Chemical profiling of illicit drugs has traditionally relied on laboratory-based techniques such as gas chromatography-mass spectrometry (GC-MS). Recent developments in portable drug testing devices have facilitated the decentralization of forensic capabilities, enabling field users to obtain rapid results. In this study, portable near-infrared (NIR) spectroscopy was evaluated for the chemical profiling of ecstasy tablets. A total of 668 tablets from 393 seizures made by Swiss law enforcement agencies were analyzed. Intra- and inter-seizure spectral variability was assessed using Euclidean distance, and a threshold value was defined to identify pairs of samples exhibiting sufficiently similar NIR profiles to be considered potentially linked. The results showed that NIR spectroscopy successfully distinguished intra- and inter-variability, with only 1.14% of inter-seizure comparisons falling below the selected threshold. Most NIR-based groupings were consistent with GC-MS chemical profiles, supporting the technique's ability to highlight spectral similarities and generate hypotheses of potential links between seizures for forensic intelligence purposes. As a further application, this study evaluates the use of NIR-based chemical profiling for early response in a drug checking context. Through a case study of eleven tablets that the drug checking service's NIR classification model failed to identify, we demonstrate the ability to link similar tablets of concern without prior knowledge of their composition. Comparing the spectra of newly analyzed specimens with previously flagged hazardous samples could enable the timely dissemination of alerts to consumers and public health authorities. The use of such rapid, non-destructive analytical methods could support more efficient decision making and contribute to harm-reduction efforts. Overall, this study supports portable NIR spectroscopy as a rapid screening approach for highlighting potential links at the pre-tableting formulation level in forensic intelligence and early-response applications.

Muscimol (MUS), a GABA-A receptor agonist naturally occurring in Amanita species, is becoming available in commercial edible products marketed for recreational use. This study described the development and validation of an new SPE- HILIC-LC-MS/MS method for the determination of MUS in post-mortem blood and its application to a polydrug user consuming also MUS candies. The method was validated over a calibration range of 0.125-10 µg/mL, with a limit of detection of 0.06 µg/mL and a limit of quantification of 0.125 µg/mL; accuracy and precision were within acceptable limits, fitting the purpose of the study. In the reported case, MUS was quantified in central blood at 0.18 µg/mL. Elevated concentrations in urine (55 µg/mL) and gastric content (24 µg/mL) were consistent with recent oral intake occurring within the perimortem timeframe. The toxicological interpretation of MUS findings remained challenging due to the absence of published reference data in literature and the concomitant presence of multiple psychoactive substances in this case. This study provided new analytical and case-related data contributing to the limited forensic literature on MUS recreational consumption.

Cocaine stands out due to its significant social problems stemming from trafficking, leading to increased violence and detrimental effects on the physical and mental health of its users. Brazil serves as a transit route for cocaine produced in Colombia, Peru, and Bolivia to Europe, with Paraná being a strategic region due to its border with Paraguay and the second-largest port in Brazil. Cocaine is gradually adulterated with cutting agents along the distribution chain, altering its chemical profile. Paradoxically, these components can create a unique chemical signature, enabling the tracing of the drug's origin and distribution routes. This study analysed 298 mid-infrared spectra of cocaine samples seized in Paraná and applied Multivariate Curve Resolution with Alternating Least Squares (MCR-ALS) to identify and characterise the main adulterants and diluents present. MCR-ALS was chosen due to its ability to decompose overlapping spectra into individual component contributions, making it ideal for analysing complex mixtures. Five distinct chemical profiles were identified, and based on their spectral interpretation and the most reported adulterants and diluents in cocaine samples, the five components were defined as: cornstarch, cocaine, caffeine, sodium bicarbonate, and creatine. The concentration profiles of the components were clustered using Hierarchical Cluster Analysis, and seven profiles of cocaine samples were identified based on the amount of cocaine and the presence of adulterants and diluents. The MCR-ALS method proved effective in identifying cocaine cutting agents via ATR-FTIR, offering a fast and reliable approach for forensic analysis that can help to identify commercial routes.

Bisulfite conversion, the conventional method for DNA methylation analysis, converts unmethylated cytosine to uracil while leaving methylated cytosine unchanged. A recently developed enzymatic conversion method employing mild reaction conditions produces DNA sequences identical to those generated by bisulfite treatment. In this method, methylated cytosine is oxidized to 5-carboxycytosine (5caC) by ten-eleven translocation dioxygenase 2 (TET2). Subsequently, cytosine is deaminated by the apolipoprotein B mRNA editing enzyme catalytic polypeptide-like (APOBEC), without affecting 5caC. These gentle reaction conditions show promise for identifying challenging forensic samples. Herein, we applied the enzymatic conversion method for the identification of body fluids. We developed a multiplex DNA methylation-based detection system and conducted a systematic comparison between enzymatic and bisulfite conversion methods. Parameters assessed included conversion efficiency, recovery, methylation detection at target CpG sites, and performance with degraded and mock casework samples. In addition, classification models were employed to enhance the interpretation of methylation data. Our findings demonstrated that the enzymatic conversion method exhibited lower conversion efficiency and recovery rate than bisulfite treatment. However, only marginal differences were observed in the accuracy of body fluid identification between the two methods. Enzymatic conversion preserved a greater number of CpG sites than bisulfite treatment in degraded samples. Moreover, when the DNA input was 10 ng, a decline in result stability was observed in bisulfite-treated samples. These results suggest that the enzymatic conversion method holds potential for challenging forensic sample identification.

The postmortem diagnosis of death from mechanical asphyxia (DMA) remains challenging owing to the absence of specific morphological signs. The present study evaluated the potential of transcription factor EB (TFEB), a master regulator of the autophagy-lysosomal pathway (ALP), as a biomarker for MA. Through transcriptomic analysis, molecular docking, molecular dynamics (MD) simulations, and an SH-SY5Y cell hypoxia model, we observed that TFEB expression was upregulated and nuclear translocation was induced during the initial phase of hypoxia. Following prolonged hypoxia (24 h), however, autophagy flux arrest and lysosomal dysfunction were observed. Mechanistically, we found that tomatidine (TA) could ameliorate these dysfunctions by activating the AMPK-TFEB signalling axis. In human brain tissue samples collected during the early postmortem interval (PMI), the MA group exhibited significantly increased TFEB and LC3 expression and decreased LAMP1 expression. ROC curve analysis demonstrated high diagnostic accuracy of TFEB for MA (AUC = 0.8526). Collectively, these findings identify TFEB and ALP-associated proteins as potential molecular markers for the postmortem diagnosis of MA.

"TransAct" is a multi-laboratory collaborative project aimed at establishing foundational work for the rationalization of research on forensic nucleic acid transfer, persistence, prevalence, and recovery (TPPR). A consortium of eight forensic laboratories systematically investigated inter- and intra-laboratory variability in the recovery and quantification of DNA from standardized mixtures of skin cells, buccal cells, and cell-free DNA (as standardized mock touch DNA samples) to improve the understanding of the factors influencing research data on DNA-TPPR. Substantial variability in absolute DNA yields (across two orders of magnitude for identical substrates and four orders of magnitude for different substrates) was observed across participating laboratories, primarily attributable to methodological heterogeneity among labs. Quantification using standard qPCR kits revealed significant differences in measured DNA concentrations depending solely on the method applied, with further variation linked to sampling techniques, and substrates. Mixture proportions of degraded low-template two-person mock touch DNA mixtures (male and female contributors) were assessed via both the ratio of the Y-chromosomal to autosomal qPCR targets and probabilistic genotyping (EuroForMix), revealing discrepancies between the two methods and laboratories. These findings underscore that TPPR data are profoundly influenced by laboratory-specific methodological differences, emphasizing the critical need for experimental standardization and shared resources to enhance the reliability of nucleic acid TPPR research and thereby improving activity level evaluation in forensic genetics. As a step towards harmonizing data collection, this work provides exemplar templates for a detailed documentation in supplementary material, covering both the collection of information on laboratory methods and the standardized display of results to facilitate consistent data reporting and future data pooling.

In odontological human identification, the dental coding system applicable to disaster victim identification software used in the International Criminal Police Organization (INTERPOL) operations provides a structured and internationally recognised framework for comparing antemortem and postmortem dental information. However, the lack of a consolidated, peer-reviewed guide to these codes can pose challenges for interpretation and practical application. This study aimed to address this gap by providing an accessible, consensus-based guide to the current INTERPOL dental coding system. The analysis focused on code classification, definition, syntax, and logical combinations for recording dental features.

Age estimation is important for the identification of unknown cadavers in forensic practice. Previous studies have shown that gut microbiota is associated with host age, but most evidence has been derived from fecal samples of living individuals. In this study, shotgun metagenomic sequencing was performed on mid-colon tissue samples from 76 cadavers to explore age-associated taxonomic and predicted functional patterns in cadaveric colon microbiota and to evaluate their potential value for forensic age estimation. After quality control and taxonomic annotation, 3980 microbial species were identified. Descriptive differences among age groups were observed in microbial composition, alpha diversity, species-enrichment patterns, co-occurrence network structure, and KEGG functional profiles; however, a multivariable PERMANOVA did not detect a statistically significant association between age group and overall species-level community composition after accounting for postmortem sampling interval, cause-of-death category, and sex. The complete random forest pipeline was re-evaluated using repeated nested five-fold cross-validation, with all data-dependent filtering, transformation, feature selection, and hyperparameter tuning restricted to the outer training data. The resulting out-of-fold performance was limited (R[2] = 0.005, MAE = 13.686 years, and RMSE = 17.741 years), and predictions showed regression toward the cohort mean. Overall, this study provides preliminary evidence that cadaveric colon microbiota contains age-associated microbial signals, but these findings should be interpreted as exploratory. Larger cohorts and independent external validation are needed before microbiome-based age prediction can be applied in forensic practice.

With unique genetic characteristics and inherent stability, circular multicopy mitochondrial DNA (mtDNA) is critical for maternal lineage tracing from degraded and trace samples, making it a sustained focus in forensic science for identifying degraded samples when nuclear DNA analysis fails, as well as in evolutionary anthropology. However, accurate and reliable mtDNA sequencing from such samples remains technically challenging. By employing rolling circle amplification (RCA) to achieve linear amplification, the DNB sequencing platform effectively avoids error accumulation, yielding sequencing results with high accuracy, low error rates, and improved uniformity even for complex sequences (e.g., GC-rich regions). In this study, we developed a whole-genome sequencing system for mtDNA using a short-overlapping amplification strategy on the DNB sequencing platform. For blood card samples, the most common sample type in forensic practice, the obtained sequencing reads showed a mean Q30 value of 97.65% (SD = 0.24%), with an average of 99.47% (SD = 0.16%) of the reads aligning to the mtDNA reference sequence rCRS, covering 99.64% of the entire mtDNA sequence. Out of the 16,569 positions, 99.06% achieved a sequencing depth greater than 10% of the mean depth. The system enables accurate calling of point-variants and length-variants, with average error rates of 0.20%, 0.09%, 0.22%, and 0.12% for the four bases A, C, T, and G, respectively; and average error rates of 7.71% and 1.10% for insertion and deletion variants, respectively. Furthermore, heteroplasmy detected at position 309 by the system, including even the third most abundant allele by sequencing depth in the MPS data, was validated by Sanger sequencing. Additionally, the system enabled balanced whole-genome mtDNA sequencing of samples subjected to high-temperature degradation with a degradation index exceeding 60, achieving a sequencing depth greater than 10% of the mean depth at 95.01% of the positions. In extremely degraded samples where effective recovery of small fragments was not possible, 93.69% of the mtDNA sequence was still obtained. The development of this system provides a balanced, accurate, and robust tool for whole-genome mtDNA sequencing in forensic practice, with potential applications in complex kinship identification, maternal lineage screening, and population genetic relationship analysis.

We critically evaluated the evidence cited by the American Academy of Pediatrics (AAP) and the Royal College of Paediatrics and Child Health (RCPCH) supporting the diagnostic significance of fractures in suspected physical child abuse. Primary studies cited by the AAP Clinical Report and the RCPCH Systematic Review were reviewed with respect to study design, methods used to classify cases as abuse, and the potential for incorporation bias and outcome misclassification. Sixty-seven studies were identified, of which 35 reported measures of diagnostic utility. All were observational, predominantly retrospective studies. No study employed an independent gold standard for the diagnosis of abuse, and all relied on imperfect reference standards that varied in their methods of case classification. The evidence demonstrated substantial methodological limitations, primarily the risks of incorporation bias and circular reasoning. Overall, the evidence underpinning current guidance on fractures in suspected child abuse is characterised by methodological flaws that limit the scientific confidence that can be placed in the reported diagnostic associations. The substantial uncertainty surrounding the diagnostic significance attributed to fractures in the evaluation of suspected child abuse should be explicitly recognised and communicated.

In the Final Report of the Commission of Inquiry into Forensic DNA Testing in Queensland, Recommendation 28 recommended that alternative procedures to the scraping method should be investigated for the recovery of biological material from crime scene exhibits for downstream DNA testing. This study forms the first phase of a larger project comparing swabbing, tape-lifting, scraping and excision of biological fluids from a fabric substrate to determine the most effective recovery procedure. Calico cotton swatches were seeded with two concentrations (neat and 1:1000) of blood, semen and saliva, samples commonly encountered in forensic casework. Each sample type was collected using the four evidence recovery methods, extracted, quantified, and the 1/1000 dilution samples were short tandem repeat (STR) profiled to assess the practical limits of each method. Excision had the greatest DNA yield with a large effect size and best STR profile quality compared to all other recovery methods for blood (p < 0.0001, ŋ[2] > 0.95), semen (p < 0.0001, ŋ[2] = 0.96) and saliva (p < 0.0001, ŋ[2] = 0.82). There were no statistical differences found between the MWE Sterile Tubed Dryswab™ Rayon swab 1028, the Puritan® 6" Standard Rayon Swab w/Polystyrene Handle - 806-PR, HazPak tape-lift or scraping for neat blood and semen. Tape-lifting had a statistically higher efficiency than scraping (p = 0.0012), the Puritan (p = 0.0046) and the MWE swabs (p = 0.0145). Results suggest that excision is the most effective biological material recovery technique for blood, semen, and saliva from 100 % white cotton.

In this work, spectral data was used to identify the species and weathering time of blow fly puparia, which helps with the estimation of the minimum post-mortem interval (PMImin) in forensic science. After a preliminary model selection stage, the two best-performing modeling approaches were compared: a classical Logistic Regression (LR) model and a more complex Convolutional Neural Network (CNN). By evaluating both models on these two tasks, we aim to assess the performance trade-offs between competitive traditional machine learning approaches and modern deep learning techniques. Moreover, beyond comparative performance results, an interpretability analysis was conducted to investigate which regions of the spectrum were most informative for weathering time estimation and species classification. These insights may guide future research in forensic entomology using IR spectroscopy by highlighting spectral regions associated with key biological changes related to ageing and species differentiation.

The identification of gunshot residues (GSR) is pivotal in forensic chemistry; however, standard confirmatory techniques, such as Scanning Electron Microscopy with Energy Dispersive X-ray Spectroscopy (SEM/EDS), are often costly, time-consuming, and centralized in major urban facilities. This study proposes a rapid and cost-effective presumptive screening framework for firearm discharge-consistent metals (Pb[2 +] and Sb[3+]), integrating Bromopyrogallol Red (BPR) colorimetry, digital image processing, multivariate chemometrics, and machine learning algorithms. One hundred samples were evaluated, including real hand swabs from shooters firing pistols, revolvers, and rifles, negative control samples ("non-shot"), and inorganic metallic standards. Digital images obtained in a standardized lighting environment were processed to extract quantitative chromatic descriptors across RGB and HSV/HSI color spaces. Exploratory analyses via Principal Component Analysis (PCA) and Non-Metric Multidimensional Scaling (NMDS) demonstrated robust class separation (PC1 + PC2 = 83.1%; NMDS stress = 0.027), with luminosity and intensity variables (V, I, R, and L) acting as primary discriminants. Supervised machine learning classifiers were developed to differentiate "shot" from "non-shot" samples, where tree-based algorithms-specifically Random Forest and XGBoost-achieved exceptional discriminatory performance, both yielding an Area Under the ROC Curve (AUC) of 0.998. Notably, the Random Forest model exhibited a minimal false-negative rate and superior probabilistic calibration, critical attributes for forensic decision-making. Executed in under 10 min per sample, this methodology provides a reliable, decentralized triaging solution to filter negative cases locally and prioritize high-probability samples for confirmatory SEM/EDS analysis.

Distinguishing between bone and non-bone material is the initial stage of forensic examination of suspect skeletal remains. This is particularly important, but more problematic, when examining disputed cremations. This forensic case study explores the application of visual, UV and elemental analysis of suspected cremated remains of pet animals. Visual analysis included sieving through the bulk for bone fragments, visual identification of bone morphology, color, texture and fragility, and reaction to UV fluorescence. Visual analysis showed that none of the 61 suspected cremations were comprised of uncontaminated bone powder. There were 19 closely resembling concrete recovered from the suspect premises, and 5 contained an assortment of marine sediment, bone-like fragments and human teeth mixed into the bulk of the suspected pet cremation. Foreign objects were present in four suspect cremations. Visual analysis was supplemented with elemental analysis with portable X-ray fluorescence (pXRF), which showed significant differences between the suspect and control cremations for: MgO, Al[2]O[3], SiO[2], P, Cl, K[2]O, Ti, Mn, Fe, Zn, Pb, and the Ca/P ratio. Control cremations could be clearly distinguished from all suspect cremations by the absence of SiO[2], a 1.8-4.3 % K[2]O content, and a 1.5-1.9% Ca/P ratio. Additionally, the Ca/P ratio of all non-bone samples clustered outside the ratio range for bone. Overall, none of the suspected cremations were comprised of uncontaminated cremated bone expected from legitimate commercial cremation practices.