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The magnitude of the diagnostic benefit conferred by performing histopathological examinations after medico-legal/forensic autopsies remains debatable. We have tried to address this issue by reviewing a series of histopathology referrals concerning medico-legal autopsies in real-world routine practice. We present an audit of the consultations provided to forensics by clinical pathologists at our institute between 2015 and 2018. Over this period, 493 post-mortem examinations were performed by forensic pathologists. Of these cases, 52 (11%) were referred for histopathology. Gross assessment was requested in 22/52 (42%) cases. Histopathology examination was performed on single organs in 15/52 (29%) cases, primarily on the lung and heart, whereas parenchymatous multi-organ analysis was carried out in 14/52 (27%) cases. Bone-marrow sampling was studied in 4/52 (8%) cases. Immunohistochemistry was needed in 16/52 (31%) cases, special stains in 9/52 (21%) cases and molecular analysis in 4/52 (8%) cases. Focusing on technical processes, standard methodology on pre-analytical procedures was changed in 10/52 (19%) cases in order to answer specific diagnostic questions. We showed that although most of the time the diagnosis is clear by the end of dissection on the basis of the macroscopic findings, histopathology can provide, modify or confirm the cause of death in many medico-legal/forensic cases. Therefore, it is desirable that forensic pathologists and clinical pathologists establish robust working relationships in a cooperative environment. We conclude that it is important to implement guidelines based on real-world routine practice in order to identify cases where histopathology can provide useful contributions, which in our experience applied to 11% of forensic cases.

According to the World Health Organization, as many as 25% of traffic accidents are linked to alcohol abuse. This study describes the results of a nine-year study performed on injured drivers ( N = 12,806) in the Verona area of Northern Italy. Blood samples were mandatorily collected on injured drivers who were admitted to the Emergency Health Care Unit of Verona Hospital between 2009 and 2017, after they had been involved in a traffic accident. Blood alcohol concentration (BAC) determination was then undertaken using a validated head space–gas chromatography–flame ionisation detector (HS-GC-FID) method. We found that 21% of drivers tested positive for alcohol (BAC ≥0.01 g/L), while 16.8% presented with BAC levels above the Italian legal limit (>0.5 g/L). Of those who had positive BACs, about 50% presented with very high BAC levels (>1.5 g/L). Daily time distribution analyses, involving 2031 alcohol-positive drivers, showed a surge between 18:00 hours and 06:00 hours (74.3%), with a specific rise during the weekend (58.9%). The percentage of alcohol-related road accidents was 20.6%, which is lower than results reported in other international studies performed over the last 20 years. However, evidence that around 50% of the positive subjects showed a BAC >1.5 g/L confirms the correlation between BAC and accident risk, which becomes even more significant at progressively increasing levels of BAC. The study highlights the need to implement further strategies to both prevent and deter the use of alcohol while driving.

The strong psychoactive effects of synthetic cannabinoids raise the need for the deeper studying of their neurometabolic effects. The pharmacokinetic properties of 5F-APINAC and its influence on metabolomics profiles associated with neurotransmission were investigated in rabbit plasma. Twelve rabbits divided into three groups received 1-mL 5F-APINAC at 0.1, 1 and 2 mg/kg. The intervention groups were compared with the controls. Sampling was performed at nine time points (0–24 h). Ultra-high-performance liquid chromatography–tandem mass spectrometry was used. The pharmacokinetics were dose-dependent (higher curve at a higher dose) with a rapid biotransformation, followed by gradual elimination within 24 h. The tryptophan concentrations abruptly decreased (p &lt, 0.05) in all tested groups, returning to the basal levels after 6 h. 5-hydroxylindole acetic acid increased (p &lt, 0.05) in the controls, but this trend was absent in the treated groups. The aspartic acid concentrations were elevated (p &lt, 0.001) in the treated groups. L-kynurenine was elevated (p &lt, 0.01) in the intervention groups receiving 1 mg/kg to 2 mg/kg. Dose-dependent elevations (p &lt, 0.01) were found for kynurenic acid, xanthurenic acid and quinolinic acid (p &lt, 0.01), whereas the anthranilic acid trends were decreased (p &lt, 0.01). The indole-3-propionic acid and indole-3-carboxaldehyde trends were elevated (p &lt, 0.05), whereas the indole-3-lactic acid trajectories were decreased (p &lt, 0.01) in the intervention groups. 5F-APINAC administration had a rapid biotransformation and gradual elimination. The metabolites related to the kynurenine and serotonergic system/serotonin pathways, aspartic acid innervation system and microbial tryptophan catabolism were altered.

Asialo-human transferrin (asialo-hTf) is a glycoform of the human serum protein transferrin characterized by the lack of the sialic acid (SA) terminal unit. It is known that glycosylation micro-heterogeneity and the presence of SA are strongly involved in protein functioning and pathophysiological activities. Some hTf glycoforms are valuable biomarkers for the detection of both genetic defects of glycosylation and/or sialoform distribution changes. The detection of the carbohydrate deficient transferrin (CDT) glycoforms is currently a widely employed method for the diagnosis of chronic alcohol abuse. The physiological significance of asialo-hTf is still unclear, despite its important biological implications. The current knowledge suggests that asialo-hTf may be involved in regulation of iron transport and release at the hepatic level, which, consequently, could strongly be affected by alcohol consumption. For these reasons, a deeper understanding of asialo-hTf structure and its physiological role is required, and an improved method of its analysis would favor the detection of both chronic abuse and other habits of alcohol intake and/or misuse. Thus, suitable analytical methods possessing higher sensitivity and specificity in comparison with the currently available techniques are certainly recommended. The present review summarizes the studies on asialo-hTf structure, roles, and detection techniques mainly in relation to its possible use as a potentially additional useful biomarker of alcohol abuse, and underlines its prospective value as a forensic and diagnostic tool.

In the context of fitness certification to hold the driving license, GGT and CDT have been used, sometimes in combination (γ-CDT), to exclude chronic alcohol abuse. The present study was carried out with the aim of comparing the power of these biomarkers as tools for the objective screening of subjects at high risk of alcohol-associated traffic injuries.288 male drivers admitted to hospital after traffic accidents were examined by determination of GGT, CDT and BAC. The degree of association of GGT, CDT and γ-CDT with BAC was analysed using non-parametric statistics.Partitioning the cases using the cut-off concentrations of 0.5 g/L for BAC (the legal limit adopted in most European countries), 55 U/L for GGT and 1.9% for CDT, a highly significant difference was found between the frequency of elevated GGT or CDT in cases where BAC was within the legal limits and those with elevated BAC values (Fisher's exact test: p 0.001). However, the calculation of the odds ratio showed a much higher increase for CDT (28 times) than for GGT (6 times) in those drivers with a BAC above the Italian legal limit in comparison with those showing a BAC within the cut-off; conversely, γ-CDT does not provide any significant advantage vs. CDT alone.Both GGT and CDT provide objective evidence of an association with the occurrence of alcohol-related severe traffic accidents, but CDT shows superior association with these events. Therefore, CDT, notwithstanding higher costs, should be preferred in a forensic/certification context.

Ranolazine is a selective inhibitor of the late inward sodium current, approved for the treatment of chronic angina. Here, a case of a possibly suicidal death due to acute ranolazine overdosing is reported. A 41-year-old woman was found unconscious by her son and was urgently admitted to the intensive care unit. She had ingested an unknown amount of ranolazine tablets. Seventeen hours after admission, the patient died. An autopsy was performed 4 days postmortem. A routine screening analysis for drugs of abuse and medicinal drugs performed by liquid chromatography (LC)–ion trap mass spectrometry (MS) on autopsy samples of biological fluids did not detect any relevant presence of toxicologically relevant compounds, but ranolazine. A quantitative analysis was then carried out by liquid chromatography--triple quadrupole (LC–QqQ) MS in order to quantify ranolazine and its major metabolite o-desmethylranolazine in biological fluids and organs. Ranolazine concentrations in biological fluids were as follows: cardiac blood, 19.5 μg/mL; femoral blood, 12.3 μg/mL; bile, 0.87 μg/mL and vitreous humor, 15.4 μg/mL. For O-desmethylranolazine, the concentrations in cardiac blood, femoral blood, bile and vitreous humor were 10.7, 9.6, 11,103 and 11.4 μg/mL, respectively. The cause of death was attributed to ranolazine overdosing. To the best of our knowledge, this is the first report of a fatality associated with ranolazine, in which the postmortem distribution of ranolazine and its metabolite has been quantitatively assessed. The present study can therefore provide useful information for interpretation of the causes and mechanisms of death in ranolazine-associated fatalities.

Diazepam is a well-known psychoactive drug widely used worldwide for the treatment of anxiety, seizures, alcohol withdrawal syndrome, muscle spasms, sleeplessness, agitation, and pre/post-operative sedation. It is part of the benzodiazepine family, substances known to primarily act by binding and enhancing gamma-aminobutyric acid (GABAShort-term (2.5 h) and medium-term (96 h) exposures to diazepam were performed at drug concentrations of 0.8, 1.6, 16, and 160 μg/L. Intervention groups were compared with a vehicle control group. Each group consisted of 20 zebrafish eggs/larvae. Metabolites related with neurotransmission were determined by ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS).Thirty-six compounds were quantified. Significantly increased tryptophan and serotonin concentrations were found in the intervention groups receiving higher doses of diazepam in 2.5 h exposure (p 0.05 control versus intervention groups). Tyrosine concentrations were higher (p 0.05) at higher concentrations in 2.5 h exposure, but lower (p 0.05) at higher concentrations in 96 h exposure. Both phenylalanine and aspartic acid concentrations were higher (p 0.05) at higher doses in 2.5 h and 96 h exposure.Short- and medium-term exposures to diazepam induce dose- and time-dependent metabolomic alterations associated with the serotonergic, dopaminergic/adrenergic, and aspartic acid neurotransmitter systems in zebrafish.

The biomarkers can provide objective and relevant information for the clinical and/or forensic diagnosis of alcohol abuse. Many researchers spent efforts not only to find new markers of alcohol abuse, but also to verify in real conditions the specificity and sensitivity limits of the existing biomarkers and to optimize the analytical methodologies for their determination. The great attention dedicated by clinical and forensic researchers is proved by the several hundreds of papers published on this subject, which can be retrieved in the main databases using the key words “alcohol abuse” and “biomarkers.” A general classification of the markers of alcohol abuse includes two sub-groups: “trait markers” and “state markers.” The most typical biomarkers for the diagnosis of alcohol abuse include gamma-glutamyltransferase, mean corpuscular volume, liver enzymes, aspartate-transaminase and alanine-transaminase, and carbohydrate-deficient transferrin (CDT). In the clinical context, CDT in longitudinal studies proved suitable for the identification of relapses after a period of abstinence.

Introduction and aim Carbohydrate Deficient Transferrin (CDT) is one of the most used biomarkers for monitoring alcohol use in pregnancy. However, its effective application in this context is hampered by the demonstrated physiological progressive increase during pregnancy (even in abstinent women) of CDT values, which in the third trimester can reach values close or exceeding the cut-offs usually adopted in clinical and forensic diagnostics. The present work was aimed at the re-assessment of CDT reference values in pregnancy. Materials and methods The CDT analysis was performed by a validated HPLC-UV Vis method on 284 serum samples of women with a physiological pregnancy and on 370 sera of non-pregnant woman from the general population (control group). All the samples were tested also for GGT for excluding alcohol abuse. The statistical analysis was performed using the MedCalc® Statistical Software. Results The re-definition of the specific reference concentrations was carried out according to the Horn and Pesce Robust Method. The resulting CDT upper reference values were 1.45%, 2.01% and 2.05% in the first, second, and third trimester, respectively. Conclusions In order to prevent the development of maternal and fetal prenatal alcohol exposure complications, the use of alcohol biomarkers, including CDT, has been proposed. However, this biomarker, in the monitoring of alcohol use in pregnancy, has so far been applied adopting the same cut-off used for general population without taking into consideration the progressive physiological increase of its value throughout the pregnancy. In the present study, a specific re-assessment of the CDT reference concentrations of each trimester is reported.

The increased diffusion of the so-called novel psychoactive substances (NPS) and their continuous change in structure andconceivably activity has led to the need of a rapid screening method to detect their biological effects as early as possible after their appearance in the market. This problem is very felt in forensic pathology and toxicology, so the preclinical study is fundamental in the approach to clinical and autopsy cases of difficult interpretation intoxication. Zebrafish is a high-throughput suitable model to rapidly hypothesize potential aversive or beneficial effects of novel molecules. In the present study, we measured and compared the behavioral responses to two novel neuroactive drugs, namely APINAC, a new cannabimimetic drug, and methiopropamine (MPA), a methamphetamine-like compound, on zebrafish larvae (ZL) and adult mice. By using an innovative statistical approach (general additive models), it was found that the spontaneous locomotor activity was impaired by the two drugs in both species: the disruption extent varied in a dose-dependent and time-dependent manner. Sensorimotor function was also altered: i) the visual object response was reduced in mice treated with APINAC, whereas it was not after exposure to MPA; ii) the visual placing responses were reduced after treatment with both NPS in mice. Furthermore, the visual motor response detected in ZL showed a reduction after treatment with APINAC during light-dark and dark-light transition. The same pattern was found in the MPA exposed groups only at the dark-light transition, while at the transition from light to dark, the individuals showed an increased response. In conclusion, the present study highlighted the impairment of spontaneous motor and sensorimotor behavior induced by MPA and APINAC administration in both species, thus confirming the usefulness of ZL as a model for a rapid behavioural-based drug screening.

New generations of synthetic cannabinoids (SCs) have recognized strong psychoactive effects, rapidly becoming potent drugs of abuse. Characterization of metabolites associated with SCs is useful for their subsequent detection in biological samples. We aimed to investigate the metabolism of 5F-APINAC, a novel SC, in human liver microsomes (HLMs) (in vitro) and in a rat model (in vivo). For the in vitro study, the standard solution of 5F-APINAC was incubated with HLMs for 1 h at 37 °C. In the in vivo study, rats received 15 mg/kg of 5F-APINAC diluted in ethanol or in dimethyl sulfoxide, while a vehicle control group received placebo. Urine was collected 3, 6, and 24 h after administration. The metabolic characterization was performed using liquid chromatography–ion trap mass spectrometry and liquid chromatography–quadrupole time-of-flight mass spectrometry. In total, 15 metabolites associated with 5F-APINAC were tentatively identified. The metabolites were classified as: 1-adamantol metabolites, oxidative defluorination metabolites, and N-fluoropentylindazole-3-carboxylic acid metabolites. The modifications included ester hydrolysis, mono-, di-, and trihydroxylation of adamantyl ring and N-fluoropentylindazole moiety, oxidation (carbonyl formation) of the N-fluoropentyl side chain, oxidative loss of fluorine, and glucuronidation, as well as combinations thereof. The predominant metabolic reaction was ester hydrolysis in both in vitro and in vivo experiments and formation of M9 (5-fluoropentylindazole-3-carboxylic acid). However, the most recommendable metabolites for proving 5F-APINAC consumption in urine were M4.1, M7, and M13. No metabolites were detected in rat’s urine 24 h after drug administration. The discovered metabolites are proposed to be incorporated into routine screening analytical methods as the urine markers of 5F-APINAC consumption. This is the first report to demonstrate the metabolism of 5F-APINAC to our knowledge.

The toxicological approach for monitoring Driving Under Influence of Drugs (DUID) requires analytical techniques with a broad spectrum of identification coupled to a high analytical sensitivity. In this context immunological methods are generally used, while GC or LC-MS are applied for the confirmation step. A different approach for drug screening is represented by the Toxtyper™ instrumentation, an LC-MS platform equipped with a high-speed ion trap mass analyzer, provided with ready-to-use protocols and a database of as many as 4500 therapeutic, toxic/illicit drugs and metabolites. The aim of the present work was to verify its performances in real conditions of drug screening of human serum in the context of DUID. To test and compare its analytical performances, four pooled serum samples were fortified with a selected panel of 47 drugs and metabolites. The agreement between the results from the ToxtyperTM and from the confirmatory techniques currently in use at the University of Verona (GC and LC-MS) was investigated by analyzing 90 real samples chosen from those routinely analyzed. The present study highlights the suitability of the ToxtyperTM for drug screening in serum with a sensitivity compatible with the needs of the DUID for all the tested compounds, with the only exception of cannabinoids.

The polysialylated isoform of the neural cell adhesion molecule (PSA-NCAM) has been shown to be a key player in neuroplastic changes and is expressed in various disorders. We investigated the PSA-NCAM expression on brain cortical tissue in a cohort of drug-related deaths. Brains from 25 drug abusers and 10 control subjects were removed at autopsy, and 2 samples of the right parietal lobe of each case were obtained. The polysialylated isoform of NCAM was evaluated on formalin-fixed and paraffin-embedded tissues. Eleven patients were polydrug abusers; 14 used a single substance. The mechanisms of death were acute respiratory failure (n = 19), cardiorespiratory failure (n = 4), acute heart failure (n = 1), and brain injury (n = 1). Toxicological analyses of blood were available for all cases, and urine and bile analyses for 19 of 25 cases. The polysialylated isoform of NCAM immunoexpression in the neuronal soma and dendritic spines was observed in 18 (72%) of 25 drug abusers and in 2 (20%) of 10 control subjects. Drug abusers were statistically more positive for PSA-NCAM than control subjects (P = 0.0082). The expression of PSA-NCAM in the parietal cortex could be an indicator of brain damage due to drug abuse, and its availability could allow the forensic pathologists to develop rapid and low-cost additional or alternative method to improve detection of drug-related deaths.

Accurate dating of bone fractures can be crucial in the context of forensic trauma analysis since it may provide essential information for the corroboration or contradiction of statements by victims or suspects in cases of domestic abuse or torture. The different stages of bone healing have been well described in the existing literature, and some previous studies attempted to define related timelines particularly in the paediatric population. However, the bone healing process can be very variable and despite the importance of the topic in the forensic field, so far little is known about the radiological appearance of bone fractures at different healing stages, and how this correlates with time, especially in an adult population. The aim of this retrospective observational study was to describe the temporal aspect of pre-defined healing stages as they appear on radiographs of tubular bone fractures in adults, and explore the effect of potential cofounding variables. A dynamic nomogram was developed as a user-friendly tool to be eventually applied in clinical or medico-legal settings. This study showed that the posttraumatic time interval (PTTI) increased progressively with the pre-defined healing stages. However, confounding factors, such as patients' age, sex, and location of the fracture need to be accounted for in the final estimation model. Further studies are needed to explore more potentially confounding variables to refine the presented outcomes. Better knowledge of the effect of different confounding variables in the dating of fracture healing will contribute to greater accuracy of PTTI estimation of bone fractures in adults.

The recent appearance of APINAC (AKB-57, ACBL(N)-018, adamantan-1-yl 1-pentyl-1H-indazole-3-carboxylate) in the market of the so-called novel psychoactive substances resulted in the need of defining its characteristics and searching its metabolites for subsequent detection in biological samples. The structure of the APINAC molecule has great similarity to the molecules of other synthetic cannabinoids. Here we report on the in vivo metabolism of APINAC using rats as an experimental model. Rat urine samples were analyzed by using gas chromatography–mass spectrometry and liquid chromatography–high resolution mass spectrometry. Data were acquired via time-of-flight mass scan, followed by Auto MS and triggered product ion scans. The predominant metabolic pathway for APINAC was ester hydrolysis yielding a wide variety of N-pentylindazole-3-carboxylic acid metabolites and 1-adamantanol metabolites. Ten metabolites for APINAC were identified, with the majority generated by hydroxylation, carbonylation, and carboxylation with or without glucuronidation. Therefore, in vivo metabolic profiles in rats were generated for APINAC. N-Pentylindazole-3-carboxylic acid, hydroxylated N-pentylindazole-3-carboxylic acid, and 1-adamantanol are likely the best targets to incorporate into analytical screening methods for drugs analysis. The presented mass spectra and retention time data may be useful for detection of these compounds in human urine.

INTRODUCTION: This study aimed to establish whether an alcoholic antiseptic, wiped or not before venipuncture, may jeopardize alcohol testing with a commercial enzymatic assay and a reference head-space gas chromatography (GC) technique. MATERIALS AND METHODS: Venous blood was collected from 23 healthy volunteers, with two sequential procedures. In the first blood collection, 2 mL of alcoholic antiseptic (0.5% chlorhexidine, 70% ethanol) were place on a gauge pad, the venipuncture site of right arm was cleaned but the antiseptic was not let to dry before phlebotomy. In the second blood collection, 2 mL of the same alcoholic antiseptic were placed on another gauge pad, the venipuncture site of left harm was cleaned and the antiseptic was accurately cleansed before phlebotomy. Ethanol was measured with a reference GC technique in whole blood and EDTA plasma, and a commercial enzymatic assay in EDTA plasma. RESULTS: No subject complained about feeling a particular itchy sensation when the alcohol was not wiped before puncturing the vein. The concentration of alcohol in all EDTA plasma samples was always lower than the limit of detection of the enzymatic assay (i.e., 2.2 mmol/L ; 0.1 g/L). Similarly, alcohol concentration was also undetectable using a reference GC technique (i.e., < 0.22 mmol/L ; 0.01 g/L) in EDTA plasma and whole blood. CONCLUSION: It seems reasonable to conclude that using ethanol-containing antiseptics before venipuncture may not be causes of spurious or false positive results of alcohol measurement at least when ideal venipunctures can be performed.

IntroductionAutophagy plays a role in various central nervous system diseases. Little is known about its molecular activation in drug addiction. Our aim was to investigate the signalling pathways of autophagy in brain tissues from drug abusers.MethodsTwenty-five drug abusers with acute lethal intoxication and 10 controls were medico-legally autopsied. Brain-tissue samples from the parietal cortex and cerebellum were obtained. Expression of LC3B, phospho-mTOR (ph-mTOR) and phospho70S6 Kinase (p70S6K) was identified in tissue microarrays, with three tissue spots per case. Blood, urine or vitreous humour were tested in all cases to identify the acute intoxication. Hair analysis was performed in 14 cases to confirm chronic intoxication; the remaining cases had a documented medical history of chronic abuse.ResultsThe autophagy marker LC3B was always positive on both the cortex and the cerebellum, stratified as strongly in 18 (72%) cases and weakly positive in seven (28%) cases. ph-mTOR was negative in all cases. The p70S6K molecule showed positivity in 14 (56%) cases on cortex tissue. The cerebellum was always negative, except for Purkinje cells. Drug abusers had statistically more double positive cases (LC3B–p70S6K) than controls ( p=0.0094).ConclusionAutophagy pathways were activated in our series, and 56% of drug abusers showed simultaneous LC3B–p70S6K immunoexpression on tissue from the parietal cortex and cerebellum. This may be of value in autopsy practice as an indicator of brain damage due to drug abuse and could serve as alternative or additional double sensitive diagnostic method to detect drug-related deaths using a tissue-based rationale.

BACKGROUND Erythrocyte mean corpuscular volume (MCV) has been used for decades as a biomarker of chronic alcohol abuse and in the treatment of alcohol dependence. More recently, it has also been adopted to investigate the fitness of subjects to hold the driving license to prevent traffic accidents. So far, however, the studies on the association of MCV with an increased risk of alcohol-associated car accidents are extremely scarce, if not totally absent. To the best of our knowledge, the present work is the first specifically aimed at studying a plausible association between elevated MCV and crash accidents correlated with alcohol abuse. METHODS A total of 6,244 drivers involved in traffic accidents underwent mandatory laboratory analyses including blood alcohol concentration (BAC) determination and MCV analysis. BAC and MCV determinations were performed by headspace gas chromatography and complete blood count, respectively. RESULTS The chi-square test evaluating the proportions of subjects with elevated MCVs (>95 fl) yielded a highly significant result (χ2 = 68.0; p 0.5 g/l). However, when considering only drivers showing BACs in the range of 0.51 to 1.5 g/l, the frequencies of elevated MCV values are fairly comparable (χ2 = 0.062, p = 0.80). In contrast, limiting the evaluation to BACs > 1.5 g/l, the frequency of elevated MCVs raised to 19.1% (χ2 = 58.9, p value

Synthetic cannabinoids are abused substances with strong psychoactive effects. Little is known about the effects on neurotransmission and the toxicity of the second-generation cannabinoid 5F-APINAC. The objective was to assess the influence of short- and long-term exposures of 5F-APINAC on metabolites associated with neurotransmission on zebrafish.Short-term ("acute", 4 h) and long-term ("chronic", 96 h) exposures to 5F-APINAC were performed at 0.001, 0.01, 0.1, 1.0 and 10 μM. Intervention groups were compared with a vehicle control. Each group n = 20 zebrafish eggs/larvae. Metabolites related to neurotransmission were determined.In chronic exposure, larvae exposed to 10 μM 5F-APINAC presented morphological and developmental alterations. GABA had the lowest concentrations at higher exposure in acute (p 0.01) and chronic (p 0.001) experiments. Glutamine showed a descending trend in the acute experiment, but an ascending trend in the chronic exposure (p 0.05). In chronic exposure, tryptophan presented an overall descending trend, but with a neat increase at 10 μM 5F-APINAC (p 0.001). Tryptamine in acute exposure presented lower (p 0.05) concentrations at higher doses. Dopamine and acetylcholine presented highest (p 0.05) concentrations in the acute and chronic exposures, but with a drop at the highest doses in the chronic experiments. In chronic exposure, xanthurenic acid decreased, except for the highest dose. Picolinic acid was increased at the highest doses in the chronic experiment (p 0.001).Short- and long-term exposures induced metabolomic alterations associated with the gamma-aminobutyric acid/glutamic acid, dopaminergic/adrenergic, cholinergic neurotransmitter systems, and the kynurenine pathway. Chronic exposure at 10 μM 5F-APINAC was associated with embryotoxicity confirmed by teratogenesis.

Essentials The reliability of platelet tests as markers of the variable bioavailability of clopidogrel is not yet defined. Kinetics of clopidogrel active metabolite (CAM) and platelet response were studied in ischemic heart disease. CAM plasma maximum concentration (Cmax) predicted vasodilator-stimulated phosphoprotein (VASP-P). Timely performed VASP-P, not an aggregation-based test, may be a surrogate for clopidogrel bioavailability. SummaryBackground The high inter-individual variability in the inhibition of platelet function by clopidogrel is mostly explained by high variability in its transformation to an active metabolite (CAM). Objective We investigated the relations between pharmacokinetics and pharmacodynamics of CAM by comparing two methods of platelet function. Methods We enrolled 14 patients undergoing percutaneous coronary interventions for non-ST-segment elevation acute coronary syndrome or inducible myocardial ischemia. Plasma concentrations of clopidogrel and CAM, phosphorylation of vasodilator-stimulated phosphoprotein (VASP-P), expressed as a platelet reactivity index (PRI) and whole-blood platelet aggregation (multiple electrode aggregometer, MEA) were measured before and after a 600-mg clopidogrel loading dose (nine time-points) and before and after 75-mg maintenance doses on days 2, 7 and 30. Results Plasma concentrations of clopidogrel and CAM were highly variable. CAM reached maximal concentration (Cmax) (median, 110.8 nm; range, 41.9–484.8) 0.5–2 h after the loading dose. A sigmoid dose‒response curve defined the relations between CAMCmax and PRI after 3 to 24 h (IC50, 459.6 nm; 95% confidence interval, 453.4–465.7; R2 = 0.82). PRI was unchanged from baseline in patients with the lowest CAMCmax (< 83 nm, n = 7), indicating low sensitivity of VASP-P. PRI values were also predicted by CAMCmax at days 2, 7 and 30. Platelet aggregation measured by MEA did not show significant relations with either PRI or with CAM pharmacokinetics at any time-point. Conclusions After 600 mg clopidogrel, VASP-P, but not whole-blood platelet aggregation measured by MEA, is almost entirely predicted by CAMCmax. VASP-P could be useful in studies aimed at investigating relations between CAM bioavailability and clinical events.

Cases of subtle fatal neck compression are often complicated by the lack of specificity of the post-mortem signs of asphyxia and by the lack of clear signs of neck compression. Herein we present a forensic case of a 45-year-old schizophrenic patient found on the floor of the bedroom of a psychiatric ward in cardiopulmonary arrest and who died after two days in a vegetative state. The deposition of the roommate of the deceased, who claimed responsibility for the killing of the victim by neck compression, was considered unreliable by the prosecutor. Autopsy, toxicological analyses, and multi-slice computed tomography (MSCT), micro computed tomography (micro-CT) and histology of the larynx complex were performed. Particularly, micro-CT analysis of the thyroid cartilage revealed the bilateral presence of ossified triticeous cartilages and the complete fragmentation of the right superior horn of the thyroid, but it additionally demonstrated a fracture on the contralateral superior horns, which was not clearly diagnosable at MSCT. On the basis of the evidence of intracartilaginous laryngeal hemorrhages and bilateral microfracture at the base of the superior horns of the larynx, the death was classified as a case of asphyxia due to manual strangulation. Micro-CT was confirmed as a useful tool in cases of subtle fatal neck compression, for the detection of minute laryngeal cartilage fractures, especially in complex cases with equivocal findings on MSCT.

Many homemade tamper processes of medical codeine formulations are available on selected “forums” on the Internet, where recreational codeine users claim to be able to purify codeine by removing additives, such as acetaminophen, to avoid or limit adverse effects. In this work, it is reported and discussed a fatal case of codeine intoxication. The findings of objects such as jars, filters, and tablets, and amounts of unknown liquid material at the death scene investigation suggested a fatal codeine intoxication after the tampering procedure called “cold water extraction.” Toxicological results obtained from the analysis of both the nonbiological material and the body fluids of the decedent integrated with the information collected at the death scene investigation confirmed the above-mentioned hypothesis. This report underlines the importance of a tight interconnection between criminalistics and legal medicine to strengthen the identification of the cause of death and the reconstruction of the event.

The chemical and biochemical analysis of bodily fluids after death is an important thanatochemical approach to assess the cause and time since death. Vitreous humor (VH) has been used as a biofluid for forensic purposes since the 1960s. Due to its established relevance in toxicology, a literature review highlighting the use of VH with an emphasis on endogenous compounds has not yet been undertaken. VH is a chemically complex aqueous solution of carbohydrates, proteins, electrolytes and other small molecules present in living organisms; this biofluid is useful tool for its isolated environment, preserved from bacterial contamination, decomposition, autolysis, and metabolic reactions. The post-mortem analysis of VH provides an important tool for the estimation of the post-mortem interval (PMI), which can be helpful in determining the cause of death. Consequently, the present review evaluates the recent chemical and biochemical advances with particular importance on the endogenous compounds present at the time of death and their modification over time, which are valuable for the PMI prediction and to identify the cause of death.

The phosphoinositide (PI) signal transduction pathway participates in liver metabolism. Abnormal activity or expression of PI-specific phospholipase C (PLC) enzymes has been described in different liver diseases. We resume the role of the PI metabolism in liver and PLC abnormalities in different liver diseases. Moreover, we present the results of PLC analyses in a normal human liver and an alcohol-damaged liver. PLC enzymes and the expression of the corresponding genes in liver biopsies from individuals deceased for complications of the alcoholic liver disease (ALD) at different stages compared with normal controls (deceased individuals with histologically normal livers without alcohol addiction anamnesis) were analyzed by using immunohistochemistry and molecular biology techniques. The expression panel of PLCs was described in normal and alcohol abuse liver. Our observations suggest that the regulation of PLC expression might be due to posttranscriptional events and that alcohol affects the epigenetic control of PLC expression belonging to PI signaling. We also describe the alternate expression of PLCB1 and PLCH1 genes in liver. Our results corroborate literature data suggesting that PLC enzymes are differently expressed in normal versus pathological liver, playing a role in the histopathogenesis of liver tissue damage. The expression and/or localization of selected PLC isoforms is especially affected in alcohol-related liver tissue histopathology. Our present observations confirm that the modulation of protein synthesis plays a role in the regulation of PLC enzymes. We also suggest that this modulation might act at the transcription level. Further studies are required to investigate related epigenetic mechanisms.

Novel psychoactive substances are intoxicating compounds developed to mimic the effects of well-established drugs of abuse. They are not controlled by the United Nations drug convention and pose serious health concerns worldwide. Among them, the dissociative drug methoxetamine (MXE) is structurally similar to ketamine (KET) and phencyclidine (PCP) and was created to purposely mimic the psychotropic effects of its “parent” compounds. Recent animal studies show that MXE is able to stimulate the mesolimbic dopaminergic transmission and to induce KET-like discriminative and rewarding effects. In light of the renewed interest in KET and PCP analogs, we decided to deepen the investigation of MXE-induced effects by a battery of behavioral tests widely used in studies of “safety-pharmacology” for the preclinical characterization of new molecules. To this purpose, the acute effects of MXE on neurological and sensorimotor functions in mice, including visual, acoustic and tactile responses, thermal and mechanical pain, motor activity and acoustic startle reactivity were evaluated in comparisons with KET and PCP to better appreciate its specificity of action. Cardiorespiratory parameters and blood pressure were also monitored in awake and freely moving animals. Acute systemic administrations of MXE, KET and PCP (0.01–30 mg/kg i.p.) differentially alter neurological and sensorimotor functions in mice depending in a dose-dependent manner specific for each parameter examined. MXE and KET (1 and 30 mg/kg i.p.) and PCP (1 and 10 mg/kg i.p.) also affect significantly cardiorespiratory parameters, systolic and diastolic blood pressure in mice.

Aim Worldwide, drug exposure in utero and in early childhood is a well-known major health issue. Hair analysis, taking into consideration its wide detection window, represents the most suitable investigative tool for these conditions. In this particular field, hair analysis of the parents, in addition to that of children, looks also important. The present study shows the results from 36 cases of suspected drug exposure in utero or in early childhood in which hair analysis of newborns/children and/or of parents has been carried. Materials and methods According a protocol established between the institute of forensic medicine and the institute of pediatrics of the university hospital of Verona, in case of suspected exposure to drugs in utero or in early childhood the physician asked the parents to give the consent to collect hair samples from the newborn or child as well as from the parents themselves to perform a toxicological evaluation of the case. In the years 2016–2018, overall 36 cases (26 in utero and 10 in early childhood) were identified, in 30 of which (20 in utero and 10 in early childhood) the parents gave the consent to collect hair samples. After digestion under acidic conditions, the hair samples underwent screening analysis by LC-MS/MS for opiates, cocaine, methadone, amphetamines, ketamine. The positive samples at the screening analysis underwent, after SPE extraction, a confirmation analysis by GC-MS. For the determination of cannabinoids, after digestion under basic conditions, the hair samples underwent LLE and GC-MS/MS analysis. In the case of parental hair analysis, a segmental study was carried out, when possible. Results Out of 20 in utero exposure cases, in 18 cases the consent to collect newborn hair samples was given. Fifteen (83%) out of these 18 cases were positive for the presence of illicit drugs: 66% for only 1 compound (5 for cannabinoids, 3 for cocaine and 2 for opiates) and 34% for 2 substances (4 for cocaine + opiates, 1 for cocaine + cannabinoids). In the two cases in which the parents did not provide the consent to collect hair samples from the newborn, it was possible to obtain hair samples from the mother, which resulted positive for cocaine and cannabinoids, respectively. Out of the 15 positive newborn cases, in 11 cases it was also possible to study the mother's hair by using a segmental analysis approach including segments referable to the pregnancy period and segments grown before the pregnancy. In all the cases, all the segments resulted positive for the presence of illicit drugs, sometimes with a decrease of concentrations in the segments referable to the last months of pregnancy. All the drugs found in the newborn hair were confirmed in the mother's hair but not vice versa. Out of the 10 early childhood cases, 7 (70%) resulted positive for the presence of illicit drugs (3 for cannabinoids, 3 for cocaine, 1 for cocaine and opiates). Out of these 7 cases, in 5 cases it was possible to study the hair of the parents which resulted positive for the same substances. Conclusions Although often criticized as affected by environmental contamination, hair analysis of newborns and children, is very useful, particularly if integrated with the Results from the parents, to study the environment were the children live and consequently to take adequate actions for the protection of the minors.

The natural mummy of Cangrande della Scala was exhumed from its tomb in the church of Santa Maria Antiqua in Verona and was submitted to a multidisciplinary study, that included archaeological, palaeopathological, palynological, toxicological and historical investigation. The body of Cangrande, still wearing his precious clothes, was in a good state of preservation. Palynological analyses demonstrated the presence of pollen grains of Digitalis sp./foxglove in the rectum content, along with Matricaria chamomilla/chamomille and Morus nigra/black mulberry. Toxicological analyses showed toxic concentrations of digoxin and digitoxin, two Digitalis glycosides, in the liver and faeces samples. Both palynological and toxicological data suggest an intoxication through the oral administration of an infusion or decoction of leaves and flowers of Digitalis. Cangrande died on July 22 1329, four day after his triumphal entrance in the city of Treviso. The sudden death was preceded by vomit and diarrhoea with fever that, according to written documents, he had contracted a few days before by “drinking from a polluted spring”. The gastrointestinal symptoms manifested by Cangrande in his last hours of life are compatible with the early phase of Digitalis intoxication and the hypothesis of poisoning is mentioned by some local historical sources. The palaeopathological analyses confirm a Digitalis poisoning. The most likely hypothesis on the causes of death is that of a deliberate administration of a lethal amount of Digitalis. Although several cases of poisoning through the use of organic substances are known from historical sources, no other direct evidences are documented in the palaeopathological literature.

Although recent Cannabis use is widely reported to be associated with drug-related traffic accidents, the evidence that Cannabis users show an increased risk of being involved in road crashes is still not unequivocally proved. The purpose of the present work is to provide an objective assessment of this hypothesis, by comparing the frequency of occurrence of positive urine analyses in drivers involved in traffic accidents (n = 1406) with that observed in a control population undergoing mandatory urine drug testing (n = 1953). Urine analyses for drugs of abuse were performed by screening immunometric techniques followed by confirmation with UHPLC-QQQ MS, adopting a cut-off concentration for THC-COOH of 15 ng/mL. A case was classified as "positive" when a driver admitted to hospital for road traffic injuries showed urine concentrations of THC-COOH higher than the cut-off. All samples showing positive results for any other controlled drug in urine or blood alcohol concentrations0.5 mg/mL were excluded from the study. Subjects positive to THC-COOH, and negative to all the other tested substances were 116 in Group 1 (8.2%) and 16 in Group 2 (0.8%). Subjects resulting negative to any tested substances were 1290 in Group 1 and 1937 in Group 2. The frequency of THC-COOH detection in the two groups was compared by using the "chi square" test, which resulted = 119.57, i.e. highly significant (P 0.01). The Odds Ratio of the two groups was =10.88, showing a high degree of association between the presence of THC-COOH in urine and the occurrence of traffic accidents (P 0.0001). The presented data, proving a high degree of association between Cannabis use and the occurrence of traffic accidents with injuries of the driver, support the use of urine testing for Cannabis in the procedures for the issuing of the driving licence, particularly in the case of subjects formerly or presently using Cannabis. This finding looks even more relevant in the present times, because of the increasing success of the policies of legalization of Cannabis for medical and non-medical purposes.

The need for investigating alcohol abuse by means of objective tools is worldwide accepted. Among the currently available biomarkers of chronic alcohol abuse, carbohydrate-deficient transferrin (CDT) is one of the most used indicator, mainly because of its high specificity. However, some CDT analytical and interpretation aspects are still under discussion, as witnessed by numerous research papers and reviews. The present article presents a critical review of the literature on CDT appeared in the period from 2007 to 2017 (included). The article is organized in the following sections: (1) introduction, (2) pre-analytical aspects (3) analytical aspects (4) diagnostic aspects (5) concluding remarks. As many as 139 papers appeared in the international literature and retrieved by the search engines PubMed, Web of Science and Scopus are quoted.

Psoriasis is a chronic skin disease associated with deregulated activation of immune cells and keratinocytes. In this study, we used the imiquimod (IMQ)-induced mouse model of psoriasis to dissect better the contribution of hematopoietic and skin-resident stromal cells to psoriasis development. The comparison of disease development in mice carrying the hematopoietic cell-specific deletion of MyD88 (Myd88fl/flVav-cre+ mice) with mice carrying the total MyD88 deficiency (Myd88−/− mice), we show that the progression of skin and systemic inflammation, as well as of epidermal thickening, was completely dependent on MyD88 expression in hematopoietic cells. However, both Myd88−/− mouse strains developed some degree of epidermal thickening during the initial stages of IMQ-induced psoriasis, even in the absence of hematopoietic cell activation and infiltration into the skin, suggesting a contribution of MyD88-independent mechanisms in skin-resident stromal cells. With the use of conditional knockout mouse strains lacking MyD88 in distinct lineages of myeloid cells (Myd88fl/flLysM-cre+ and Myd88fl/flMRP8-cre+ mice), we report that MyD88 signaling in monocytes and Mϕ, but not in neutrophils, plays an important role in disease propagation and exacerbation by modulating their ability to sustain γδ T cell effector functions via IL-1β and IL-23 production. Overall, these findings add new insights into the specific contribution of skin-resident stromal vs. hematopoietic cells to disease initiation and progression in the IMQ-induced mouse model of psoriasis and uncover a potential novel pathogenic role for monocytes/Mϕ to psoriasis development.

Dextromethorphan is the d-isomer of 3-methoxy-N-methylmorphinan (methorphan), a synthetic analogue of codeine. It interacts with the σ-opioid receptors with resulting antitussive activity, while it does not show any significant affinity for the μ and δ opioid receptors, which are responsible for analgesic and central nervous system depressant effects. On this basis, d-methorphan is widely used all over the world as an over-the-counter cough suppressant drug. Indeed, there are hundreds of cough and cold preparations containing d-methorphan alone or in combination with analgesic (acetaminophen, acetyl salicylic acid), decongestant (phenylephrine, pseudoephedrine), anthistaminic (chlorpheniramine, brompheniramine, pheniramine), and/or expectorant mucolytic agents. In large doses, d-methorphan and its metabolite dextrorphan antagonize the actions of excitatory amino acids on N-methyl-d-aspartate (NMDA) receptors as do phencyclidine and ketamine. This action is responsible for the hallucinogenic and dissociative effects experienced by the subjects who abuse d-methorphan. Moreover, both d-methorphan and its metabolite dextrorphan are reported to inhibit reuptake of serotonin, and to have competitive 5HT1 agonist activity. On this basis, d-methorphan abuse could lead also to serotonin syndrome. Actually, many papers report fatal and non-fatal cases of abuse of d-methorphan especially among teenagers. Levomethorphan is the l-isomer of 3-methoxy-N-methylmorphinan and, unlike d-methorphan, is a potent narcotic analgesic. For this reason it is not commercially available and in many countries, including Italy, it is listed as a controlled substance. Moreover, the hepatic O-demethylation of l-methorphan produces levorphanol, which is a pure opioid agonist used to treat severe pain. Levorphanol has the same properties as morphine with respect to the potential for habituation, tolerance, physical dependence and withdrawal syndrome, but it is 4–5 times more potent than morphine and has a longer half-life. Starting from 2010, the Italian National Early Warning System (NEWS, Department of Antidrug Policy, Presidency of the Council of Ministers) collected numerous warnings from national collaborating centres (forensic laboratories, law enforcement, health services, research centres) related to heroin containing methorphan. Moreover, the use of methorphan as adulterant is reported in recent scientific literature. Both notifications and scientific papers often report methorphan as an adulterant, since the analytical methods used for heroin analysis cannot distinguish between the two stereoisomers. On the other hand, some papers report the identification of dextromethorphan because the gas chromatography–mass

Background The determination of Carbohydrate Deficient Transferrin (CDT) in a forensic context should be based on the use of a screening technique followed, for the “positive samples”, by a confirmatory technique. The aim of this study was to compare the two most used automated screening methods for CDT analysis, immuno-nephelometric assay (INA) and multi-capillary electrophoresis (mCE), with a validated HPLC procedure, used as confirmation test, in order to re-evaluate the cut-off concentrations of the screening methods. Methods 195 serum samples underwent CDT analysis by using the N Latex CDT direct immuno-nephelometric assay, the multicapillary system Capillarys™ and an anion exchange HPLC method with UV-visible detection at 460 nm developed and validated at our laboratories. Statistical analyses were performed by using Bland–Altman plots and ROC curves. Results and discussion The 95% limits of agreement were ± 0.94% when comparing INA and HPLC and ± 0.60% when comparing mCE and HPLC. The ROC analysis of both INA and mCE, using HPLC as the reference method, showed that no false negative results were found when the cut-off was fixed to 1.2% for mCE and to 2.3% for INA. Conclusions The study showed a good agreement among CDT determinations carried out either with mCE or INA or HPLC. However, the usual cut-offs of both mCE (1.3%) and INA (2.5%) should be lowered to minimize false negatives at the screening analysis.

To analyze the morphology and depth of stab wounds experimentally produced on human legs amputated for medical reasons using multislice computed tomography (MSCT) and magnetic resonance imaging (MRI) after the instillation of a single contrast medium solution (CMS). For morphological analysis, MSCT and MRI scans were performed before and after the instillation of CMS into the wound cavity. Depth measurements were performed on the sagittal view only after CMS instillation. Subsequently, each wound was dissected using the layer-by-layer technique and the depth was measured by a ruler. One-way between-groups pairwise analysis of variance (ANOVA) and Bland–Altman plot analysis were used for comparing radiological and anatomical measurements. Unenhanced MSCT images did not identify the wound channels, whereas unenhanced MRI evidenced the wound cavity in 50 % of cases. After the instillation of CMS, both MSCT and MRI depicted the wound channel in all the investigated stabbings, although the morphology of the cavity was irregular and did not resemble the shape of the blade. The radiological measurements of the wounds’ depth, after the application of CMS, exhibited a high level of agreement (about 95 % at Bland–Altman plot analysis) with the anatomical measurements at dissection. A similar systematic underestimation, however, has been evidenced for MSCT (average 11.4 %; 95 % CI 7–17) and MRI (average 9.6 %; 95 % CI 6–13) data after the instillation of CMS with respect to wound dissection measurements. MSCT and MRI after the instillation of CMS can be used for depicting the morphometric features of stab wounds, although depth measurements are affected by a slight systematic underestimation compared to layer-by-layer dissection.

In this study, a rapid and simultaneous separation of 12 synthetic cannabinoids and Δ(9)-tetrahydrocannabinol (Δ(9)-THC) in herbal blends was obtained by means of nano-liquid chromatography (nano-LC). The nano-LC experiments were performed in a 100μm i.d. capillary column packed with Cogent(®) bidentate C(18) silica particles for 25.0cm. All compounds were resolved using an isocratic elution mode in less than 30min. A mobile phase containing ACN/MeOH/H(2)O/formic acid 69/5/25/1 (v/v/v/v) was employed for the chromatographic separation. The developed analytical method was validated in terms of precision, linearity, sensitivity and accuracy. Under optimal nano-LC-UV conditions, the resulting RSD percentages for intra-day and inter-day repeatability, related to retention time and peak area, were below 2.98 and 6.40%, respectively. Limits of detection and quantification were 0.2 and 0.5μg/mL, respectively, for all the studied compounds. Linearity was assessed in the concentration range of interest for all analytes with determination coefficients r(2)≥0.9975. The method was then applied to the determination of synthetic cannabinoids in herbal blends. Quantitative analyses of the cannabimimetic compounds in six products showed that there was a wide difference in the concentration of the studied compounds among different products. Further, the nano-LC system was coupled with a mass spectrometer measuring the MS and MS-MS spectra to unequivocally identify the cannabinoids present in smoking mixtures.

The diatom test is widely used by forensic pathologists as proof of drowning, notwithstanding some criticisms mainly concerning the occurrence of false-positive results (presence of diatoms in the tissues of subjects who died from causes other than drowning). The aim of the present study was to verify the claimed inaccuracy of the method caused by an excessive rate of false-positives related to inadvertent exposure to diatoms of the general population. The study was carried out to investigate the presence of diatoms in the tissues (lungs and sternum) of subjects who died from causes other than drowning. Two groups of cadavers that underwent an autopsy at the Institute of Forensic Medicine of the University of Verona were included in the study. Group A comprised 45 individuals who died from causes other than drowning, whereas Group B comprised 20 bodies which had been recovered from water. The extraction of the diatoms was performed by incubation of samples in nitric acid for 48 hours at 60°C. The analysis of the samples from Group A showed the absence of diatoms in both lung and sternum samples. In Group B all lung samples showed the presence of diatoms, whereas only six sternum samples were shown to contain diatoms. The difference between Groups A and B was statistically highly significant. The absence of diatoms in the samples collected from Group A falsified the hypothesis that false-positive results from the diatom test may occur due to diatoms entering living bodies through the respiratory and/or digestive tracts via air, water or food, supporting the validity of the diatom test as proof of drowning.

Background: Interest has grown into the use of multidetector computed tomography (CT) and magnetic resonance imaging as an adjunct or alternative to the invasive autopsy. We sought to investigate these possibilities in postmortem CT scan using an innovative virtual autopsy approach. Methods: Twenty-five postmortem cases were scanned with the Philips Brilliance CT-64 and then underwent traditional autopsy. The images were interpreted by two blinded forensic pathologists assisted by a radiologist with the INFOPSY® Digital Autopsy Software System which provides three-dimensional images in Digital Imaging and Communications in Medicine format. Diagnostic validity of virtual autopsy (accuracy rate, sensitivity, specificity, and predictive values) and concordance between the two forensic pathologists (kappa intraobserver coefficients) were determined. Results: The causes of death at traditional autopsies were hemorrhage due to traumatic injuries (n = 8), respiratory failure (5), asphyxia due to drowning (4), asphyxia due to hanging or strangulation (2), heart failure (2), nontraumatic hemorrhage (1), and severe burns (1). In two cases, the cause of death could not be ascertained. In 15/23 (65%) cases, the cause of death diagnosed after virtual autopsy matched the diagnosis reported after traditional autopsy. In 8/23 cases (35%), traditional autopsy was necessary to establish the cause of death. Digital data provided relevant information for inferring both cause and manner of death in nine traumatic cases. The validity of virtual autopsy as a diagnostic tool was higher for traumatic deaths than other causes of death (accuracy 84%, sensitivity 82%, and specificity 86%). The concordance between the two forensic pathologists was almost perfect (>0.80). Conclusions: Our experience supports the use of virtual autopsy in postmortem investigations as an alternative diagnostic practice and does suggest a potential role as a screening test among traumatic deaths.

Carbohydrate-deficient transferrin (CDT) is a well-recognized highly specific marker of chronic alcohol abuse. The association of CDT with alcohol-related traffic accidents was evaluated to objectively validate the use of this marker for certifying the physical fitness for driving license regranting after its confiscation for drunk driving.The study was carried out on 468 injured drivers (InjDr), who underwent mandatory blood alcohol concentration (BAC) and drug analysis in biological fluids. The InjDr group was divided into 2 subgroups on the basis of BAC legal limit adopted in Italy (BAC ≤ 0.5 g/l: InjDr1 ; BAC0.5 g/l: InjDr2 ). The control group (CntDr) included 236 subjects holding safety-sensitive job positions and undergoing mandatory toxicological analyses. The determination of BAC in blood and CDT in serum were performed using validated analytical methods based on head-space gas chromatography and high-performance liquid chromatography, respectively.The evaluation of CDT distribution in the 3 groups (CntDr, InjDr1 , InjDr2 ) showed that CDT distribution in the InjDr1 group was similar to that observed in the CntDr group (p = 0.159) and different from that observed in the InjDr2 group (p0.001). Partitioning the CDT data of each group into "CDT positives" and "CDT negatives" on the basis of the cut off (1.90%), it was possible to calculate the odds of the 3 groups and then the odds ratios. The odds ratio of InjDr1 versus CntDr was 4.56 (p = 0.158), whereas the odds ratio of InjDr2 versus CntDr was 132 (p0.001). Furthermore, a dose-response effect was found only when comparing InjDr2 with CntDr.The data of the present study strongly support the use of the CDT test to evaluate the risk of a subject to be involved in a road accident while driving under the influence of alcohol.

Most assays for carbohydrate-deficient transferrin (CDT) (1)(2)(3) use cartridge extraction of CDT isoforms followed by immunoassay (4)(5). In 2001 the US Food and Drug Administration cleared the %CDT turbidimetric immunoassay (TIA; Axis Shield Plc) for detection of sustained and harmful alcohol use. Another method, based on anion-exchange HPLC separation of the CDT isoforms with direct detection at 460 nm [selective for the iron–transferrin (Tf) complex], was first developed by Jeppsson et al. (6) and adopted with minor changes by several authors, who reported advantages over immunoassays in terms of analytical selectivity, accuracy, and precision (7)(8)(9)(10). Recently released commercial reagents for HPLC analysis (Recipe®) offer advantages in interlaboratory analytical standardization. Capillary zone electrophoresis (CZE) with dynamically coated capillaries and direct ultraviolet detection at 200 nm (11)(12)(13)(14)(15)(16)(17) has also been successfully applied to CDT determination in human serum (2), and a CZE method with a proprietary capillary coating is commercially available (CEofix® CDT; Analis). Validation studies for each of these techniques have been reported in the literature, but no direct comparison studies have been published. We compared 3 commercial methods, based on TIA, CZE, and HPLC, focusing on their application to the diagnosis of chronic alcohol abuse in the forensic environment and in other areas requiring high diagnostic reliability and objectivity. CDT immunoassays were performed with a commercial reagent set (%CDT TIA) from Axis Shield on a BN2 nephelometer (Dade Behring), according to the manufacturer’s instructions. The procedure included iron saturation followed by CDT extraction onto anion-exchange disposable cartridges and a TIA. CZE was performed on a P/ACE MDQ capillary electropherograph (Beckman Coulter) with uncoated fused-silica capillaries [50 cm (total length) × 50 μm (i.d.)] obtained from Polymicro …

gamma-Hydroxybutyric acid (GHB) is a central nervous system (CNS) depressant and hypnotic which, in recent times, has shown an increasing abuse either as recreational drug (due to its euphoric effects and ability to reduce inhibitions) or as doping agent (enhancer of muscle growth). Analogues of GHB, namely gamma-butyrolactone (GBL) and 1,4-butanediol (1,4-BD), share its biological activity and are rapidly converted in vivo into GHB. At present, GHB and analogues are placed in the Schedules of Controlled Substances. Numerous intoxications in GHB abusers have been reported with depressive effects, seizures, coma and possibly death. The purpose of the present work was the development of a rapid analytical method based on capillary zone electrophoresis for the direct determination of GHB in human urine and serum at potentially toxic concentrations. Analytical conditions were as follows. Capillary: length 40 cm (to detector), 75 microm i.d.; buffer: 5.0 mM Na(2)HPO(4), 15 mM sodium barbital adjusted to pH 12 with 1.0 M NaOH; voltage: 25 kV at 23 degrees C; indirect UV detection at 214 nm; injection by application of 0.5 psi for 5 s. alpha-Hydroxyisobutyric acid was used as internal standard (IS). Sample pretreatment was limited to 1:8 dilution. Under these conditions, the sensitivity was approximately 3.0 microg/ml (signal-to-noise ratio3). Calibration curves prepared in water, urine and serum were linear over concentration ranges 25-500 microg/ml with R(2)/=0.998. Analytical precision was fairly good with R.S.D.0.60% (including intraday and day-to-day tests). Quantitative precision in both intraday and day-to-day experiments was also very satisfactory with R.S.D./=4.0%. No interferences were found neither from the most common "drugs of abuse" nor from endogenous compounds. In conclusion, capillary electrophoresis can offer a rapid, precise and accurate method for GHB determination of biological fluids, which could be important for screening purposes in clinical and forensic toxicology.

An enantioselective CE-based determination of methorphan and its main metabolites in blood is described. Enantiomeric separations were carried out in 50cm×50μm (ID) uncoated fused silica capillaries, using a background electrolyte composed of 150mM sodium phosphate pH 4.4 added with 5mM 2-(hydroxypropyl)-β-cyclodextrin and methanol 20% (v/v), at a constant voltage of 25kV. Sample injections were performed under field amplified sample stacking conditions. Detection was by recording UV absorbance at the wavelength of 200nm. Linearity of response was assessed within a concentration range from 25 to 500ng/mL for dextrometorhan, levomethorphan and their main metabolites (namely dextrorphan and levorphanol, respectively). Folcodine was used as internal standard. Under these conditions, the limit of quantification resulted 25ng/mL for each one of the analytes. The intra-day and inter-day precision, in terms of coefficient of variation (CV) were below 3.7% and 14.9 % for migration times and peak areas, respectively. The present method was successfully applied to the analysis of post-mortem blood samples from ten subjects died for heroin overdoses. Among the samples "positive" for methorphan (n=4), the d-enantiomer was found in concentrations ranging from 214 to 1282ng/mL. The concentration of its main metabolite dextrorphan in the same samples ranged from 49 to 389ng/mL.

The natural mummy of prince Cangrande, Lord of Verona, Italy (1291–1329 AD) was studied. Two samples were taken: rib bone and muscle. These samples were cleaved with trypsin and analysed by liquid chromatographic methods coupled to mass spectrometry (Q-TOF, ion-trap). Special attention was devoted to nonenzymatic protein modification––the deamidation of asparagine and glutamine. A huge amount of collagen was determined in the tissues of the mummy (covering over 80 % of the sequence)––collagen type I was identified in the rib bone and collagen types I and III in the muscle. A high overall percentage of asparaginyl and glutaminyl residues were deamidated (up to 92 %). In agreement with the literature we can suppose that the deamidation of really old samples (at least 100-years-old) is mainly dependent on the burial conditions and/or thermal age and cannot serve as a precise “molecular clock”.

The current analytical technology for the determination of New Psychoactive Substances in biological samples is still largely inadequate, because the immunoassays are unsuitable for the detection of most of these compounds and the use of traditional gas chromatography–mass spectrometry techniques is hampered by the lack of chromatographic standards and mass fragmentation patterns. Taking advantage of the molecular recognition capability of high-resolution mass spectrometry, the present work aimed to apply liquid chromatography-quadrupole-time of flight mass spectrometry for the rapid identification of New Psychoactive Substances in the hair, a peculiar tissue which “keeps memory” of the recent history of drug intake of the subject. All the samples were screened for the presence of 50 different New Psychoactive Substances (synthetic cannabinoids, cathinones and phenethylamines), substances that had been reported officially by the National Early Warning System in the period 2009–2011. Among the 435 samples analyzed, 8 were found “positive” for the following compounds: JWH-018, JWH-073, JWH-081, JWH-250, JWH-122, in a broad range of concentrations (0.010–1.28 ng/mg). Results strongly support the use of hair analysis to monitor the diffusion of new psychoactive drugs in the community.

The present paper describes an integrated diagnostic strategy to check the physical fitness of subjects, formerly users of illicit drugs, to obtain a driving license, after having quit their addiction. According to the Italian law, applicants for a driving license with a history of drug abuse must give evidence to have quit this behaviour and to show no risk of relapse in the future. To prove this, at our institute, they undergo medical examination, hair analysis and a urinalysis program on eight seriate samples, collected over about 40 days. About 700 subjects per year are investigated with this strategy. The hair samples are screened for opiates (morphine), cocaine and ecstasy, the most abused illicit substances in our region, by using commercial radioimmunoassays adopting cut-off levels of 0.1 ng/mg. All positive samples and about 10% of negatives are confirmed by high-performance liquid chromatography. Further confirmation of results can be carried out by capillary electrophoresis (and/or GC/MS or MS/MS). In 1998, the prevalence of positives for morphine, cocaine and ecstasy was 4.8, 11.3 and 2.6%, respectively. In this year, for the first time, the percentage of hair samples positive for cocaine was greater than that for opiates. The results of this integrated diagnostic strategy are presented and discussed, with particular emphasis on the comparison between hair analysis on a single sample and seriate urinalyses (on eight samples).

Hair analysis is often used in forensic toxicology to study, retrospectively, chronic exposure of individuals to drugs, and consequently newborn hair may become an ideal sample to study intrauterine exposure to xenobiotics as well as to endogenous compounds. As a tool to investigate a supposed maternal thyroxine (T4) supply to the congenital hypothyroid fetus, we devised to use the analysis of T4 extracted from newborn hair. In the present paper, the analytical method based on T4 extraction from hair followed by a radioimmunoassay is described. To verify the nature of the T4-like immunoreactive material present in newborn hair, it was further studied by HPLC fractionation with radioimmunoassay of the eluted fractions. On the basis of a clear correspondence between retention times of T4 standard and T4-immunoreactive compound extracted from hair, we assigned this immunoreactive material to T4. Then, we determined T4 hair concentrations in 19 control newborns at birth and 12 congenital hypothyroid infants at 22 days of life. Values obtained from hypothyroid infants (31.47+/-8.8 pg/mg(hair), mean+/-S.D.) were not significantly lower than those obtained from healthy newborns at birth (36.10+/-13.2 pg/mg(hair)). Such results are in agreement with the hypothesis of a maternal supply of thyroxine to the fetus through placental crossing.

The importance of the chiral analysis of amphetamine-related substances in both clandestine preparations and biological samples is widely recognized. For this purpose, capillary electrophoresis was successfully applied by several authors, but only few reports concerned ring-substituted amphetamines, which represent the main components of "ecstasy", a widely abused "recreational" substance. In the present work, the simultaneous chiral analysis of ephedrine, amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), 3-4-methylenedioxyamphetamine (MDA) and 3,4-methalenedioxyethylamphetamine (MDE) is reported, by using capillary electrophoresis with native beta-cyclodextrin (15 mM) as the chiral selector. After preliminary tests at different pH values (phosphate buffer 100 mM, pH 2.5-9.0) and with bare or coated fused-silica capillaries, the optimized conditions were: pH 2.5 phosphate, uncoated capillary (45 cm x 50 microm inner diameter), potential 10 kV. Detection was either by fixed wavelength (200 nm) or multiwavelength (190-400 nm) UV absorbance. Under these conditions, good resolution was obtained for all the analytes, with excellent chiral selectivity and efficiency. The sensitivity for the individual enantiomers was better than 0.2 microg/mL, analytical precision was characterized by relative standard deviation values < 0.8% (< or = 0.15% with internal standardization) for migration times intra-day and < 2.0% (< or = 0.54% with internal standardization) day-to-day; linearity, in the range 0.156-40 microg/mL, and accuracy were also satisfactory. After a simple liquid-liquid extraction, urine samples could be analyzed with a sensitivity well below the recommended NIDA cut-off of 500 ng/mL. For hair samples, it was necessary to increase the sensitivity by applying a field-amplified sample stacking procedure, which allowed the chiral determination of MDA, MDMA and MDE at concentrations occurring in real samples from ecstasy users, with the possibility of recording UV spectra of the peaks.