Forensic medicine represents a critical socio-health observatory for socially marginalized populations. This study provides the first systematic retrospective forensic analysis of mortality among people experiencing homelessness in Italy. We examined 104 decedents undergoing autopsy in Milan between 2014 and 2024. The annual number of autopsied homeless decedents increased over the study period, peaking in 2023-2024. The majority of deaths occurred in indoor public spaces (45/104, 43.3%), with the remainder in hospitals or green areas, reflecting extreme social marginalization and environmental exposure. Anamnestic data were available for 56/104 cases (53.8%), of whom 42 (75%) had chronic medical conditions, while psychiatric or psychosocial histories were known for 43/104 cases (41.3%). These figures likely underestimate the true burden due to fragmented healthcare access and incomplete documentation. Causes of death were classified as natural (25/104, 24.0%), non-natural (17/104, 16.4%), or undetermined (62/104, 59.6%). Natural deaths, predominantly in middle-aged adults, illustrate accelerated health deterioration associated with homelessness, while non-natural deaths reflect heightened exposure to hazardous urban environments. Undetermined deaths - mainly acute circulatory insufficiency without macroscopic findings - highlight substantial gaps in post-mortem evaluation. Toxicological and histological analyses were rarely performed, revealing procedural and structural constraints that impede accurate cause-of-death determination in socially vulnerable and clinically complex populations. These findings uncover preventable and identifiable patterns of mortality, emphasize the forensic and public health value of comprehensive post-mortem investigation, and call for integrated strategies combining proactive healthcare delivery, harm reduction, and standardized forensic protocols to improve diagnostic accuracy and strengthen mortality surveillance in homeless populations.

Salt mummification is an exceptionally rare phenomenon, resulting from the hygroscopic and antimicrobial properties of saline environments that inhibit microbial decomposition and promote tissue desiccation. While archaeological examples are well known, no modern forensic case has been described to date. We present the first forensic case of natural salt mummification observed in southern France, where a male body was discovered encrusted in a ∼4 cm-thick crystalline salt layer. External examination revealed a completely desiccated, brown, leathery body with preserved facial and appendicular morphology, allowing biological profiling. Autopsy showed extensive tissue desiccation compatible with preservation in a highly saline environment, with heterogeneous preservation patterns combining mummification and focal putrefactive changes, including complete cerebral liquefaction.Toxicological analyses of visceral samples were negative for drugs and toxic substances. DNA and fingerprint analyses were performed but did not result in identification. Based on environmental reconstruction of salinity dynamics, evaporation rates, and local climate conditions, provided contextual information supporting a tentative post-mortem interval hypothesis.This case is unique due to its occurrence in a warm, humid, anthropogenic saline environment and not in a salt mine context. This resulted in a hybrid pattern of desiccation and putrefaction not previously described in modern forensic literature. The case highlights the relevance of environmental analysis and multidisciplinary investigation for identification and post-mortem interval estimation in "complex" bodies.

Blood-bearing footwear impressions represent an important source of information in Bloodstain Pattern Analysis (BPA), yet the dynamics of blood depletion during consecutive walking sequences remain poorly characterized. This proof-of-concept pilot study compared two blood depletion models generated by contact between a patterned shoe sole and a passive blood pool on a non-porous surface: a continuous walking sequence and an interrupted partial-drying sequence incorporating a 2-3-minute period of partial drying after the first step. Both models consisted of an initial passive pooling phase (Step 0) followed by ten consecutive transfer events. Each experimental condition was independently performed twice (n = 2 complete sequences per condition; four complete sequences overall). High-resolution calibrated digital images were acquired for each step and analyzed using custom Python/OpenCV-based morphometric algorithms. Parameters evaluated included blood transfer area, spatial distribution of residual microdeposits, and progressive degradation of the tread pattern. Residual blood reactivity was additionally assessed using the Combur Test on fresh and dried traces before and after rehydration with physiological saline solution. In the continuous sequence, transfer area decreased markedly during the first transfer events, declining from approximately 160 cm[2] at Step 0 to 60-70 cm[2] at Step 1, 20-30 cm[2] at Step 2, and 10-20 cm[2] at Step 3. Despite this substantial quantitative reduction, the overall footwear geometry remained recognizable for a relatively large number of consecutive transfer events. In contrast, the interrupted partial-drying sequence exhibited earlier morphological degradation. Following partial drying, residual blood deposits became more localized and less spatially dispersed; however, footprint interpretability declined sharply around Step 4, which was treated as an experiment-specific transition and not as a universal threshold. Beyond this stage, the tread pattern progressively lost interpretability despite the continued presence of detectable blood residues. Combur Test results were generally negative on completely dried traces after approximately 24 h. However, rehydration with physiological saline restored test positivity even during advanced stages of depletion and on the shoe outsole itself, indicating persistence of presumptive heme reactivity even after substantial loss of footprint interpretability. Overall, the findings suggest that depletion of blood-bearing footwear impressions is a multifactorial morphodynamic and biochemical process whose observed expression differed between the two experimental conditions. Persistence of biologically detectable blood does not necessarily coincide with persistence of the footprint as a morphologically interpretable entity. In particular, the interrupted partial-drying condition showed an apparent dissociation between biochemical persistence and morphological persistence, with marked loss of footwear recognizability beyond Step 4 despite the continued presence of detectable blood residues. These findings suggest that apparently degraded or non-identifiable footwear impressions may retain forensically relevant biological evidence, highlighting the importance of integrating morphological and biochemical approaches in the interpretation of blood-bearing footwear traces.Trial registration: not applicable.

Estimation of the postmortem submersion interval (PMSI) remains challenging in bodies recovered from aquatic environments, particularly after prolonged submersion and in the presence of adipocere. We report the cases of two divers who disappeared during the same accident in the Mediterranean Sea in December 1993 and were recovered 30 years later at depths of 103 and 113 m, 13 m apart. Both bodies underwent whole-body postmortem computed tomography, external examination, autopsy, toxicological analyses, and genetic identification. Both showed advanced skeletonization predominantly affecting the upper body, associated with persistence of adipocere-like tissues in the pelvic regions and lower limbs. However, the quality of soft-tissue preservation differed markedly: in X1, residual tissues were largely disorganized and consisted of yellowish-to-brown aggregates, whereas X2 showed more extensive preservation of the skin, hair, and discernible muscle masses in the lower limbs. X1 wore a two-piece semi-dry wetsuit, whereas X2 wore a full dry suit; these differences in preservation are discussed in relation to the characteristics of the diving equipment and local microenvironmental conditions. Pelvic tissues compatible with adipocere showed attenuation values ranging from - 100 to - 40 Hounsfield units. Genetic identification was achieved from muscle in X1 and from femoral bone in X2; toxicological analyses were negative. These observations illustrate regional and interindividual variability in preservation after the same 30-year marine PMSI and highlight the limitations of estimating PMSI solely on the basis of the macroscopic appearance of the bodies.

Single, fragmented, and pulp-free teeth are challenging specimens for forensic age estimation, particularly when conventional morphological indicators are unavailable or compromised. DNA methylation provides a molecular approach for age prediction and may be useful for degraded dental samples. In this study, we established and evaluated methylation-based age-prediction models using sectioned pulp-free teeth as simulated forensic specimens. In this study, we prioritized 10 previously reported age-associated CpG loci that have been validated in skeletal tissues, dental tissues, and ultimately constructed methylation-based age-prediction models using nine CpG sites significantly correlated with chronological age in incised and pulp-free teeth (n = 128). Before model construction, the effects of tooth types and sex were assessed. Neither tooth types nor sex significantly modified the methylation-age relationship according to ANOVA-based interaction analysis, with P values of 0.340 and 0.468, respectively. Adding methylation-by-tooth types or methylation-by-sex interaction terms also did not significantly reduce prediction error, with P values of 0.210 and 0.156, respectively. Age estimation models were developed using partial least squares regression, ridge regression, elastic net regression, and support vector regression with radial basis function kerne (SVR-RBF), with hyperparameters optimized via ten-fold cross-validation to prevent overfitting and ensure stability and generalizability. Ridge regression provided the best balance between fitting and generalization, with test-set R[2] = 0.749 and MAE = 6.112 years. Paired analysis of pulp-retained and pulp-free teeth showed no significant difference in overall prediction error, but cg22507023 differed significantly between the two groups. Supplementary validation on paired teeth of distinct positions from 12 randomly selected individuals confirmed no intra-individual differences in CpG methylation level and predicted age. Overall, this study demonstrates that DNA methylation analysis can provide useful molecular evidence for forensic age estimation in pulp-free teeth and supports unified modeling without stratification by tooth types or sex.

Forensic age estimation in living individuals is essential in legal and migratory contexts, particularly when assessing whether an individual has reached the age of majority. The method proposed by Roberts et al. evaluates the relative distal root canal width (RCW) of mandibular molars, a parameter measurable only after complete apical closure. This method has not yet been validated in a Spanish population, making its regional evaluation essential due to known population-specific variations in dental maturation. A retrospective cross-sectional study was conducted on 576 digital orthopantomographs of individuals aged 16-24 years presenting with complete apical closure of the mandibular molars LL6, LL7, and LL8. Distal root canal patterns were classified into categories A, B, C, and U. Intra- and inter-observer agreements, association with legal adulthood, diagnostic accuracy, and receiver operating characteristic curves were evaluated. Reproducibility was high (kappa > 0.80). Category RCW-C was the only pattern significantly associated with legal adulthood (odds ratio = 2.58; p = 0.006), showing high specificity (84.6%) and positive predictive value (94.2%), but low sensitivity (31.9%). Area under the curve values ranged from 0.665 in males to 0.616 in females, indicating modest discriminative performance. In conclusion, RCW-C is a specific and reproducible indicator associated with legal adulthood in individuals with fully developed mandibular molars. However, its limited sensitivity underscores that the method should be used only as a complementary tool within a multimodal forensic age estimation protocol.

The choice between short-read and long-read sequencing represents a critical trade-off in forensic mitochondrial DNA (mtDNA) analysis. In this study, Illumina short-read sequencing (SRS) and QitanTech Nanopore long-read sequencing (LRS) platforms were systematically compared using data from 177 individuals across 39 maternal pedigrees. The results demonstrated that LRS, which utilized a single-amplicon enrichment strategy, delivered superior coverage uniformity (median 100%), effectively reducing the coverage variability typically associated with SRS multiplex PCR. Moreover, LRS improved the discrimination of interference from nuclear mitochondrial DNA segments (NUMTs) by spanning homologous regions with longer reads. In contrast, the SRS data showed pronounced off-target alignment, which was notably enriched on chromosomes 17 and 2 because of spurious sequence homology. However, although both platforms produced concordant consensus haplotypes, SRS exhibited higher accuracy for low-level heteroplasmy detection. Pedigree-based validation revealed that the current LRS workflow produced background-noise signals that were misclassified as heteroplasmy at a 10% allele frequency threshold. Consequently, we propose a complementary workflow: employing LRS for structural characterization and NUMT exclusion while relying on SRS for high-fidelity quantification of heteroplasmy. This integrated strategy offers a robust solution for forensic casework involving complex or low-input samples.

Animal scavenging is frequently encountered in forensic investigations. The identification of scavenging is crucial, as scavengers have the potential to disrupt forensic contexts significantly by disarticulating body elements and consuming portions of bone. Determining the anatomical areas more commonly affected by scavengers can assist in recognizing the presence of scavenging activity in forensic contexts. Past forensic cases, submitted to the Forensic Anthropology Unit of the National and Kapodistrian University of Athens Medical School for analysis between 1999 and 2023, were analyzed. Of the 256 cases examined, 62 exhibited signs of carnivore scavenging, while 194 presented no such evidence. To evaluate the impact of scavenging on human decomposition, the data collected from these cases were analyzed and compared. This comparison aimed to identify and assess any statistically significant differences in preservation and environmental conditions between scavenged and non-scavenged remains. The results indicate that scavenging is more likely to occur in rural settings and areas characterized by low population density. A lower prevalence of upper limb bones was reported in cases exhibiting signs of scavenging. Furthermore, this study identified specific anatomical regions that are preferentially affected by scavenger activity. Given the relative scarcity of similar research in Europe, the present study provides valuable insights into scavenging patterns in Greece and the broader European context.

The October 7, 2023 terrorist attack in Israel resulted in over 1,200 casualties, creating unprecedented disaster victim identification (DVI) challenges. Traditional ante-mortem (AM) reference methods using personal items proved insufficient as entire families were affected. We implemented a novel strategy utilizing archived medical specimens from Ministry of Health repositories to construct a self-direct-reference antemortem (AM) DNA database. This study systematically evaluates factors affecting STR profiling success from 151 archived clinical specimens (2003-2023): histological samples (FFPE blocks/cytology slides, n = 131), diagnostic swabs (n = 8), and blood/serum (n = 12). Four variables were assessed: tissue source, extraction method, incubation protocol, and sample age. DNA was amplified using Investigator 24plex Kit, success measured by informative autosomal STR loci obtained (maximum 21). Descriptive and non-parametric inferential statistical analyses were applied to compare profiling outcomes across variable conditions, confirming statistically significant differences between sample types and methods. This study demonstrates that archived medical specimens represent reliable, viable and valuable sources of AM references with success rates ranging from 67 to 100% of mean locus recovery (proportion), depending on tissue type and processing optimization. The findings emphasize the potential of these specimens especially when traditional materials are unavailable and providing actionable guidance for global DVI preparedness.

Moderate-density single nucleotide polymorphisms (SNPs) (nearly 2000 SNPs) have shown great forensic application value in Chinese Han. However, the forensic efficacy of these SNPs still requires validation in more populations. In this study, we evaluated the performance of these moderate-density SNPs combined with machine learning (ML) method for identifying first- to fourth-degree kinships in Chinese Yunnan Zhuang (CYZ) group. Moreover, we integrated genetic data from 135 populations spanning nine geographic regions obtained from public datasets to conduct comprehensive population genetic analyses. The present results showed that these SNPs were effective in identifying first- to third-degree kinships from unrelated individuals. The density curves of Log10 likelihood ratio (Log10LR) and cumulative identity by state (CIBS) for first- to second-degree kinships and unrelated individuals were completely separated. When the Log10LR thresholds were set at -4 and 4 to distinguish the third-degree kinship from unrelated individuals, the system power was 86.40% with error rate of 0. The K-Nearest Neighbor model using Log10LR and CIBS feature sets performed best in classifying first- to fourth-degree kinships, with F1 score of 0.9781. Population genetic analyses revealed that CYZ group exhibited the closest genetic affinity with geographically adjacent populations, particularly the southern Chinese minorities (Dai, Miao, and Tujia groups) and the Kinh from Vietnam in Southeast Asia. Principal component, phylogenetic tree and ADMIXTURE analyses revealed significant differences in the genetic structures among East Asian subpopulations, especially the minorities from southern and northern China, suggesting that these SNPs had potential to distinguish these subpopulations. Multinomial LASSO analysis was used to screen 688 SNPs to discriminate five East Asian subpopulations. Using these selected SNPs, a partial least squares-discriminant analysis model based on hierarchical classification approach and an Elastic Net model based on flat multi-classification approach both achieved overall accuracies above 0.9 in classifying five East Asian subpopulations. This study demonstrated that the combination of moderate-density SNPs with ML method could provide an effective solution for kinship identification and fine-scale discrimination of East Asian subpopulations.

Next-generation sequencing (NGS) has facilitated the widespread use of DNA metabarcoding in fields such as biodiversity, population research, and food adulteration. To improve the identification of animal-derived DNA in samples containing degraded and mixed DNA, a DNA metabarcoding system targeting the mitochondrial Cytb, COI, 16 S rRNA, and 12 S rRNA genes using targeted NGS (tNGS) was developed for vertebrate species. In line with the International Society for Forensic Genetics (ISFG) recommendations for non-human DNA analysis, the forensic performance of the novel assay was systematically evaluated, including assessments of sensitivity, mixtures, inhibitors, degraded DNA, processed meat products, and case sample studies. Sensitivity studies showed that 10 pg of genomic DNA per reaction from a single species was sufficient to detect at least one marker for all tested species, whereas 100 pg enabled reliable species identification using all four markers. For mixed samples with a total DNA input of 1 ng, all ten species were accurately identified in an equimolar mixture. Minor components were also detected in binary mixtures at a 99:1 ratio (1% minor contributors). Notably, the assay recovered fragmented DNA from experimentally degraded samples and highly processed food matrices. Analysis of casework samples further demonstrated that the tNGS-based multiplex assay enables broad-spectrum vertebrate identification and holds great practical value for curbing wildlife trafficking, mitigating food fraud, and facilitating biodiversity monitoring.

Historical and post-conflict mass grave programmes increasingly recover genetic profiles from skeletal remains, yet final identification may remain limited when family reference collection, post-mortem analysis, matching and re-analysis are organised as separate projects. This evidence-informed framework development study presents the Kinship-Centred Continuous Identification Framework for settings in which the possible victim set is open or uncertain. Framework development is defined as the integration of established evidence and operational standards into a time-ordered system with explicit decision points, feedback pathways and governance requirements; individual components are not claimed as novel. A purposive evidence map covering database inception to 9 May 2026 identified recurrent operational bottlenecks across legal medicine, forensic genetics, humanitarian forensic action, archaeology, anthropology, disaster victim identification and transitional justice. The synthesis produced seven design principles and ten implementation components. The framework begins before excavation with mandate clarification, a provisional candidate-victim list, genealogical triage and prioritised family reference collection. It incorporates commingling-aware sampling, profile-informativity classification, question-led marker selection, direct and programme-wide matching, trained human interpretation and multidisciplinary reconciliation. Paterna Cemetery (Valencia, Spain), containing mass graves associated with executions during the Francoist repression, served as the case-generating example; published programme-level evidence illustrates why grave assignment, candidate lists and genealogies must remain revisable. The framework also requires periodic re-examination of direct-reference options and unresolved profiles. Its central premise is that skeletal degradation and progressive loss of highly informative relatives are coupled constraints. Legal medicine services should therefore move from episodic exhumation support toward accountable, consent-based and continuously updated identification infrastructure.

In societies following the paternal transmission of surnames, their distribution should keep the same trail as that of Y-chromosome lineages. We evaluated this hypothesis by establishing the Y-STR haplotypes of two hundred and thirty-two individuals from the Argentine Northwest, distributed among one hundred and forty-eight surnames. We compared these two sources of information, searching for matches within and through surnames, establishing Median-Joining networks and estimating their time of divergence. We defined two nodal haplotypes (Native American and European), surrounded by the rest. We found cases in which surnames matched Y-STR haplotypes and also recognized identical genetic lineages involving different surnames. These findings suggest that the irregular transmission of surnames in the Argentine Northwest has been higher than that found in other countries. Likewise, similarities between both kinds of lineages should not be taken for granted.