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- Quo vadis, BGA? A collaborative EDNAP exercise on the challenges and progress in forensic biogeographical ancestry inference.Publication . Diepenbroek, Marta; Amorim, António; Anslinger, Katja; Arora, Natasha; Amory, Christina; Ballard, David; Bekaert, Bram; Bouakaze, Caroline; Afonso Costa, Heloísa; Decorte, Ronny; Ewers, Lena; Fagerholm, Siri Aili; Garcia, Oscar; van der Gaag, Kris; Gysi, Mario; Haas, Cordula; Hollard, Clemence; Kling, Daniel; Palencia-Madrid, Leire; Pereira, Vania; de la Puente, María; Ruiz-Ramírez, Jorge; Sadam, Maarja; Sidstedt, Maja; Mogensen, Helle Smidt; Staadig, Adam; Stoljarova-Bibb, Monika; Court, Denise Syndercombe; Tillmar, Andreas; Tvedebrink, Torben; Xavier, Catarina; Weiler, Natalie; Parson, Walther; Phillips, ChristopherThere is a broad consensus that forensic tests for the prediction of externally visible characteristics (EVC) and analysis of biogeographic ancestry (BGA) of an individual are technically reliable. However, interpretation of the results and population-specific genotype distribution patterns remains challenging. EVC and BGA analyses provide valuable information for population genetics studies and as investigative leads for criminal cases, as well as for historical and contemporary identification tests. However, inaccurate or incorrect predictions, for example, from subjective bias in the interpretations made, have the potential to misdirect police investigations. The legal situation regarding EVC and BGA testing varies by country: ranging from countries where it is explicitly prohibited, to those without specific regulations on biogeographic ancestry prediction, and others that have already enacted laws governing its use. The reluctance to utilize these analyses is not only due to legal restrictions and data protection concerns, but also to initial limited sets of sufficiently comprehensive forensic DNA assays. Forensic BGA marker panels typically contain up to ∼300 SNPs. This relatively small number of genetic markers, along with limited reference population data, complicates the interpretation of results from donors of unknown origin. This paper presents the results of a collaborative EDNAP study, which, for the first time, evaluated the approach to reporting EVC and BGA data between international laboratories. For the study, DNA from nine individuals with self-reported ancestry was collected and analysed using various forensic panels differing in the number and composition of ancestry-informative markers genotyped, comprising: the Precision ID mtDNA Whole Genome Panel, the VISAGE Basic Tool and the VISAGE Enhanced Tool for Appearance and Ancestry Prediction, and the Ion AmpliSeq™ PhenoTrivium Panel. To ensure full data protection, all SNP genotypes and uniparental marker haplotypes obtained were not shared with third parties. Instead, the genetic data were analysed using a range of commonly used population analysis software packages. These analysis outcomes were then distributed to twelve European forensic laboratories (both academic and law enforcement institutions), who were asked to prepare reports based on their interpretation of the phenotypes and ancestry they inferred from the analysis data. A questionnaire sent alongside the genetic information, aimed to evaluate which difficulties were encountered by the participants in processing the BGA analysis data they were given.
- Deaths in custody: Physical restraint, excited delirium and medico-legal investigation – A systematic reviewPublication . Dias, Alexandra Gonçalves; Cainé, LauraBackground/objectives: Deaths in custody represent a critical intersection between health care, law enforcement and human rights. By depriving an individual of liberty, the State assumes an enhanced duty to protect life and ensure access to adequate medical care. Any death occurring in custodial settings must therefore be regarded as potentially suspicious until clarified through a prompt, independent and technically robust investigation. Methods: This systematic review synthesizes and critically examines scientific evidence on deaths in police and prison custody, with particular emphasis on physical restraint, pathophysiological mechanisms, and medico-legal investigation practices. A systematic search of PubMed and Scopus (2010–2025) was conducted in accordance with PRISMA 2009 guidelines. Results: The evidence indicates that deaths in custody are predominantly multifactorial, resulting from the interaction between physical restraint, intense physiological stress, intoxication with psychoactive substances and individual vulnerabilities such as obesity or underlying cardiac disease. The concept of “excited delirium” remains highly controversial, lacking validated diagnostic criteria or specific biomarkers and is frequently used as a diagnosis of exclusion in medico-legal contexts. Physical restraint, particularly prolonged prone positioning with thoracic or cervical compression, may precipitate metabolic acidosis and restraint-associated cardiac arrest, even in the absence of significant hypoxemia. Experimental studies in healthy volunteers fail to reproduce realworld custodial conditions and should not be used to negate associated risks. Conclusions: Rigorous and independent medico-legal investigation, including complete autopsy, toxicological analysis and contextual reconstruction, is essential for accurate causal attribution and institutional accountability. These findings have direct implications for forensic practice, death certification and the prevention of avoidable deaths in custodial settings.
- Beyond the core: The role of supplementary short tandem repeats in forensic geneticsPublication . Ramos, Vitória; Ferreira da Silva, Benedita; Fadoni, Jennifer; Amorim, António; Cainé, LauraConventional forensic DNA profiling predominantly utilizes established core sets of au-tosomal short tandem repeats (STRs), such as the FBI’s Combined DNA Index System (CODIS) and the European Standard Set (ESS). While these panels are effective for stand-ard forensic casework, they may be inadequate in more demanding scenarios, including severely degraded samples, complex multi-contributor mixtures, and kinship cases with deficiencies, where enhanced discriminatory capability is crucial. Additional non-core STR loci address these challenges while maintaining the non-coding, phenotypically un-informative nature that ensures the legal and ethical acceptability of forensic genetic evi-dence in court. This review assesses the forensic applications, population genetic param-eters, validation requirements, and ethical considerations associated with non-core STR loci. A representative supplementary STR panel is presented as a case study to illustrate both the forensic value and the analytical requirements associated with the implementa-tion of extended non-core STR systems. Challenges in implementation were identified in areas such as nomenclature standardization, backward compatibility with existing na-tional databases, and geographic gaps in population reference data. The review concludes that a hybrid strategy, which retains core capillary electrophoresis (CE)-based profiling for routine casework and employs extended non-core panels for complex cases, represents the most practical path for the field.
- Forensic Evaluation of the Ion AmpliSeq MH-74 Microhaplotype Panel in the Portuguese PopulationPublication . Nascimento, Rui; Afonso Costa, Heloísa; Cunha, Eugénia; Amorim, AntónioBackground/Objectives: Microhaplotypes have emerged as promising genetic markers for forensic applications, combining low mutation rates with high polymorphic information. However, population data remain limited for several regions, including Portugal. This study aimed to generate population data for the Ion AmpliSeq™ MH-74 panel in a Portuguese population and evaluate its performance for forensic identification and kinship analysis. Methods: A total of 237 unrelated individuals were genotyped using massively parallel sequencing on the Ion GeneStudio S5 platform. Allele frequencies and forensic parameters were estimated. Population structure was assessed using principal component analysis (PCA) based on reference data from MicroHapDB. Kinship performance was evaluated through simulations of full siblings, half siblings, and first cousins and compared with STR markers using likelihood ratios (LR) and error rates. Results: The MH-74 panel showed high genetic diversity (mean He = 0.615; mean PIC = 0.565) and strong forensic efficiency, with very high combined power of discrimination and exclusion and a random match probability of 3.86 × 10−55. PCA revealed clustering consistent with global population structure, with the Portuguese population grouping within European populations. Microhaplotypes produced higher log10(LR) values for related pairs compared to STRs, particularly for closer relationships, with lower false negative rates. Conclusions: The MH-74 panel demonstrates high discriminatory power and reliable performance for kinship analysis in the Portuguese population, supporting the use of microhaplotypes as an effective complement to STR markers in forensic genetics.
- Genetic identification of human skeletal remains in forensic context: a reviewPublication . Cainé, Laura; Henriques, Madalena; Rohovska, Adelina; Sousa, Bárbara; Afonso Costa, Heloísa; Correia Dias, Maria Helena; Rodrigues, Joana; Franco, Magda; Mukan, Olena; Nascimento, Rui; Vânia Mofreita; Amorim, AntónioBackground/Objectives: Genetic identification of human skeletal remains plays a pivotal role in forensic investigations when other traditional or primary methods are not appropriate. Decomposition, storage and environmental conditions often leave the skeletal structure as the only basis for identification. This review synthesizes current methodologies and technological advances in damaged DNA extraction and analysis, emphasizing the forensic relevance of skeletal remains for genetic identification. Methods: A comprehensive literature analysis highlights the basis of genetic identification; sampling that considers intrinsic and extrinsic factors influencing the DNA yield and its quality; pre-treatment methods; extraction protocols that are suitable for its sensitivity; genetic marker panels that allow for human identification; and statistical evaluation and analysis of the results. The last chapter demonstrates the real-world impact of genetic identification on historical cases, underscoring its broader significance in legal, humanitarian, and socio-historical contexts, supporting a critical evaluation of best practices, methodological robustness, and ethical considerations within the field. Results: Teeth, femur and the petrous portion of temporal bone are the main samples used for genetic analysis. STR profiling and mitochondrial DNA are the gold standard markers for skeletal human identification. Minimally destructive protocols that enhance a high DNA yield are chosen, with silica-based methods being highlighted in the extraction protocols. Next-Generation Sequencing techniques have also improved analytical outcomes, by enabling high-throughput data generation, increased coverage depth, nucleotide-level sequence data, and high-level multiplexing of genetic targets. Conclusions: This review provides a comprehensive framework for researchers and practitioners seeking to optimize genetic identification workflows in forensic sciences and bioarcheology. These methodological advances have significantly increased identification success rates, especially in cases involving degraded or limited skeletal remains. Reviews such as this one help us to identify methodological gaps, ethical concerns, and future research directions, thereby establishing best practices when working with highly degraded skeletal material, supporting more reliable, standardized, and legally defensible applications of genetic identification in forensic, archeological, and humanitarian contexts.
- Detection and quantification of selected cannabinoids in hair samples by liquid-liquid extraction and LC-MS/MSPublication . Antunes, Mónica; Simões, Susana; Fonseca, Suzana; Franco, João; Barroso, Mário; Gallardo, EugeniaCannabis remains the most used illicit drug worldwide, with rising use linked to medical and recreational decriminalization. This has driven the development of analytical techniques to detect cannabinoids in biological matrices. Hair offers key advantages due to its non-invasive collection, extended detection window, stability, and easy storage. This study presents the development and validation of a robust method for extracting natural cannabinoids from hair, following ANSI/ASB 2019, FDA, and Society of Hair Testing guidelines. Hair samples were washed with methanol and cut into small pieces. Approximately 20 mg of hair was incubated with 1 M NaOH and methanol (30 min, 50 °C). The mixture was acidified with acetic acid and underwent liquid-liquid extraction using hexane/ethyl acetate (90/10, v/v). The organic phase was evaporated and reconstituted in 1-pentanol/methanol (50/50, v/v). Analysis was conducted by LC-MS/MS using multiple reaction monitoring (MRM) and triple-stage mass spectrometry (MS³). The method was selective, specific, precise, and linear, with working ranges of 5-2000 pg/mg for THC, CBN, and CBD; 50-2000 pg/mg for THC-OH; and 0.2-20 pg/mg for THC-COOH. Ion suppression was observed but did not affect sensitivity, with LLOQs and LODs from 0.2 to 50 pg/mg. Over 25 hair samples from university students tested positive for cannabis. THC ranged from 5.9 to 2430.7 pg/mg; one sample had THC-OH above LLOQ (61.4 pg/mg); THC-COOH ranged from 0.3 and 36.4 pg/mg; CBN from 5.7 to 461.0 pg/mg; and CBD from 5.7 to 850.2 pg/mg. Results aligned with self-reported use, confirming the method's forensic suitability.
- Development and validation of a simple and fast method for routine analysis of new synthetic opioids and hallucinogens in whole blood using protein precipitation and UHPLC-MS/MSPublication . Pereira, Joana R.P.; Antunes, Mónica; Neng, N.R.; Mustra, Carla; Franco, João; Fonseca, SuzanaIn forensic toxicology, the rapid and reliable detection of emerging synthetic opioids and hallucinogens is crucial for case investigations and public health monitoring. This work describes the development, optimization and validation of a simple, fast and sensitive methodology for the simultaneous analysis of 6 new synthetic opioids (carfentanil, fentanyl, isotonitazene, metonitazene, norfentanyl, and sufentanil) and 2 hallucinogens (lysergide [LSD] and mescaline), together with the main LSD metabolite 2-oxo-3-hydroxy-lysergide [LSD-OH], in whole blood samples by liquid chromatography coupled to tandem mass spectrometry. Under optimized experimental conditions, linearity was verified between 0.1 and 20 ng/mL for all analytes except mescaline (2.5-500 ng/mL), with r2 > 0.99 for 1/x weighting, and no significant carryover or matrix effects were observed. Good precision (% RSD < 13 %) and trueness (% Bias within ± 20 %) values were achieved. The estimated limit of quantification (LOQ) was 0.1 ng/mL for all compounds except mescaline (2.5 ng/mL). Authentic forensic samples were also analyzed, and positive samples for fentanyl, norfentanyl, and sufentanil were identified. The proposed methodology allows the simultaneous analysis of compounds from different families of psychoactive substances, in both postmortem and in vivo samples, using only 50 µL of whole blood. The demonstrated speed, simplicity, and effectiveness make it particularly advantageous for routine implementation in forensic toxicology laboratories.
- Medico-legal examination of sexual assaulted victims unable to consent in Portugal: ethical decision makingPublication . Santa-Rosa, Bárbara; Silvestre, Margarida; Vieira, Duarte Nuno; Corte Real, FranciscoMedical decision-making is a complex task in any field. In the medico-legal examination of victims that have (allegedly) been sexually assaulted there are many specific variables and features influencing the decision. It is essential to complement the clinical intervention with a forensic approach. Clinical parameters such as the victim's physical and cognitive state along with circumstantial information such as the elapsed time from the event and the type of abuse (described or suspected) grant different levels of priority to the forensic medical assessment. In such cases, forensic medical doctors or other medical doctors responsible for attending to the victim may have to decide whether to perform the examination prior to a judicial analysis of the case if consent cannot be obtained. This implies the need to deliberate about performing the examination and/or reporting the case to legal authorities. This article discusses the forensic medical decision-making process in cases of alleged recent sexual assault of victims who are legally unable to consent or unable to consent for other reasons. We aimed to identify possible ethical problems that can arise in this context and discuss which elements should be considered by medical doctors when making decisions about such cases. The Portuguese legal framework of medico-legal examinations is analyzed. The authors also make considerations about reporting these cases from a legal point of view. The discussion turns to an ethical perspective where possible ethical problems arising from medical deliberation are identified. Issues about legally incompetent victims and incompetent victims due to other reasons are addressed. A decision-making tree, based on the problems identified, is proposed.
- Reading between the lines. Older people’s maltreatment: from detection to reporting in primary healthcarePublication . Frazão, S.; Morais, C.; Norton, P.; Pinto, P. Vieira; Magalhães, T.The issue of older people’s maltreatment (OPM) is gaining increased attention worldwide because of its significant short and long-term severe consequences. Healthcare professionals play a crucial role in detecting, reporting, and managing these cases, but many difficulties in these procedures exist, and underreporting is very frequent. Thus, the aim of this study was to identify the extent of and difficulties in detecting and reporting suspected OPM cases by general practitioner physicians in Portugal. A cross-sectional survey was conducted among general practice physicians (GPPs) to assess their perceptions, experiences, and challenges in detecting and reporting older people maltreatment (OPM). The 13-item questionnaire explored sociodemographic factors, responsibility, confidence, reporting practices, and training needs, providing an updated insight into OPM management in primary healthcare. It was anonymous and conducted through a self-report questionnaire distributed in paper format to GPPs working in the National Health Service in the Northern of Portugal. We received 356 completed questionnaires for further analysis. The findings indicate that, on average, physicians suspect fewer than one OPM case per year; only 32% suspected at least one case in the previous year, although 55.3% and 54.5% of the physician’s revealed confidence in identifying signs of physical abuse and neglect, respectively. The ambiguity of the psychological symptoms was identified as a great challenge in detection (50.3%). Only 36.5% of the physicians were acquainted with the procedures for reporting suspected cases; however, of the 32% who suspected at least of one case in the previous year, only 39.5% reported all or at least some suspected cases; 16.9% chose not to report the case when requested by the victim or his/her caregivers. Most (91.6%) expressed a willingness to adopt protocols to assist them in managing such cases. A correlation was found between physicians’ satisfaction with OPM reporting training during undergraduate and postgraduate education and their ability to report (p=0.024), as well as with the number of reported cases (p=0.003). Thus, the detection and reporting of OPM by physicians remain major challenges that need to be overcome. Further research and greater awareness of the importance of these procedures are essential to ensure victims’ protection, safety, and health.
- General data protection regulation: an algorithmic proposal for forensic photographyPublication . Cura, Mariana; Loureiro, Ricardo; Marcelino, Pedro; Rodrigues, Vanessa; Andrade, José PauloSince 2018, the General Data Protection Regulation (GDPR) has regulated personal data within the scope of the European Union. With the exponential technological advancements in mobile photography, it is crucial to expose forensic professionals to this body of law to maintain good practices for fieldwork and scientific research in this field. GDPR, as far as its application in forensic photography, can be broken down into four pillars: informed consent of the subject, acceptable image capture practices (data), data storage and security at rest, and data transfers and security in transit. All these pillars have different approaches currently in use by forensic professionals. However, only some of them are permitted under the law. We present the appropriate ways to proceed with smartphone photography while remaining in compliance and maintaining the ability to share data critical to fieldwork and scientific research. In addition, some of the common pitfalls are described. An algorithm is proposed to facilitate compliance with European regulations relating to personal data, as applied to mobile forensic photography. The same flow chart can be used in other countries with different regulations concerning health data, privacy, and security issues.