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Computed tomography (CT)-guided percutaneous needle biopsy of the lung is a well-recognized and relatively safe diagnostic procedure for suspicious lung masses. Systemic air embolism (SAE) is a rare complication of transthoracic percutaneous lung biopsies. Herein, we present a case of an 81-year-old man who underwent CT-guided percutaneous needle biopsy of a suspicious nodule in the lower lobe of the right lung. Shortly after the procedure, the patient coughed up blood which prompted repeat CT imaging. He was found to have a massive cardiac air embolism. The patient became unresponsive and, despite resuscitation efforts, was pronounced dead. The pathophysiology, risk factors, clinical features, radiological evidence, and autopsy findings associated with SAE are discussed, which may, in light of the current literature, assist with the dilemma between assessing procedural complications and medical liability. Given the instances of SAE in the setting of long operative procedures despite careful technical execution, providing accurate and in-depth information, including procedure-related risks, even the rarest but potentially fatal ones, is recommended for informed consent to reduce medicolegal litigation issues.

The relevance of the problem of urine tampering is well-known in forensic toxicology, with sample dilution being the most used method to cheat toxicological controls. Among the criteria to assess urine integrity, the quantification of creatinine probably represents the most popular method. The present paper presents a simple and low-cost analytical device for on-site creatinine determination as first-line screening for urine dilution. The proposed microfluidic devices were designed as a three-dimensional origami pattern. The device included three colorimetric reactions based on picric acid (PA-based reagent), 3,5-dinitrobenzoic acid (DNBA-based reagent), and Nessler's reagent. The last one, to the best of our knowledge, has never been used before for creatinine determination. In order to assure the highest ease and economy of operation, the color detection and data processing were performed using a built-in smartphone camera and the associated software. The optimized device showed a detection limit of 0.02 g/L. The proposed method was used for the qualitative screening for urine dilution of 48 samples, showing a diagnostic sensitivity and specificity for PA-based, DNBA-based and Nessler's reagent of 83.3%-80.0%, 72.2%-70.0%, and 100.0%-93.3% respectively, versus reference enzymatic method adopting a cut-off of 0.2 g/L. In conclusion, the present preliminary study shows that the proposed device could be a useful tool for on-site screening for urine tampering at the time of sample collection for toxicological testing.

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.

Headspace gas chromatography with a flame ionization detector (HS-GC–FID) is a well-established approach for determining blood alcohol concentration, including in cadaveric specimens. Although the integrity of blood specimens can be adequately guaranteed after the sampling, the quantification of ethanol in cadaveric blood can be affected by postmortem fermentative phenomena occurring between the time since death and the sampling of biofluids. The vitreous humor is less affected by putrefactive phenomena allowing compound determination and its use as an alternative biological matrix. The present work aimed to develop and validate a method using the salting-out effect and based on HS-GC–FID for the determination of ethanol in the vitreous humor. The reported analytical method is based on a simple vitreous humor pre-treatment consisting of a dilution (1:9) with a solution of 2.5 mol/L K2CO3 and 0.0012 mol/L tert-butanol (internal standard). After 1 min of incubation, part of the specimen evaporated in the headspace (2,000 µL) is injected into the chromatographic system and analyzed in isothermal mode (40°C), with a chromatographic time of 1.6 min. The method was validated in terms of selectivity, the lowest limit of detection, intraday and total imprecision, and trueness (bias). The determination of ethanol in the vitreous humor and blood was carried out in 75 cases. The correlation between the two matrices was confirmed in 61 cases. However, 14 vitreous humor specimens showed lower ethanol concentrations, and in the related blood specimens, it was possible to identify the signal of n-propanol, a typical product of postmortem microbial fermentation, that justifies the excess of ethanol in the blood specimens.

It is well known that traffic injuries still represent one of the main causes of death and that high blood alcohol concentrations while driving significantly increase the occurrence of accidents. However, only limited literature on the correlation between chronic alcohol abuse and accident risk is available. The aim of the present study was to investigate the hypothesis of an association between elevated concentrations of carbohydrate deficient transferrin (CDT) and the occurrence of alcohol-related traffic accidents.The analytical determinations of BAC and CDT were performed following certified methods in HS-GC-FID and HPLC, respectively. For BAC, 0.50 g/L was used as cut-off, whereas 2.0% was used for CDT, according to the standardisation proposed by IFCC. A total of 929 drivers, tested for BAC at the time of hospital admission after a traffic accident, were classified into two groups: InjDr 1 (BAC ≤ 0.50 g/L) and InjDr 2 (BAC0.50 g/L); all drivers were also tested for CDT.InjDr 1 included 674 individuals, only 2.5% showing a CDT above the cutoff, whereas InjDr 2 group consisted of 255 subjects, 28.6% testing positive for CDT (Odds Ratio 15.5). When subdividing the InjDr group into increasing classes of CDT, a steady increase in the percentage of BAC-positive drivers was appreciated. Moreover, average BAC was found to parallel each class of CDT.The reported data strongly support the use of CDT as a biomarker of increased risk of alcohol-related traffic accidents in the procedures of re-granting of the driving license upon confiscation for "drink driving".

The increase of the carbohydrate-deficient transferrin (CDT) as results of an heavy intake of alcohol for at least two weeks, is a well-known biochemical modification since the middle '70s. Notwithstanding the first commercial kit for the diagnosis of chronic alcohol abuse based on this biomarker was commercially accessible already thirty years ago, only expensive analytical methods are currently available for its determination. The present paper shows a new approach intrinsically sensitive and specific, based on a specific derivatization of transferrin, and not requiring sophisticated instrumentation.The proposed procedure is based on a selective chelation of terbium (III) by transferrin followed by detection using an characteristic Fluorescence Resonance Transfer Energy (FRET) phenomenon (ex 298 nm - em 550 nm).The proposed procedure showed a limit of detection of 2.5 pmol/mL and a reproducibility intra-day and inter-days15% and 20%, respectively. The results obtained analyzing 40 serum samples using the developed method, were compared with those obtained with HPLC-Vis and an RConsidering its main features (low-cost, ease of operation, minimum need of instrumentation) the present method is suitable for application in screening contexts and in non-strictly regulated environments (e.g. clinical diagnosis) as well as in developing countries or remote areas.

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.

In recent years, a significant increase of reports about suicidal cases due to intentional sodium nitrite intake has been described. In the forensic pathology context, the strategy to approach intoxication cases by sodium nitrite, without any preliminary information or hint, is not straightforward. Indeed, in a number of cases the lack of crime scene data and/or specific pathological signs makes difficult the identification of nitrite poisoning. Moreover, the analytical determination of nitrite in blood is challenging, due to its rapid oxidization to nitrate by hemoglobin. Although several methods have been proposed for the clinical analysis of nitrate and/or nitrite in biological samples, none of these is specifically focused on the determination of these ions in cadaveric samples. Consequently, the diagnosis of nitrite fatal intoxication is still based on methemoglobin analysis. The present paper reports the optimization and validation of an analytical method of capillary ion analysis (CIA) with UV detection, for the determination of nitrite and nitrate in biological fluids and its application to two authentic cases of death by nitrite intake. The analyses were carried out in a bare fused-silica capillary (75 µm inner diameter) using 100 mM sodium tetraborate (pH 9.24) as background electrolyte and applying a voltage of − 15 kV between the capillary ends. The detection was obtained by direct UV absorption recorded at 214 nm wavelength. Bromide was used as the internal standard. Linearity was established in the range of 0.25–5 mmol/L). Reproducibility (intraday and day-to-day) was characterized by relative standard deviations (RSDs) 14.7% for peak areas. The method was applied to the determination of nitrite and nitrate in two real forensic cases, where high concentrations of nitrate were found in cadaveric blood samples (6.5 and 4.4 mmol/L, respectively). Nitrite was found only in trace amounts, due to the instability of this ion in cadaveric blood where it is oxidized to nitrate. The present method represents a new tool for the direct and rapid determination of nitrite and nitrate in cases of forensic interest, and thus offers a diagnostic tool more sensitive and precise than the need methemoglobin analysis.

The present study investigated the morphology of fresh and brine-cured table olives (TOs) as well as the changes that occur when drupes are attacked by the fruit fly Bactrocera oleae. Morphological analyses were performed using light microscopy (LM) and environmental scanning electron microscopy coupled with energy dispersive spectroscopy (ESEM-EDS). The LM analysis was carried out with visible light to evaluate sections stained with either PAS or Azan mixtures as well as unstained sections observed at fluorescence microscopy. The results of the analyses showed that: i) Azan and PAS staining played a useful complementary role, increasing the information provided by the histological analysis. Indeed, in both fresh and brine-cured TOs, epidermal layers and mesocarpal cells were clearly revealed, including sclereid cells. The histological analysis allowed also to identifying the presence of secoiridoid-biophenols (seco-BPs) in both cell walls and vacuoles, as well as in the drupe regions that had been attacked by fruit flies, where they were found at higher concentrations; ii) in fresh and brine-cured olives, the excitation at 480 nm revealed the distribution of the fluorophores, among which the seco-BP are enclosed; iii) the ESEM-EDS analysis revealed the natural morphology of fresh olives, including the dimensions of their cell layers and the size and depth of the mechanical barriers of suberized or necrotic cells around the larva holes. In addition, the elemental composition of regions of interest of the drupe was determined in fresh and brine-cured TOs. The results highlighted the effectiveness of combined use of LM and ESEM-EDS in order to obtain a picture, as complete as possible, of the structural morphology of TOs. Such analytical combined approach can be used to support multidisciplinary studies aimed at the selection of new cultivars more resistant to fly attack.

Post-mortem changes of ions in the body fluids have been proposed as an objective tool for inferring the time of death. In particular, the post-mortem increase of potassium concentrations in the vitreous humour has gained great attention in the literature. On the other hand, ammonium, another ion released in post-mortem processes, has received much less attention, potentially due to unresolved analytical issues using current clinical chemistry methods. This paper presents an application of a new analytical approach based on capillary electrophoresis providing the simultaneous analysis of potassium and ammonium ions in the vitreous humour. In addition, to assess the consistency of the post-mortem increase of ammonium concentrations in the vitreous humour, the determination of this ion in the vitreous humour of the two eyes of the same body at the same post-mortem interval has been verified. Vitreous humour was collected from 33 medico-legal cases where the time of death was known exactly. Prior to analysis, all samples were diluted 1:20 with a 40 μg/mL solution of BaCl2 (internal standard). In the study of the variability of the ammonium concentration between the two eyes, no statistically significant differences were found, supporting the hypothesis of an even post-mortem increase of the ion concentrations in this particular biological fluid. Significant correlations of potassium and ammonium ions with the post-mortem interval were found, with r2 of 0.75 and 0.70, respectively.

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 seed morphology of three Pseudocereal Grains (PSCg), i.e. quinoa (Chenopodium quinoa Willd, Chenopodiaceae), buckwheat (Fagopyrum esculentum Moench, Polygonaceae) and amaranth (Amaranthus caudatus L., Amaranthaceae) was studied by light microscopy (LM) and Environmental Scanning Electron Microscopy coupled with Energy Dispersive Spectroscopy (ESEM-EDS). LM was used with visible light to evaluate either unstained sections or sections stained with Azan mixture and with fluorescent light. The aim of the study was to compare the architecture of the three seeds in order to connect their morphology with nutrient localization. The Azan staining allowed for the visualization of the seed coat, the embryo - with its shoot apical meristem - and the radicle cell layers, whereas the use of fluorescent microscopy identified the cells rich in phenolic compounds. Finally, the ESEM-EDS analysis revealed that the seed coat of the quinoa was thinner than that of amaranth or buckwheat. In all PSCg, starch granules appeared to be located in large polygonal cells, surrounded by a thin cell wall. Several globoids of proteins were observed in the embryo cells. In the radicle section, the vascular bundles of the procambium were evident, while Amaranth only showed a consistent layer of calcium crystals, located between the embryo and the perysperm. The morphological differences of the three PSCg were discussed in the context of their structural resistance to processing technologies which impact on nutritional value of derived foods.

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.

Summary In this study, we compare the histological localisation of phenols and β-glucan in six cereal cross sections. The concentrations of β-glucan and total phenols in whole grain flour (WGF) were also investigated to provide their nutrient content in grams. Cross sections of durum, soft, einkorn and emmer wheat, oats and barley were stained with Azan Trichrome (ATH) and periodic acid–Schiff (PAS) to characterise the morphology and cytohistology of the living cells in the aleurone, subaleurone and germ, as well as the a-nucleated cells in the starchy endosperm. Phenol localisation in the outer layers was performed by fluorescence microscopy due to the presence of ferulic acid. β-Glucan, examined with Calcofluor White staining, was found to be located primarily in the subaleurone layer and starchy endosperm of oats and barley. Our findings shed light on why WGF nutritional value is markedly reduced when the bran is removed through milling.

Most of the on-site approaches for inferring of the post-mortem interval are still based on observative data from the direct body inspection, whereas, objective and quantitative analyses, such as potassium in the vitreous humor, are require laboratory instrumentation and skilled personnel. The present paper presents a simple and low cost analytical method suitable for use at the crime scene for inferring the time since death. The method uses a microfluidic paper-based device (μPAD) for the determination of ammonium in the vitreous humor (VH) based on the selective interaction between the ammonium and the Nessler's reagent. The color change was measured in terms of “RGB distance” by using a simple and free smartphone application. The optimized device showed a limit of detection of 0.4 mmol L−1, with between days precision less than 9.3% expressed as relative standard deviation, and accuracy between days from 94.5% to 104.5%. The selectivity of the Nessler's reaction was tested towards the main vitreous humor compounds, and no significant interferences were found. This paper-based analytical device was successfully used for the determination of ammonium ion in VH samples from forensic autopsies. The results obtained with the proposed method, although for a limited number of cases (n = 25), showed a close correlation with the data obtained with an instrumental analysis based on capillary electrophoresis. Moreover, in order to make the evaluation of results as simple as possible, a direct correlation between the color intensity, expressed as RGB distance, and the post-mortem interval was studied and a significant correlation was found (R2 > 0.78). In conclusion, the present preliminary study showes that the proposed device could be an additional tool to the traditional methods for a more accurate, although still presumptive, estimation of the time of death directly at the crime scene.

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

Proteins and their modifications of the natural mummy of Cangrande della Scala (Prince of Verona, Northern Italy, 1291–1329) were studied. The nano-LC-Q-TOF analysis of samples of rib bone and muscle from the mummy showed the presence of different proteins including Types I, III, IV, V, and XI collagen, hemoglobin (subunits alpha and beta), ferritin, biglycan, vitronectin, prothrombin, and osteocalcin. The structure of Type I and Type III collagen was deeply studied to evaluate the occurrence of modifications in comparison with Type I and Type III collagen coming from tissues of recently died people. This analysis showed high percentage of asparaginyl and glutaminyl deamidation, carbamylation and carboxymethylation of lysine, as well as oxidation and dioxidation of methionine. The most common reaction during the natural mummification process was oxidation—the majority of lysine and proline of collagen Type I was hydroxylated whereas methionine was oxidated (oxidated or dioxidated). To the best of our knowledge, this is the first study which reports the protein profile of a natural mummified human tissue and the first one which describes the carbamylation and carboxymethylation of lysine in mummified tissues.

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.

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.

The aim of the present work was to develop a novel in-house mixed-mode SPE sorbent to be used for the HPLC-Ion TrapMS determination of 16 basic drugs in urine. By using a computational modelling, a virtual monomer library was screened identifying three suitable functional monomers, methacrylic acid (MAA), itaconic acid (IA) and 2-acrylamide-2-methylpropane sulfonic acid (AMPSA), respectively. Three different sorbents were then synthetized based on these monomers, and using as cross-linker trimethylolpropane trimethacrylate (TMPTMA). The sorbent characterization analyses brought to the selection of the AMPSA based phase. Using this novel in-house sorbent, a SPE-HPLC-Ion TrapMS method for drug analysis in urine was validated proving to be selective and accurate and showing a sensitivity adequate for toxicological urine analysis. The comparison of the in-house mixed-mode SPE sorbent with two analogous commercial mixed-mode SPE phases showed that the first one was better not only in terms of process efficiency, but also in terms of quality-price rate. To the best of our knowledge, this is the first time in which an in-house SPE procedure has been applied to the toxicological analysis of a complex matrix, such as urine.

Background and aim The non-glycosylated glycoform of transferrin (Tf), known as asialo-Tf, was not selected (in favor of disialo-Tf) as the measurand for the standardization of carbohydrate deficient transferrin (CDT) determination because of a lower diagnostic sensitivity provided with the currently available analytical procedures for sera. However, asialo-Tf could provide an additional value to disialo-Tf in the CDT analysis employed in forensic toxicology contexts. The present work aimed at developing an easy sample preparation based on PEG precipitation in order to improve the detectability of asialo-Tf in capillary electrophoresis (CE). Methods Equal volumes (35 μL) of serum and of 30% PEG-8000 were mixed and briefly vortexed. After centrifugation, the supernatant was iron saturated with a ferric solution (1:1, v/v). The mixture was analyzed in CE for asialo-Tf and disialo-Tf determination. Results PEG-8000 precipitation allowed the improvement of the baseline in the electropherograms in terms of interferences reduction particularly in the asialo-Tf migration region. The detection of asialo-Tf was possible in 89% of samples with disialo-Tf above the cut-off limit, whereas only 16% of them showed asialo-Tf by employing the traditional sample preteatment. Conclusions Asialo-Tf represents an additional value to disialo-Tf as a biomarker of alcohol abuse in forensic toxicology.

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.

After attending this presentation, attendees will be aware of a new application of micro-Computed Tomography (CT) for dealing with the identification of the signs of pressure of the neck in cases of subtle manual strangulation. This presentation will impact the forensic science community by suggesting a novel approach for the identification of laryngeal fractures by means of micro-CT analysis. It is widely accepted that there are no specific or fully determinate signs of asphyxia. In cases of subtle fatal neck compression, postmortem findings on external examination of a body as well as on autopsy may vary considerably depending on the type of violent trauma, the strength of the resistance exerted by the victim, and on the intensity and duration of neck compression. Based on the detection of a combination of morphological hallmarks such as external injuries to the skin, hyoid or laryngeal fractures, hemorrhages of the overlying soft tissues, and subconjuctiveal petechiae, the diagnosis of homicidal neck compression is generally accepted. A case is presented of a 45-year-old schizophrenic patient found on the floor of the bedroom of a psychiatric ward in cardiopulmonary arrest. The patient was successfully resuscitated; nevertheless, brain death was declared after two days in a vegetative state. The roommate of the deceased, who was also schizophrenic, stated that “the voice of God told me to kill my roommate” and death occurred after a prolonged scuffle. The body showed only vague signs of violence at external inspection (i.e., a subtle bruise on the forehead and a minute scratch on the left arm). At autopsy, the identification of subepicardial petechiae and moderate pulmonary emphysema were not useful for the diagnosis of asphyxial death because these signs might be related to resuscitative maneuvers with prolonged artificial ventilation. Since focal hemorrhagic infiltration of the tracheal mucosa and the superior horns of the thyroid cartilage were observed, a CT scan of the entire larynx was performed, with the goal of identifying laryngeal fractures. Moreover, both superior horns of the thyroid cartilage were analyzed thorough a micro-CT, a radiological technique for tissue analysis with a spatial resolution of a few microns. The 3D reconstruction allowed for identification of multiple cartilage fragments of the larynx. Consequently, the cause of death was identified as asphyxia due to manual strangulation. Considering that micro-CT analysis is able to generate images with a high level of detail compared to conventional radiological techniques, in cases of subtle fatal neck compression this type of investigation could be useful for the morphological identification of comminuted bilateral fractures. This may be particularly useful in cases involving children and young adults where fractures or cartilage damages may be minimal or absent as a consequence of the elasticity of the ligaments around the larynx and hyoid bone as well as the early stage of ossification.

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.

This paper puts forward a new method for the transferrin (Tf) glycoform analysis in body fluids that involves the formation of a transferrin-terbium fluorescent adduct (TfFluo). The key idea is to validate the analytical procedure for carbohydrate-deficient transferrin (CDT), a traditional biochemical serum marker to identify chronic alcohol abuse. Terbium added to a human body-fluid sample produced TfFluo. Anion exchange HPLC technique, with fluorescence detection (λ exc 298 nm and λ em 550 nm), permitted clear separation and identification of Tf glycoform peaks without any interfering signals, allowing selective Tf sialoforms analysis in human serum and body fluids (cadaveric blood, cerebrospinal fluid, and dried blood spots) hampered for routine test. Serum samples (n = 78) were analyzed by both traditional absorbance (Abs) and fluorescence (Fl) HPLC methods and CDT% levels demonstrated a significant correlation (p

Continued progress in chronic alcohol abuse investigation requires the development of less invasive procedures for screening purposes. The application of finger-prick and related dried blood spots (fpDBS) for carbohydrate deficient transferrin (CDT) detection appears suitable for this aim. Therefore, the goal of this project was to develop a screening method for CDT using fpDBS with CZE analysis. Blood samples prepared by finger-prick were placed on DBS cards and left to air dry; each dried fpDBS disc was shredded into small pieces and suspended in acid solution (60 μL of HCl 120 mmol/L). After centrifugation (10 min at 1500 × g), the collected sample was adjusted to pH 3.5. After an overnight incubation, the pH was neutralised and an iron rich solution was added. After 1 h, CZE analysis was carried out. A group of 47 individuals was studied. Parallel serum samples were collected from each investigated subject and the %CDT for each sample was measured using HPLC and CZE techniques. The fpDBS transferrin sialo isoform electropherograms were similar to those obtained with serum. Moreover, fpDBS CZE CDT percentage levels demonstrated significant statistical correlation with those obtained from serum for both HPLC and CZE %CDT (p < 0.01; r2 = 0.8913 and 0.8976, respectively), with %CDT from 0.8 to 13.7% for fpDBS and from 0.7 to 12.7% for serum. The newly developed fpDBS procedure for CDT analysis provides a simple and inexpensive tool for use in population screening.

Cocaine is one of the most used psychomotor stimulants all over the world. On this basis, the interest for the pharmacological activity and the pharmacodynamic and pharmacokinetic aspects of this drug is very prominent in both clinical and forensic toxicological environments. The review presents and discusses 65 scientific publications covering all the aspects of cocaine toxicokinetic, including absorption, distribution, metabolism and elimination of the drug. Particular attention has been dedicated to the studies on the disposition of the drug in alternative biological matrices, such as oral fluid, hair, fetus fluids and tissues, and sweat. In fact, in the last years the use of these matrices has been proposed in clinical and forensic drug analysis in order to obtain additional information to that which can be obtained by analyzing the traditional biological matrices, such as blood and urine.

Capillary electrophoresis coupled to time-of-flight mass spectrometry was used in the present work for the determination of therapeutic and abused drugs and their metabolites in the hair of subjects undergoing addiction treatments, in order to monitor their compliance to therapy. For this purpose a rapid, qualitative drug screening method was adopted based on capillary electrophoresis hyphenated with time-of-flight mass spectrometry, which had earlier been developed and validated for the forensic-toxicological analysis of hair, limitedly to illicit/abused drugs [1]. Sampling of hair was carried out in order to refer to a time window of about two months from the date of sampling (i.e. 2cm ca. from cortex). A single extraction procedure was applied, allowing the determination in the hair matrix of "drugs of abuse" referred to the past abuses, and therapeutic drugs prescribed in the detoxification program as well as their metabolites. Analyte identification was based on accurate mass measurements and comparison of isotope patterns, providing the most likely matching between accurate mass value and elemental formula. Small molecules (

The present article reviews and comments the applications of capillary electrophoresis in the different areas of forensic sciences covering the time from the second half of 2009 until the first part of 2011, being the latest update of previous reviews covering the years from 2001 to 2009. Numerous articles reporting applications of capillary electrophoresis to analytical problems of potential interest for the forensic researchers and scientists can be found in the most qualified journals of analytical chemistry, analytical biochemistry, pharmacology, toxicology, laboratory medicine, human genetics, etc. However, the present review has been focused on discussing only the most relevant examples of analytical applications of capillary electrophoretic and electrokinetic techniques published in the following fields: (i) illicit and abused drugs, (ii) ions and small molecules of forensic interest, (iii) proteins and peptides of forensic interest, (iv) dyes and inks, (v) forensic DNA. The present review collects and comments on 60 references.