From flesh to bones: Multi-omics approaches in forensic science.

Recent advancements in omics techniques have revolutionised the study of biological systems, enabling the generation of high-throughput biomolecular data. These innovations have found diverse applications, ranging from personalised medicine to forensic sciences. While the investigation of multiple aspects of cells, tissues or entire organisms through the integration of various omics approaches (such as genomics, epigenomics, metagenomics, transcriptomics, proteomics and metabolomics) has already been established in fields like biomedicine and cancer biology, its full potential in forensic sciences remains only partially explored. In this review, we have presented a comprehensive overview of state-of-the-art analytical platforms employed in omics research, with specific emphasis on their application in the forensic field for the identification of the cadaver and the cause of death. Moreover, we have conducted a critical analysis of the computational integration of omics approaches, and highlighted the latest advancements in employing multi-omics techniques for forensic investigations.

Why identification matters: an explorative study on six cases of family reunification.

The escalating phenomenon of migration, accompanied by a disturbing surge in associated tragedies, has persistently violated internationally protected human rights. Absence of physical evidence, namely the presence of adequately identified corpses, may impede the full enjoyment of human rights and-in some cases-the course of justice as it obstructs the initiation of legal proceedings against individuals implicated in causing such catastrophes. It also presents administrative obstacles, as death certificates are indispensable in legitimizing statuses like orphanhood and widowhood. Family reunification, particularly for orphans, plays a significant role for those attempting to reconnect with their relatives all over the world. Likewise, for mothers, the acknowledgment of their marital status or widowhood can be a pathway to regain their marginalized right to social life. To elucidate this issue, we analyzed six representative cases from the tragic October 3, 2013, shipwreck near the Italian island of Lampedusa, where 366 individuals were retrieved dead from the sea. These cases underscore the practical challenges involved, highlighting the compelling need for continued efforts to ensure that this burdensome problem transcends from being a mere ethical, moral, and legal discourse. Although considerable progresses, these cases also reveal that substantial work still lies ahead. There is a pressing need for improved mechanisms to certify kinship ties, which are often the limiting factor in many reunifications, and can hinder the granting of custody to children. The severity and far-reaching implications of this problem necessitate thoughtful attention and action, especially considering the ongoing escalation in migration and related fatalities.

The Role of Molecular Investigations in Estimating the Time since Deposition (TSD) of Bloodstains: A Systematic Review of the Literature.

At many crime scenes, investigators are able to trace and find traces of blood. For many years, it was believed that such traces could only be subjected to genetic investigations, such as those aimed at comparing DNA profiling with a suspect to verify his identity, and that it was therefore not possible to backdate the traces. In recent years, various works have used experimental models to investigate the possibility of identifying markers and methodologies for estimating the time since deposition (TSD) of bloodstains. Despite the results, these methods are still not part of standard procedures, and there is no univocal analysis methodology. In this work we carried out a systematic literature review of all the papers published in the last ten years on this topic, comparing the experimental models created. This review demonstrates the potential that different molecular approaches, such as transcriptomics, metabolomics, proteomics, and spectrometry, can have in the analysis of TSD, with notable sensitivity and specificity. This paper also analyzes the intrinsic and extrinsic limits of these models and emphasizes the need to continue research work on this topic, considering the importance that this parameter can assume in forensic investigations against a suspect.

Potential Role of mRNA in Estimating Postmortem Interval: A Systematic Review.

Although the postmortem interval estimation still represents one of the main goals of forensic medicine, there are still several limitations that weigh on the methods most used for its determination: for this reason, even today, precisely estimating the postmortem interval remains one of the most important challenges in the forensic pathology field. To try to overcome these limitations, in recent years, numerous studies have been conducted on the potential use of the mRNA degradation time for reaching a more precise post mortem interval (PMI) estimation. An evidence-based systematic review of the literature has been conducted to evaluate the state of the art of the knowledge focusing on the potential correlation between mRNA degradation and PMI estimation. The research has been performed using the electronic databases PubMed and Scopus. The analysis conducted made it possible to confirm the potential applicability of mRNA for reaching a more precise PMI estimation. The analysis of the results highlighted the usefulness of some mRNAs, such as ß-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, especially in short time frames, within a few hours or days of death. The matrices on which these analyses were conducted were also analyzed, resulting in less exposure to the external environment, including the heart, brain, and dental pulp. The major limitations were also reported, including the short time intervals analyzed in most of the articles, the lack of mathematical models, and the failure to report the error rate between the mRNA degradation time and PMI. Given the still small number of published articles, the lack of globally recognized standardized methods, and the numerous techniques used to evaluate the mRNA degradation times, numerous and larger studies are still necessary to reach more solid and shared evidence.

Sex estimation by volumetric evaluation of the maxillary canine using cone-beam computed tomographic images.

BACKGROUND: Teeth can be a reasonable part of sex estimation in case of being the single evidence available or in doubt although they are not as accurate as bones in sex estimation. The size of teeth is greater in males than in females. In this study, it was aimed to determine cut-off values of the tooth volume (TV) and root volume (RV) of the maxillary canines and to evaluate the validity of the method for discriminating between males and females. METHODS: Cone beam computed tomography (CBCT) images of 250 individuals aged 18 to 63 years with equal age and sex distribution were assessed retrospectively. The sample divided into reference set including 168 images of 84 females and 84 males and validation set including 82 images of 48 females and 34 males. Receiver operating characteristic (ROC) curve analysis, and Youden's index were used to determine cut-off values of the volumetric measurements and to test the discriminative performance of the method. RESULTS: The optimal cut-off values of TV ≥ 581 mm3 and RV ≥ 334 mm3 for estimating sex were established from the reference set. The sensitivity (Se), specificity (Sp), and accuracy (Ac) were 0.77 for TV ≥ 581 mm3 while they were 0.82, 0.77, and 0.79 for RV ≥ 334 mm3. When we tested the previously derived cut-off values of TV of the maxillary canine, the Se, Sp, and Ac were found to be respectively 0.71, 0.90, and 0.82 for TV ≥ 619 mm3 and 0.97, 0.27, and 0.56 for TV ≥ 510 / 460 mm3 (right/left). CONCLUSIONS: The identified cut-off values of TV and RV of the maxillary canines can be used to estimate sex with moderate accuracy when the only evidence available is teeth or in case of doubt. The method's applicability should be assessed for different populations.

Digital matching of palatal rugae patterns for forensic identification in edentulous denture wearers.

Identifying edentulous victims in forensic contexts poses a significant challenge. It has been reported that having a denture to reproduce and compare Palatal Rugae (PR) patterns is crucial for identifying edentulous individuals, yet there are no validated protocols for conducting this procedure. In this study, a new method was developed and validated for plaster molding of the internal surface of upper dentures, along with a protocol involving focus-stacked photographs and reference scales. Thirty-eight edentulous subjects participated in the study, obtaining plaster models of the patients (PM), and their dentures (AM) were standardized. The AM/PM model was highlighted and photographed with ten shots in different z-focuses for each model, using standard lighting and aligning the position of the PR perpendicular to the photographic axis using tubular spirit levels and modeling clay. The images were processed using the stacking technique and analyzed by three observers through the proposed protocol using Adobe® Photoshop®. The results were analyzed based on the intra-observer and inter-observer agreement levels, with a 95% confidence interval. This study demonstrated high-precision intra-observer and inter-observer agreement (k = 1) in the matching of Palatal Rugae (PR) and maxillary morphology obtained from participants and their dentures. The protocol is simple, cost-effective, and precise. It enables standardization of the technique for obtaining plaster models, and the exposure of PR and photographic protocol minimizes the presence of artifacts in the images, thereby reducing the likelihood of errors and promoting the reproducibility of both the recording technique and the comparison of the PR.

The rights of migrants to the identification of their dead: an attempt at an identification strategy from Italy.

Europe is turning a blind eye on a humanitarian disaster unfolding at its doorsteps, with thousands of migrants dying unidentified in Mediterranean waters. Since 2014, Italy has been struggling in an almost indifferent international scenario to identify its dead migrants. Despite the lack of sufficient resources, of the difficulties in collecting post mortem data from the disseminated bodies, and of the problems of contacting and collecting ante mortem information from relatives, it has been proven, with a series of pilot studies, that not only can these bodies be identified but that relatives are also looking for their loved ones and need death certificates. This article focuses on the administrative limbo and lack of regulations obliging single states to engage in appropriate procedures to maximise identification.

Applications of artificial intelligence in forensic sciences: Current potential benefits, limitations and perspectives.

In recent years, new studies based on artificial intelligence (AI) have been conducted in the forensic field, posing new challenges and demonstrating the advantages and disadvantages of using AI methodologies to solve forensic well-known problems. Specifically, AI technology has tried to overcome the human subjective bias limitations of the traditional approach of the forensic sciences, which include sex prediction and age estimation from morphometric measurements in forensic anthropology or evaluating the third molar stage of development in forensic odontology. Likewise, AI has been studied as an assisting tool in forensic pathology for a quick and easy identification of the taxonomy of diatoms. The present systematic review follows the PRISMA 2020 statements and aims to explore an emerging topic that has been poorly analyzed in the forensic literature. Benefits, limitations, and forensic implications concerning AI are therefore highlighted, by providing an extensive critical review of its current applications on forensic sciences as well as its future directions. Results are divided into 5 subsections which included forensic anthropology, forensic odontology, forensic pathology, forensic genetics, and other forensic branches. The discussion offers a useful instrument to investigate the potential benefits of AI in the forensic fields as well as to point out the existing open questions and issues concerning its application on real-life scenarios. Procedural notes and technical aspects are also provided to the readers.

Optimization of InnoXtract™ extraction and purification system for DNA extraction from skeletal samples.

The InnoXtract™ extraction and purification system is a purification method designed for DNA extraction from low-template samples, specifically rootless hair shafts. Its ability to successfully capture highly fragmented DNA suggests its suitability for use with other challenging sample types, including skeletal remains. However, the lysis and digestion parameters required modifications to successfully optimize the method for this sample type. A two-part digestion was developed utilizing a homebrew digestion buffer (0.5 M EDTA, 0.05% Tween 20, and 100 mM NaCl) and a supplemental lysis with the Hair Digestion Buffer included in the InnoXtract™ kit. Additionally, the magnetic bead volume was modified to improve DNA recovery from these challenging samples. With the altered protocol, the quality and quantity of DNA recovered from InnoXtract™ extracts were comparable to another commercial skeletal extraction method (PrepFiler™ BTA). This modified extraction method successfully purified sufficient amounts of quality DNA from a variety of skeletal samples to produce complete STR profiles. Successful STR typing from surface decomposition, burned, cremated, buried, and embalmed remains indicates the potential of this new method for challenging human identification and missing-person cases.

Third molar maturity index (I3M) assessment according to different geographical zones: a large multi-ethnic study sample.

Identification of living undocumented individuals highlights the need for accurate, precise, and reproducible age estimation methods, especially in those cases involving minors. However, when their country of origin is unknown, or it can be only roughly estimated, it is extremely difficult to apply assessment policies, procedures, and practices that are accurate and child-sensitive. The main aim of this research is to optimize the correct classification of adults and minors by establishing new cut-off values for four different continents (Africa, America, Asia, and Europe). For this purpose, a vast sample of 10,701 orthopantomographs (OPTs) from four continents was evaluated. For determination and subsequent validation of the new third molar maturity index (I3M) cut-off values by world regions, a cross-validation by holdout method was used and contingency tables (confusion matrices) were generated. The lower third molar maturity indexes, from both left and right side (I3ML and I3MR) and the combination of both sides (I3ML_I3MR) were calculated. The new cut-off values, that aim to differentiate between a minor and an adult, with more than 74.00% accuracy for all populations were as follows (I3ML; I3MR; I3ML_I3MR, respectively): Africa = (0.10; 0.10; 0.10), America = (0.10; 0.09; 0.09), Asia = (0.15; 0.17; 0.14), and Europe = (0.09; 0.09; 0.09). The higher sensitivity (Se) was detected for the I3ML for male African people (91%) and the higher specificity (Sp) of all the parameters (I3ML; I3MR; I3ML_I3MR) for Europeans both male and female (> 91%). The original cut-off value (0.08) is still useful, especially in discriminating individuals younger than 18 years old which is the goal of the forensic methods used for justice.

Exploring the Potential of Proteome Analysis as a Promising Tool for Evaluation of Sudden Cardiac Death (SCD) in Forensic Settings: A Literature Review.

Sudden cardiac death (SCD) represents a global emergency, with a high number of cases affecting all age groups every year. The prevention of these fatal events requires an accurate knowledge of etiology and pathogenesis, which can vary. Autopsy is an indispensable tool in cases of SCD for diagnostic purposes, as well as for judicial and preventive purposes for family members. Despite the completion of all routine post-mortem investigations, it is often complicated for the forensic pathologist to define the triggering cause of these events. The study of the proteome is proving to be extremely promising in the field of human cardiovascular disease. This paper aims to offer a literature review on the study of the proteome in post-mortem cadaveric biological samples obtained from SCD cases. The aim of this work is to outline the state of the art of the scientific advances that protein analysis can offer in the diagnosis of SCD and the limits that various studies have traced up to now. In conclusion, the work defines the future perspectives of this field in SCD, suggesting strategies to overcome the reported limits and improve the diagnostics of these events.

Chronology of permanent teeth mineralization in Brazilian individuals: age estimation tables.

BACKGROUND: Age is important in forming the uniqueness of individuals. When chronological age is not available, age estimation is required, particularly in court cases. The mineralization chronology of permanent teeth is a valuable tool for age estimation of subadults. This study aimed to evaluate the mineralization stages of permanent teeth of Brazilian subjects from imaging exams, using the classification by Moorrees et al. modified by the authors, to verify the existence of correlation between the chronology of mineralization stages and sex and to prepare numerical tables of the chronology of dental mineralization stages for Brazilian individuals. METHODS: Digital panoramic radiographs of 1100 living Brazilian individuals of both sexes, aged between 2 and 25 years, born between 1990 and 2018, from the image bank of a Dental Radiographs and Documentations clinic located in the city of Araraquara, SP, Brazil. The images were evaluated according to the level of crown and root development and classified according to the stages proposed by Moorrees et al. (Am J Phys Anthropol 21: 205-213, 1963) adapted by the authors. All analyses were performed in the R software. Descriptive and exploratory analyses were performed on all data. For intra- and inter-examiner analyses, the rate of agreement and Kappa statistics at a 95% confidence interval were used. Kappa was interpreted according to Landis and Koch. RESULTS: Only upper and lower canines showed significant differences between the sexes (p < 0.05), with higher average ages for men. The findings were presented in tables, as well as age estimates with 95% confidence intervals for each mineralization stage and each tooth. CONCLUSION: In the present study, we evaluated the mineralization stages of permanent teeth of Brazilian subjects from digital panoramic radiographs and found no correlation between the chronology of mineralization stages and sex, except for canines. From the obtained results, numerical tables of the chronology of dental mineralization stages were prepared.

Automated wound segmentation and classification of seven common injuries in forensic medicine.

In forensic medical investigations, physical injuries are documented with photographs accompanied by written reports. Automatic segmentation and classification of wounds on these photographs could provide forensic pathologists with a tool to improve the assessment of injuries and accelerate the reporting process. In this pilot study, we trained and compared several preexisting deep learning architectures for image segmentation and wound classification on forensically relevant photographs in our database. The best scores were a mean pixel accuracy of 69.4% and a mean intersection over union (IoU) of 48.6% when evaluating the trained models on our test set. The models had difficulty distinguishing the background from wounded areas. As an example, image pixels showing subcutaneous hematomas or skin abrasions were assigned to the background class in 31% of cases. Stab wounds, on the other hand, were reliably classified with a pixel accuracy of 93%. These results can be partially attributed to undefined wound boundaries for some types of injuries, such as subcutaneous hematoma. However, despite the large class imbalance, we demonstrate that the best trained models could reliably distinguish among seven of the most common wounds encountered in forensic medical investigations.

Applications of Raman spectroscopy in the analysis of biological evidence.

During the past few decades, Raman spectroscopy has progressed and captivated added attention in the field of science. However, the application of Raman spectroscopy is not limited to the field of forensic science and analytical chemistry; it is one of the emerging spectroscopic techniques, utilized in the field of forensic science which in turn could be a supporting tool in the law and justice system. The advantage of Raman spectroscopy over the other conventional techniques is that it is rapid, reliable, and non-destructive in nature with minimal or no sample preparation. The quantitative and qualitative analysis of evidence from biological and non-biological origins could easily be performed by using Raman spectroscopy. The forensic domain is highly complex with multidisciplinary branches, and therefore a plethora of techniques are utilized for the detection, identification, and differentiation of innumerable pieces of evidence for the purpose of law and justice. Herein, a systematic review is carried out on the application of Raman spectroscopy in the realm of forensic biology and serology considering its usefulness in practical perspectives. This review paper highlights the significance of modern techniques, including micro-Raman spectroscopy, confocal Raman spectroscopy, surface-enhanced Raman spectroscopy, and paper-based surface-enhanced Raman spectroscopy, in the field of Raman spectroscopy. These techniques have demonstrated notable advancements in terms of their applications and capabilities. Furthermore, to comprehensively capture the progress in the development of Raman spectroscopy, all the published papers which could be retrieved from the available databases from the year 2007 to 2022 were incorporated.

Maxillary sinus in gender determination: a morphometric analysis using cone beam computed tomography.

Gender determination of skeletal remains is an important forensic procedure in the identification process. Maxillary sinus remains intact even when the skull and other bones may be badly disfigured. Cone beam computed tomography (CBCT) can provide precise information about complex anatomical structures, as it is characterized by rapid volumetric image acquisition with high resolution. The present study aimed to assess the accuracy of maxillary sinus measurements using cone beam computed tomography in gender determination and to develop a formula using discriminate function analysis. Bilateral maxillary sinus images (left and right) were acquired for 200 patients (100 females and 100 males) and different parameters (width, length, height, area, perimeter, and volume) were measured and evaluated. Mean and standard deviation of both maxillary sinuses measurements were calculated and compared. The data was subjected to discriminative statistical analysis and analyzed using an unpaired t-test. The difference between all these variables was statistically significant between males and females. Based on discriminant analysis, the most pronounced variable in the differentiation of gender groups was maxillary sinus height. Gender assessment was established correctly with an accuracy of 75% for females and 64% for males with an overall accuracy of 69.5%. Adding other independent variables to the model did not result in an improvement in overall accuracy. Cone beam computed tomography measurement of maxillary sinus can be used as an aid in forensic anthropology for gender determination.

Classification of rib fracture types from postmortem computed tomography images using deep learning.

Human or time resources can sometimes fall short in medical image diagnostics, and analyzing images in full detail can be a challenging task. With recent advances in artificial intelligence, an increasing number of systems have been developed to assist clinicians in their work. In this study, the objective was to train a model that can distinguish between various fracture types on different levels of hierarchical taxonomy and detect them on 2D-image representations of volumetric postmortem computed tomography (PMCT) data. We used a deep learning model based on the ResNet50 architecture that was pretrained on ImageNet data, and we used transfer learning to fine-tune it to our specific task. We trained our model to distinguish between "displaced," "nondisplaced," "ad latus," "ad longitudinem cum contractione," and "ad longitudinem cum distractione" fractures. Radiographs with no fractures were correctly predicted in 95-99% of cases. Nondisplaced fractures were correctly predicted in 80-86% of cases. Displaced fractures of the "ad latus" type were correctly predicted in 17-18% of cases. The other two displaced types of fractures, "ad longitudinem cum contractione" and "ad longitudinem cum distractione," were correctly predicted in 70-75% and 64-75% of cases, respectively. The model achieved the best performance when the level of hierarchical taxonomy was high, while it had more difficulties when the level of hierarchical taxonomy was lower. Overall, deep learning techniques constitute a reliable solution for forensic pathologists and medical practitioners seeking to reduce workload.

The role of legal medicine professionals in preventing pregnancy and sexually transmitted infections among female victims of sexual assault.

Sexual violence can have an overwhelming impact on the victim's physical and mental health; the consequences include unintended pregnancy and sexually transmitted infections (STIs). Therefore, the examiners must assess victims for possible pregnancy and sexually transmitted infections as a part of the sexual assault examination. This article aims to orient the medico-legal examiners towards their role in preventing unintended pregnancy and sexually transmitted infections among victims of sexual assault. Prompt detection of pregnancy or STIs is critical, as any delay would adversely affect the successful administration of emergency contraception and post-exposure prophylaxis (PEP) against human immunodeficiency virus (HIV) and other sexually transmitted infections.

The role of PMCT for the assessment of the cause of death in natural disaster (landslide and flood): a Sicilian experience.

In this report, the authors provide a contribution of PMCT in assessing the cause of death due to natural disasters. Here, the PMCT findings of 43 subjects who died during both landslide and flood were described. The post-mortem imaging revealed, clearly, traumatic injuries and/or the presence of foreign material in airways allowing to assess the cause of death of each subject, together with external inspection and the collected circumstantial data. Particularly, the PMCT has been helpful for characterization and localization of the clogging substance in airways providing findings on bronchial branches involvement. Moreover, the investigation offered detailed data on skeletal injuries in all anatomic districts and put in evidence both the precise fracturing site and the characteristics of fracture stubs for each bone fracture. This report supports the recommendation of the virtual autopsy in a case with several victims, as in natural disasters, and its role as an alternative diagnostic investigation when the standard autopsy is not feasible.

Quantifying the potential of morphological parameters for human dental identification: part 1-proof of concept.

In forensic identification, lack of eccentric characteristics of intact dentitions hinders correct ante-mortem/post-mortem (AM/PM) matching. It remains unclear which morphological dental parameters hold strong potential as identifiers. This study aimed to establish a method to quantify and rank the identifying potential of one (or a combination of) continuous morphological parameter(s), and to provide a proof of concept. First, a statistic was defined that quantifies the identifying potential: the mean potential set (MPS). The MPS is derived from inter-observer agreement data and it indicates the percentage of subjects in the AM reference dataset who at least need to be considered to detect the correct PM subject. This was calculated in a univariate and a multivariate setting. Second, the method was validated on maxillary first molar crowns of 82 3D-digitally scanned cast models. Standardized measurements were registered using 3D modeling software (3-Matic Medical 12.0, Materialise N.V., Leuven, Belgium): tooth depth, angles between cusps, distances between cusps, distances between the cusps, and the mesial pit. A random sample of 40 first molars was measured by a second examiner. Quantifying and ranking the parameters allowed selecting those with the strongest identifying potential. This was found for the tooth depth (1 measurement, MPS = 17.1%, ICC = 0.879) in the univariate setting, and the angles between cusps (4 measurements, MPS = 3.9%) in the multivariate setting. As expected, the multivariate approach held significantly stronger identifying potential, but more measurements were needed (i.e., more time-consuming). Our method allows quantifying and ranking the potential of dental morphological parameters as identifiers using a clear-cut statistic.

European Council of Legal Medicine (ECLM) on-site inspection forms for forensic pathology, anthropology, odontology, genetics, entomology and toxicology for forensic and medico-legal scene and corpse investigation: the Parma form.

Further to a previous publication by the European Council of Legal Medicine (ECLM) concerning on-site forensic and medico-legal scene and corpse investigation, this publication provides guidance for forensic medical specialists, pathologists and, where present, coroners' activity at a scene of death inspection and to harmonize the procedures for a correct search, detection, collection, sampling and storage of all elements which may be useful as evidence, and ensure documentation of all these steps. This ECLM's inspection form provides a checklist to be used on-site for the investigation of a corpse present at a crime or suspicious death scene. It permits the collection of all relevant data not only for the pathologist, but also for forensic anthropologists, odontologists, geneticists, entomologists and toxicologists, thus supporting a collaborative work approach. Detailed instructions for the completion of forms are provided.