followed by the commonly-used CNNs (1.77-1.93 years), the linear model (1.94 years), and the hybrid neural network CoAt Net (2.01 years). In DL models, SE Net 18 was the best-performing DL model with similar results to SVM in the normal test set and achieved an MAE of 2.08 years in the external variant test. For age classification, all the models exhibit superior performance in the classification of 18-, 20-, 21-, and 22-year thresholds with limited value in the 16-year threshold. Both ML and DL models produce desirable performance in BAE based on medial clavicle CT.

The Identified Skeletal Collection of the School of Legal Medicine: a contemporary osteological collection housed in Universidad Complutense de Madrid, Spain.

ABSTRA: Osteological collections are an important resource for the development of methods to assist in the study of skeletal remains in archeological and/or forensic contexts. The aim is to describe the current characteristics of the Identified Skeletal Collection of the School of Legal Medicine and its historical context. The Identified Skeletal Collection of the School of Legal Medicine of the Complutense University of Madrid consists of 138 male and 95 female individuals, born between 1880 and 1980 and deceased between 1970 and 2009. The minimum age of the sample is perinatal and the maximum age is 97 years. The collection is an essential tool for forensic research, given that its population characteristics can be extrapolated to those of present-day Spain. Access to this collection offers unique teaching opportunities as well as provides the information necessary to develop various lines of research.