Epigenetic analyses in forensic medicine: future and challenges.

The possibility of using epigenetics in forensic investigation has gradually risen over the last few years. Epigenetic changes with their dynamic nature can either be inherited or accumulated throughout a lifetime and be reversible, prompting investigation of their use across various fields. In forensic sciences, multiple applications have been proposed, such as the discrimination of monozygotic twins, identifying the source of a biological trace left at a crime scene, age prediction, determination of body fluids and tissues, human behavior association, wound healing progression, and determination of the post-mortem interval (PMI). Despite all these applications, not all the studies considered the impact of PMI and post-sampling effects on the epigenetic modifications and the tissue-specificity of the epigenetic marks.This review aims to highlight the substantial forensic significance that epigenetics could support in various forensic investigations. First, basic concepts in epigenetics, describing the main epigenetic modifications and their functions, in particular, DNA methylation, histone modifications, and non-coding RNA, with a particular focus on forensic applications, were covered. For each epigenetic marker, post-mortem stability and tissue-specificity, factors that should be carefully considered in the study of epigenetic biomarkers in the forensic context, have been discussed. The advantages and limitations of using post-mortem tissues have been also addressed, proposing directions for these innovative strategies to analyze forensic specimens.

Artificial intelligence in healthcare: why not apply the medico-legal method starting with the Collingridge dilemma?

Technology has greatly influenced and radically changed human life, from communication to creativity and from productivity to entertainment. The authors, starting from considerations concerning the implementation of new technologies with a strong impact on people's everyday lives, take up Collingridge's dilemma and relate it to the application of AI in healthcare. Collingridge's dilemma is an ethical and epistemological problem concerning the relationship between technology and society which involves two approaches. The proactive approach and socio-technological experimentation taken into account in the dilemma are discussed, the former taking health technology assessment (HTA) processes as a reference and the latter the AI studies conducted so far. As a possible prevention of the critical issues raised, the use of the medico-legal method is proposed, which classically lies between the prevention of possible adverse events and the reconstruction of how these occurred.The authors believe that this methodology, adopted as a European guideline in the medico-legal field for the assessment of medical liability, can be adapted to AI applied to the healthcare scenario and used for the assessment of liability issues. The topic deserves further investigation and will certainly be taken into consideration as a possible key to future scenarios.

Expression of matrix metalloproteinase-9 (MMP-9) in human skin within 1 hour after injury through immunohistochemical staining: a pilot study.

Matrix metalloproteinase-9 (MMP-9) is involved in tissue remodeling and in skin wound healing. The present study focuses on the MMP-9 expression in epidermal wound healing within 1 h after injury, to test whether MMP-9 can be used to estimate the time of injury in forensic practice.A sample consisting of 5 individuals undergoing surgery was analyzed. With the consent of the patients, sections of skin were removed from the surgical wound at predefined time intervals. For each subject, 8 sections were taken, one for each time interval defined at 0 '- 1' - 3 '- 5' - 10 '- 15' - 30 '- 60' minutes. The specimens were immunostained with MMP-9, and the number of positively stained cells was examined.The number of positively stained cells showed an increasing trend as a function of time. Less than 30 positively stained cells were found in all cases within 3 min. At the post-infliction time of 5 min, the number of positively stained cells exceeded 30 in 3 out of 5 cases. The number of MMP-positive cells exceeded 40 in all cases in over 10 min.In the light of these results, the count of MMP-9 positive cells might be a useful marker in the wound-age estimation within 1 h in forensic setting. More research is required to collect more samples and to compare samples from the hyperacute phase with those from several days after injury.

Forensic species identification: practical guide for animal and plant DNA analysis.

The importance of non-human DNA in the forensic field has increased greatly in recent years, together with the type of applications. The molecular species identification of animal and botanical material may be crucial both for wildlife trafficking and crime scene investigation. However, especially for forensic botany, several challenges slow down the implementation of the discipline in the routine.Although the importance of molecular analysis of animal origin samples is widely recognized and the same value is acknowledged to the botanical counterpart, the latter does not find the same degree of application.The availability of molecular methods, especially useful in cases where the material is fragmented, scarce or spoiled preventing the morphological identification, is not well known. This work is intended to reaffirm the relevance of non-human forensic genetics (NHFG), highlighting differences, benefits and pitfalls of the current most common molecular analysis workflow for animal and botanical samples, giving a practical guide. A flowchart describing the analysis paths, divided in three major working areas (inspection and sampling, molecular analysis, data processing and interpretation), is provided. More real casework examples of the utility of non-human evidence in forensic investigations should be shared by the scientific community, especially for plants. Moreover, concrete efforts to encourage initiatives in order to promote quality and standardization in the NHFG field are also needed.

Current status of undergraduate teaching in forensic & legal medicine in Europe.

The European Council of Legal Medicine (ECLM) is the body established in 1992 to represent practitioners forensic & legal medicine and is composed of delegates of the countries of the European Union (EU) and from other countries which form part of Europe to a current total of 34 member countries. The aims of this study were to determine the current status of undergraduate forensic & legal medicine teaching in the curriculum of medical studies in ECLM countries and to use the results of this study to determine whether it would be appropriate to develop new guidelines and standards for harmonising the content of undergraduate forensic medicine training across ECLM member countries. A detailed questionnaire was sent to all individuals or organisations listed on the ECLM contact database. Responses were received from 21 of 33 countries on the database. These responses showed considerable emphasis on undergraduate teaching of forensic medicine in all countries with the exception of Belgium and the United Kingdom. There was great general consistency in the subjects taught. The data from this survey provide a baseline which should assist in developing a strategy to harmonise forensic & legal medicine undergraduate training in member countries of the ECLM. The ECLM is now in a good position to establish a pan-European working group to coordinate a consensus document identifying an appropriate and modern core undergraduate forensic medicine curriculum that can be presented to the medical education authorities in each country, and which can be adapted for local requirements, based on available personnel, the forensic medicine structure in the country, and most importantly, the needs of the local population.

Applications of forensic anthropology methodology: accuracy of virtual face reproductions performed on the Tenchini collection.

Facial reproduction validation uses the methodology typical of forensic anthropology to confirm the accuracy of three-dimensional reproductions of faces. Achieving high accuracy in virtual facial reproductions is still under study. In the present paper, the Tenchini collection, which contains wax reproductions of prisoners' faces and their skulls, was used. By creating facial reproductions on skulls from this collection, the result was compared with the real face of the person to whom the skull belongs. The three-dimensional volume of each examined skull of Tenchini collection was acquired via CT scan and photogrammetry. Subsequently, the virtual reproduction of each skull was processed using three-dimensional graphics software (ZBrush, Pixologic®). The morphological comparison parameters used in the field of personal identification by forensic anthropology were used. The blind procedure required the operator to see the mask subsequent to the facial reproduction phase, so that the facial reproduction cannot exploit the information that knowledge of the mask would have produced. The parameters used in the study of facial reproductions have shown discrepancies between the characteristics of the masks and the facial reproductions, partly expected because due to the lack of soft tissue on the skull. However, a high degree of accuracy in the facial reproductions performed due to the applied methodology was documented. The present study allowed us to observe how these parameters can be useful to study the accuracy of facial reproduction and identify what difficulties are encountered in producing a result close to the real appearance.

Determination of ethyl glucuronide in hair and self-reported alcohol consumption in university students.

Young individuals constitute an intriguing population, as their drinking habits are notably shaped by their perception of their peers' alcohol consumption. Nonetheless, excessive alcohol intake can have detrimental effects on academic performance, interpersonal relationships, and the risk and severity of accidents. This study reported the first data involving students enrolled from three universities on a voluntary basis for alcohol consumption evaluation. Alcohol consumption was assessed through questionnaires and EtG quantification in hair (hEtG) carried out by liquid chromatography-mass spectrometry (LC-MS/MS) analysis after a solid-phase extraction (SPE) purification step. The results of our study demonstrated that 77.1% of samples tested negative for hEtG or displayed hEtG ≤ 5 pg/mg. Particularly, the student population was not characterized by samples with hEtG indicative of chronic excessive consumption (hEtG ≥ 30 pg/mg). No significant association was identified between biological sex, among the degree course/the year attended, nor in relation to BMI or smoking/coffee consumption. Among the obtained results, it was worth noting that the comparison of self-reporting abstinence from tobacco and coffee accounted for 65.3% and 16.7%, respectively, while only 2.8% of the total declared abstinence from alcohol. The current study has uncovered a significant level of interest among students in this analysis and its interpretation. This suggests that implementing public health promotion activities within a university setting could be beneficial.

Third trimester intrauterine fetal death: proposal for the assessment of the chronology of umbilical cord and placental thrombosis.

The timing of umbilical cord and placental thrombosis in the third trimester intrauterine fetal death (TT-IUFD) may be fundamental for medico-legal purposes, when it undergoes medical litigation due to the absence of risk factors. Authors apply to human TT-IUFD cases a protocol, which includes histochemistry and immunohistochemistry (IHC) for the assessment of thrombi's chronology. A total of 35 thrombi of umbilical cord and/or placenta were assessed: 2 in umbilical artery, 6 in umbilical vein, 15 in insertion, 10 in chorionic vessels, 1 in fetal renal vein, 1 in fetal brachiocephalic vein. Thrombi's features were evaluated with hematoxylin-eosin, Picro-Mallory, Von Kossa, Perls, and immunohistochemistry for CD15, CD68, CD31, CD61, and Smooth Muscle Actin. The estimation of the age of the thrombi was established by applying neutrophils/macrophages ratio taking into consideration, according to literature, the presence of hemosiderophagi, calcium deposition, and angiogenesis. To estimate an approximate age of fresh thrombi (< 1 day), a non-linear regression model was tested. Results were compared to maternal risk factors, fetal time of death estimated at autopsy, mechanism, and cause of death. Our study confirms that the maternal risk factors for fetal intrauterine death and the pathologies of the cord, followed by those of the placental parenchyma, are the conditions that are most frequently associated with the presence of thrombi. Results obtained with histological stainings document that the neutrophile/macrophage ratio is a useful tool for determining placental thrombi's age. Age estimation of thrombi on the first day is very challenging; therefore, the study presented suggests the N/M ratio as a parameter to be used, together with others, i.e., hemosiderophagi, calcium deposition, and angiogenesis, for thrombi's age determination, and hypothesizes that its usefulness regards particularly the first days when all other parameters are negative.

Ventricular androgenic-anabolic steroid-related remodeling: an immunohistochemical study.

BACKGROUND: Several fatal cases of bodybuilders, following a myocardial infarction after long exposure to androgenic-anabolic steroids (AAS), are reported. In recent years, evidence has emerged of cases of heart failure related to AAS consumption, with no signs of coronary or aorta atherosclerosis. This study aims to further investigate the pathogenesis of the ventricular AAS-related remodeling performing immunohistochemistry (IHC). METHOD: In order to examine innate immunity activity and myocytes and endothelial cell apoptosis, IHC analyses were performed on heart tissue of two cases of bodybuilders who died after years of supratherapeutic use of metelonone and nandrolone and where no atherosclerosis or thrombosis were found, using the following antibodies: anti-CD68, anti-iNOS, anti-CD163, anti-CD 15, anti-CD8, anti-CD4, anti-HIF1 α, and in situ TUNEL staining. RESULTS: Results confirm the experimental findings of recent research that, in the absence of other pathological factors, if intensive training is combined with AAS abuse, myocytes and endothelial cells undergo apoptotic alterations. The absence of inflammatory reactions and the presence of an increased number of M2 macrophages in the areas of fibrotic remodeling confirm that the fibrotic changes in the heart are apoptosis-related and not necrosis-related. CONCLUSIONS: In conclusion, the study indicates that, in very young subjects with chronic hypoxia-related alterations of the heart, signs of a heart failure in the other organs and a history of AAS abuse, death can be ascribed to progressive heart failure due to the direct apoptotic cardiac and endothelial changes produced by AAS.

Back to the Future - Part 1. The medico-legal autopsy from ancient civilization to the post-genomic era.

Part 1 of the review "Back to the Future" examines the historical evolution of the medico-legal autopsy and microscopy techniques, from Ancient Civilization to the Post-Genomic Era. In the section focusing on "The Past", the study of historical sources concerning the origins and development of the medico-legal autopsy, from the Bronze Age until the Middle Ages, shows how, as early as 2000 BC, the performance of autopsies for medico-legal purposes was a known and widespread practice in some ancient civilizations in Egypt, the Far East and later in Europe. In the section focusing on "The Present", the improvement of autopsy techniques by Friedrich Albert Zenker and Rudolf Virchow and the contemporary development of optical microscopy techniques for forensic purposes during the 19th and 20th centuries are reported, emphasizing, the regulation of medico-legal autopsies in diverse nations around the world and the publication of international guidelines or best practices elaborated by International Scientific Societies. Finally, in "The Future" section, innovative robotized and advanced microscopy systems and techniques, including their possible use in the bio-medicolegal field, are reported, which should lead to the improvement and standardization of the autopsy methodology, thereby achieving a more precise identification of natural and traumatic pathologies.

Back to the Future - Part 2. Post-mortem assessment and evolutionary role of the bio-medicolegal sciences.

Part 2 of the review "Back to the Future" is dedicated to the evolutionary role of the bio-medicolegal sciences, reporting the historical profiles, the state of the art, and prospects for future development of the main related techniques and methods of the ancillary disciplines that have risen to the role of "autonomous" sciences, namely, Genetics and Genomics, Toxicology, Radiology, and Imaging, involved in historic synergy in the "post-mortem assessment," together with the mother discipline Legal Medicine, by way of its primary fundament, universally denominated as Forensic Pathology. The evolution of the scientific research and the increased accuracy of the various disciplines will be oriented towards the elaboration of an "algorithm," able to weigh the value of "evidence" placed at the disposal of the "justice system" as real truth and proof.

Are mast cells implicated in asphyxia?

In a previous immunohistochemical (IHC) study, we documented the reaction of lung tissue vessels to hypoxia through the immunodetection of HIF1-α protein, a key regulator of cellular response to hypoxic conditions. Findings showing that asphyxia deaths are associated with an increase in the number of mast cell (MC)-derived tryptase enzymes in the blood suggests that HIF1-α production may be correlated with MC activation in hypoxic conditions. This hypothesis prompted us to investigate the possible role of pulmonary MC in acute asphyxia deaths. Lung of 47 medico-legal autopsy cases (35 asphyxia/hypoxia deaths, 11 controls, and 1 anaphylactic death) were processed by IHC analysis using anti-CD117 (c-Kit) antibody to investigate peri-airway and peri-vascular MC together with their counts and features. Results showed a significant increase in peri-vascular c-kit(+) MC in some asphyxia deaths, such as hanging, strangulation, and aspiration deaths. A strong activation of MC in peri-airway and peri-vascular areas was also observed in lung samples from the anaphylaxis case, which was used as a positive control. Our study points to the potential role of MC in hypoxia and suggests that an evaluation of MC in the lungs may be a useful parameter when forensic pathologists are required to make a differential diagnosis between acute asphyxia deaths and other kinds of death.

[Not Available]. / Il consenso informato in Terapia Occupazionale: proposta di una modulistica.

UNLABELLED: The legislative developments that led to the Three-year Degree of the Health Professions poses any health professional in the position of having to comply with the ethical and legal duty to obtain valid informed consent from the patient prior to treatment. In the present work, attention was focused on the figure of the occupational therapist. MATERIALS AND METHODS: Informed consent forms have been developed according to the specific disease from which the patient undergoing occupational therapy is affected. The following categories of sick were identified: amputation, developmental age, orthopedy, spinal cord injury, neurology, psychiatry, geriatry and oncology. RESULTS AND CONCLUSION: The consent forms are particularly well suited to obtaining valid consent from the patient and, at the same time, allow the occupational therapist to obtain all the information he/she needs to carry out the treatment in safety. This results improved patient compliance to therapy by facilitating a better empathic relationship with the therapist.

Molecular analysis of different classes of RNA molecules from formalin-fixed paraffin-embedded autoptic tissues: a pilot study.

For a long time, it has been thought that fresh and frozen tissues are the only possible source of biological material useful to extract nucleic acids suitable for downstream molecular analysis. Recently, for forensic purpose such as personal identification, also fixed tissues have been used to recover DNA molecules, whereas RNA extracted from such material is still considered too degraded for gene expression studies. In the present pilot study, we evaluated the possibility to use forensic formalin-fixed paraffin-embedded (FFPE) samples, collected at autopsy at different postmortem intervals (PMI) from four individuals, to perform advanced molecular analyses. In particular, we performed qualitative and quantitative analyses of total RNAs extracted from different FFPE tissues and put expression profiles in relation with the organ type and the duration of PMI. Different classes of RNA molecular targets were studied by real-time quantitative RT-PCR. We report molecular evidence that small RNAs are the only RNA molecules still detectable in all the FFPE autoptic tissues. In particular, microRNAs (miRNAs) represent a consistent, stable, and well-preserved molecular target detectable even from tissue sources displaying signs of ongoing putrefaction at autopsy. In this pilot study, we show that miRNAs could represent a highly sensitive and potentially useful forensic marker. Amplification of specific miRNAs using paraffin-embedded blocks could facilitate retrospective molecular analysis using specific forensic-archived tissues chosen as most suitable according to PMI, and this approach would address molecular evidence in forensic cases in which fresh or frozen material is no longer available.

[Informed consent in physiotherapy: proposal of a form]. / Il consenso informato in fisioterapia: proposta di una modulistica.

UNLABELLED: The aim of the work is to highlight the need, the duty and the obligation also for the physiotherapist to obtain a valid informed consent of the patient. MATERIALS AND METHODS: The authors, starting with the informed consent forms that already exist for physicians, offer four modules tailored to the needs of the physiotherapist, specific to each field of rehabilitation. RESULTS AND CONCLUSIONS: Such informed consent may be very useful to physiotherapists to fulfill the obligations of giving information and obtaining consent from patients. At the same time it allows physiotherapists to obtain all information they need about patient's condition and permit patient to understand the proposed treatment and adhere to it.

Migrating granulomatous chronic reaction from hyaluronic acid skin filler (Restylane): review and histopathological study with histochemical stainings.

BACKGROUND: A unique case is presented in whom an allergic reaction to Restylane filler, associated with migrating granulomas, persisted despite medical interventions. A histopathological study was requested for evidence at court. METHODS: Hematoxylin-eosin, alcian blue and colloidal iron staining were applied to skin sample biopsies obtained 5 months and 3 years after the hyaluronic acid (HA) injection. RESULTS: The histological staining highlighted the presence of the filler inside the foreign body granuloma and in the derma of a biopsy obtained after 5 months; a small amount of filler was discovered within a granulomatous reaction 3 years after the injection. CONCLUSIONS: Smaller fragments of HA display inflammatory, angiogenic and immune-stimulatory activities. Intradermal skin testing before the start of HA filler therapy, and before each subsequent injection, may prevent legal implications for the plastic surgeon. Informed consent to skin tests should be obtained.

Drug-facilitated sexual assault followed by femicidal chloroform poisoning and suffocation: A case-report of criminal responsibility.

INTRODUCTION: Criminal responsibility evaluation represents one of the most controversial and debated issues in forensic psychiatry. Although clear procedures have been recommended, little research exists on decision-making process by forensic psychiatrists. We present a case assessing the criminal responsibility of a murderer who committed femicide as a result of chloroform poisoning and suffocation after a drug-facilitated sexual assault. MATERIALS AND METHODS: A.S., a 30-year-old female, was found dead in the home of S.P., a 50-year-old male. S.P. recounted killing A.S. by forced inhalation of chloroform, when the woman had experienced sensory clouding following unintentional ingestion of Zolpidem, a hypnotic agent. A multidisciplinary approach was taken to resolve the case. Autopsy, histological, genetic, and toxicological examinations were performed by a forensic pathologist, while a digital forensic examiner analysed electronic devices. A pool of three forensic psychiatrists and two psychologists was asked to assess the mental state of S.P. at the time of the crime. RESULTS AND CONCLUSIONS: The cause of death of A.S. was identified as a lethal chloroform intoxication in altered consciousness caused by Zolpidem, while homicidal suffocation was also described. Mobile forensics demonstrated that S.P. had videotaped the crime scene, clearly revealing that A.S. had been sexually assaulted by S.P. before dying. Criminal responsibility of S.P. was evaluated through various psychological tests and seven interviews with the accused, each lasting an average of 180 min. Specialists concluded that S.P. could not be exempted from being responsible for the homicide.

The forensic imaging technique of age progression used within missing people cases: The Italian Missing Children association realizing age progression for Missing Child Kenya.

The age progression technique allows to update identification photos of individuals who have been missing for years. This method is based on the study of the facial variation and on the dynamics of facial growth. Knowledge about changes in the distribution of facial fat and the tone of the cartilage allows realistic predictions of the facial appearance after many years. Beside these elements, age progression is also supported by similar characters between family members. This is why photos of the missing and family members are used to create an age progression, making the new identikit accurate. Non-profit associations are crucial for the dissemination of information on the problem of missing persons. In Italy, the Italian Missing Children association provides for free an age progression service with the support of forensic anthropology. With the activation of a protocol, consisting of guidelines, and a form for the request for age progression by family members, the Italian Missing Children has started a collaboration with Missing Child Kenya. It allowed to disseminate up-to-date photos of the presumed current appearance of the missing children in Kenya. Thus, in the present study some cases are reported, in which age progression technique was applied. The photos provided by Italian Missing Children were shown in video documentaries made by Missing Child Kenya, allowing to continue the dissemination of age progression photo during the Covid quarantines and to keep the population active in the search for the missing.

Father figure: Study of the age progression process from old pictures and its value in forensic sciences.

The application of age progression to the search for missing persons demonstrates its usefulness in being able to reconstruct the aged face based on the current age of a person who has been missing for many years. Age progression takes into account facial physiognomic elements and their changes over time. Based on these premises, the present study aims to investigate the evolution of somatic features, from childhood to adulthood, of one of the authors' father using photos at different ages of him. Sixteen photos were collected depicting an individual of Italian origin between the age of 1 and 72. The photos were processed in Adobe© Photoshop software to graphically highlight the shape development of individual physiognomic elements. The shape of the face, eyebrows, eyes, nose, mouth, and cheeks were analysed in detail using a morphological approach. The ageing processes that occurred in this case were discussed on the basis of the literature. The present study demonstrates the in-depth analysis of the dynamics of facial growth in a single case. Nonetheless, aging is different for each individual, so further studies are necessary to observe facial development on as many subjects as possible.

Medically assisted suicide in Italy: Recent legal developments on a controversial topic.

On 16th June 2022, the first case of lawful 'medically assisted suicide' took place on Italian soil. This event is a result of decade-long debates on informed consent and end-of-life care stimulated by medical jurisprudence. The authors first retrace the crucial moments that allowed this to happen and underline the problems still to be solved. The cases of DJ Fabo, Davide Trentin, Mario and Fabio Ridolfi are discussed, signalling how they influenced the path implemented by Italian jurisprudence.