Francesco Ausania

Estimating the post‐mortem interval (PMI) remains a major challenge in forensic practice, as conventional thanatological signs are strongly influenced by biological and environmental variables. Immunohistochemistry is a complementary strategy for identifying tissue biomarkers with time‐dependent post‐mortem persistence. In this retrospective study, we assessed the immunohistochemical expression of Caspase‐8, Annexin A1, B‐cell lymphoma 2 (Bcl‐2), and Nuclear Factor kappa B (NF‐κB) in brain, heart, and liver samples from 32 autopsies with known PMI (1.38–23 days). Cases were stratified into four PMI groups: < 2 days, ≥ 2 to < 5 days, ≥ 5 to ≤ 8 days, and > 8 days. Immunoreactivity was scored semi‐quantitatively as weak (1+) or strong (2+). Caspase‐8 showed the highest overall positivity and the longest persistence, remaining detectable up to 22 days in heart, 16 days in brain, and 14 days in liver, with complete positivity across tissues up to 8 days. Annexin A1 displayed last positivity at 6.09 days in brain and 7.63 days in heart and liver, being consistently negative beyond 8 days. Bcl‐2 expression was restricted to myocardium and detected only in very early PMI cases (< 2 days), whereas NF‐κB was consistently negative. These findings support a tissue‐specific, multiparametric model for PMI stratification.

Digital pathology has transformed anatomic pathology by enabling whole-slide imaging, remote review, reproducible annotation, image analysis, and the development of artificial intelligence-based decision-support tools. In forensic pathology, however, the transition from glass slides to digital forensic histopathology remains in its early stages, despite recent evidence supporting the feasibility and diagnostic reliability of whole-slide imaging in medico-legal practice. The subsequent crucial step involves not merely digitizing forensic slides but also developing curated, multicentric, legally governed, and controlled-access datasets comprising forensic histopathological images. Such datasets are essential for education, interobserver harmonization, external quality assessment, and the development of reproducible quantitative tools suitable for forensic applications. This perspective paper argues that forensic pathology should regard datasets as scientific infrastructure rather than as passive image collections. An artificial intelligence-ready forensic histopathology dataset should include whole-slide images, standardized annotations, technical acquisition parameters, histological processing information, forensic metadata, quality-control records, reference standards, uncertainty labels, and explicit governance rules. Although important lessons can be drawn from anatomic pathology datasets, forensic pathology presents specific challenges, including low case numbers, post-mortem artifacts, heterogeneous sampling, variable fixation and preservation conditions, judicial and privacy concerns, and differences in national regulation of post-mortem data. We therefore propose a literature-informed conceptual framework for the development of forensic histopathology datasets, intended for forensic pathologists, researchers, institutional stakeholders, and AI tools developers. The framework is intended to support the development of shared resources for education, research, and AI development and validation, thereby providing a foundation for trustworthy and scientifically defensible computational applications in forensic pathology.

In forensic autopsies, tissue samples are routinely collected, fixed in formalin, and embedded in paraffin (FFPE) to ensure long-term preservation for histological examination. In some cases, these specimens may represent the only remaining biological material available for postmortem genetic analysis. However, the use of FFPE samples in forensic genetics poses significant challenges, as formalin fixation compromises DNA integrity and reduces the likelihood of obtaining complete genetic profiles. Moreover, relatively few experimental studies in the scientific literature have addressed the integration of forensic pathology requirements with those of forensic genetics. To address this gap, we designed a pilot study to systematically evaluate the impact of formalin fixation time on genetic analysis outcomes. FFPE liver and lung samples from 11 individuals undergoing forensic autopsy were examined. Fixation times ranged from 3 to 33 days, and three sections from each FFPE block were used for DNA extraction. Femoral blood samples collected during autopsy were analyzed in parallel as reference samples. DNA extraction was performed using the QIA amp DNA Mini Kit. Genomic DNA was amplified using the Power Plex Fusion 6 C System, and analysis was performed on the GeoStudio Genetic Analyzer for HID. Preliminary results indicate that complete genetic profiles can be obtained after fixation for up to 11 days, with a progressive decrease in correctly typed alleles as fixation time increases. Overall, the study experimentally demonstrates a correlation between DNA degradation, as reflected in the quality of genetic profiles, and the duration of formalin fixation.

Cardiovascular disease is a leading cause of mortality, with coronary artery disease accounting for over 60% of cases in adults. Accurate quantification of coronary stenosis in forensic autopsies is crucial for determining causality between pathological findings and death, but is hindered by subjective visual assessments and inter-observer variability. This study aimed to develop an AI-driven tool using whole-slide images for objective stenosis measurement in forensic investigations. From 98 anonymized H&E-stained autopsy slides (234 coronary sections), 103 high-quality regions of interest were selected and split into training (n = 82), validation (n = 14), and test (n = 7) datasets. Annotations delineated lumen, internal elastic lamina, and external elastic lamina using QuPath. A SegFormer-B0 transformer model was trained with data augmentation, weighted cross-entropy loss, and AdamW optimization. Post-processing enforced anatomical structural hierarchy and generated hybrid confidence maps. On validation dataset, agreement with ground truth was excellent (MAE 3.22% points; RMSE 4.00; MAPE 6.07%; r = 0.986; ICC = 0.986), with slight underestimation. Bland–Altman bias was − 1.36 pp (95% LoA − 9.01 to 6.28 pp), indicating performance across severities. On the test set, accuracy improved (MAE 1.01 pp; RMSE 1.50; MAPE 2.11%; r = 0.998; ICC = 0.995) and outperformed three pathologists’ visual estimates (MAE 16.11, 8.18, 4.79 pp). Bland–Altman bias for the model was − 0.82 pp with tight limits of agreement (− 3.48 to 1.83 pp). Total inference time was 199.08 s for seven cases (28.44 s/image). WSI-based transformer pipeline enables rapid, auditable, and reproducible coronary stenosis measurement, reducing inter-observer variability and supporting standardized interpretation in forensic investigations. The online version contains supplementary material available at 10.1007/s00414-026-03800-6.

In high-income countries, humans are continuously exposed to indoor and outdoor air pollution. Chronic exposure to these airborne solids and gases from natural or artificial sources is related to higher mortality. The objective of this work is to critically assess whether the association between indoor air pollution and death can support robust causal inference from a strict medico-legal perspective. We conducted a narrative review of existing literature on reported health consequences, autopsy and histopathological findings potentially linked to indoor air pollution exposure, and dose–response relationships and examined their role in criminal liability in Western countries. Despite prevention measures and regulations, establishing criminal liability for indoor air pollution remains arduous beyond a reasonable doubt given associative epidemiological evidence, translational biases, and non-specific autopsy findings. Further research on non-linear models and targeted forensic investigations is warranted.

Background The Investigator Argus Y-28 QS Kit is a 27-locus, six-dye multiplex, which includes six rapidly mutating Short Tandem Repeats (RM Y-STRs). Methods A new text file containing the optimized panel and bin sets for GeneMapper ID-X Software v1.6 has been prepared. In addition, an internal validation study was conducted on the Investigator Argus Y-28 QS kit using the SeqStudio™ Genetic Analyzer for HID. Results The full results of that study are presented here, including data on precision, sensitivity, reproducibility, stutter determination, and reduction volume analysis. A concordance study and three case reports are documented. Finally, the kit was successfully used to conduct a proficiency test. Conclusions The obtained findings achieved a satisfactory performance of the Investigator Argus Y-28 QS Kit on the SeqStudio™ Genetic Analyzer for HID.

Background: Nowadays, exotoxic substance intake is among the most frequently employed methods of suicide. Self-poisoning is quite common among psychiatric patients treated in hospitals. Psychotropic drugs used for suicide include phenothiazines. Promazine hydrochloride (Talofen©) is an alpha-lytic phenothiazine neuroleptic with a high affinity for histaminergic H1 receptors and a low affinity for dopaminergic D2, serotoninergic 5-HT, alpha1-adrenergic, and muscarinic receptors, which may explain its potent sedative effect. The most common adverse effects include extrapyramidal syndromes, weight gain, orthostatic hypotension, QTc prolongation, convulsions, delirium, and psychosis. Rare adverse events include the potential occurrence of autoimmune syndromes and vasculitis. Methods: We report herein the delayed death of a 59-year-old woman due to cardiocirculatory arrest on an arrhythmic basis in the context of vasculitis of the small pulmonary vessels and prolongation of the QTc interval secondary to voluntary acute intoxication with promazine hydrochloride. Results: The incident occurred in a psychiatric patient with a history of prior self-harming acts. Histological investigations revealed wavy fibers in the heart, a lymphocytic granulocyte infiltrate in the walls of small- and medium-caliber vessels, and spotty perivascular deposition of histiocyte-macrophage cells in the lungs. Immunophenotypic investigations showed the prevalence of CD15+ and T-CD3+ elements, thus identifying a small vessel vasculitis. These findings were consistent with the literature regarding adverse events following the intake of promazine hydrochloride, although vasculitis is rare. Conclusions: Thus, while QTc lengthening and arrhythmic incidents are widely reported events associated with promazine hydrochloride use, the development of a rare condition such as pulmonary vasculitis undoubtedly played a synergistic and decisive stressogenic role in the genesis of the cardiac event, leading to irreversible functional arrest.

Pathology has benefited from the rapid progress of image-digitizing technology during the last decade. However, the application of digital whole slide images (WSI) in forensic pathology still needs to be improved. WSI validation is crucial to ensure diagnostic performance, at least equivalent to glass slides and light microscopy. The College of American Pathologists Pathology and Laboratory Quality Center recently updated internal digital pathology system validation recommendations. Following these guidelines, this pilot study aimed to validate the performance of a digital approach for forensic histopathological diagnosis. Six independent skilled forensic pathologists from different forensic medicine institutes evaluated 100 glass slides of forensic interest (80 stained with standard hematoxylin and eosin, 20 with special staining), including different organs and tissues, with light microscopy (Olympus BX51, Tokyo, Japan). Glass slides were scanned using the Aperio GT 450 DX Digital Slides Scanner (Leica Biosystems, Nussloch, Germany). After two wash-out weeks, forensic pathologists evaluated WSIs in front of a widescreen using computer devices with dedicated software (O3 viewer, O3 Enterprise, Zucchetti, Trieste, Italy). Side-by-side comparisons between diagnoses performed on tissue glass slides versus WSIs were above the threshold stated in the validation guidelines (mean concordance of 97.8%). CSUQ Version 3 questionnaire showed high satisfaction for all pathologists (mean result: 6.6/7). Our institutional digital forensic pathology system has been validated for practical casework application. This approach opens new scenarios in practical forensic casework investigations, such as sharing live histological ex-glass slides online, as well as educational and research perspectives, with improving impacts on the whole daily workflow.

Ethanol is the psychoactive substance identified most frequently in post‐mortem specimens. Unfortunately, interpreting post‐mortem ethanol concentrations can be difficult because of post‐mortem alcohol redistribution and the possibility of post‐mortem alcohol neogenesis. Indeed, in the time interval between death and sample collection, the decedent may be exposed to non‐controlled environments for an extended period, promoting microbial colonization. Many authors report that in the presence of carbohydrates and other biomolecules, various species of bacteria, yeast, and fungi can synthesize ethanol and other volatile substances in vitro and in vivo. The aim of this study was to study the impact of several variables on microbial ethanol production as well as develop a mathematical model that could estimate the microbial‐produced ethanol in correlation with the most significant consensual produced higher alcohol, 1‐propanol. An experimental setup was developed using human blood samples and cadaveric fragments incubated under strictly anaerobic conditions to produce a novel substrate, “cadaveric putrefactive blood” mimicking post‐mortem corpse conditions. The samples were analyzed daily for ethanol and 1‐propanol using an HS‐GC‐FID validated method. The formation of ethanol was evaluated considering different parameters such as putrefactive stage, blood glucose concentration, storage temperature, and storage time. Statistical analysis was performed using the Mann–Whitney non‐parametric test and simple linear regression. The results indicate that the early putrefactive stage, high blood glucose concentration, high temperature, and time of incubation increase microbial ethanol production. In addition, the developed mathematical equation confirms the feasibility of using 1‐propanol as a marker of post‐mortem ethanol production.