Cannabinoid Stability in Postmortem Brain Samples Stored at Different Temperatures.

Drug stability is an important concern of forensic toxicological testing, particularly postmortem (PM) samples that may be stored for an extensive period of time before analysis. In PM toxicology, the complex assessment of analyte stability in biological matrices can profoundly impact the interpretation of toxicological results and the outcome of forensic casework. The aim of this work is to assess the stability of ∆9-tetrahydrocannabinol (THC), cannabidiol, 11-hydroxy-THC (11-OH-THC), 11-nor-9-carboxy-∆9-THC (THCCOOH) and 11-nor-∆9-THC-9-carboxylic acid glucuronide (THCCOOH-glucuronide) in brain stored at three different temperatures (4°C, -20°C and -70°C) up to over 12 months (390 days) in order to establish the best storage condition for preventing potential drug degradation during the storage period. Brain is suitable and useful for xenobiotic concentrations and is a valuable specimen in the interpretation of PM toxicological results. In our study, pooled brain specimens were spiked at low and high concentrations to evaluate the change in concentration over time. Stable compounds were quantified within ±20% of the target concentration (the mean concentration resulting from the initial analysis). According to stability criteria, our preliminary findings revealed that all the cannabinoids studied are stable in frozen brain samples (-20°C and -70°C) for over 12 months: all the analytes' concentrations remained unaffected during storage over time, with the analytical variation staying within ±20%. On the contrary, under refrigeration conditions (4°C), 11-OH-THC, THCCOOH and THCCOOH-glucuronide were instable. Authentic brain samples, collected from eight cases during the autopsy, were analyzed, and the stability was evaluated. This study provided new data on cannabinoid stability in brain. The stability of the brain samples, both in spiked samples and in authentic caseworks, highlights the importance of the brain as a valid testing matrix when retesting is required after a long period of time or when laboratories are faced with backlog.

Two caseworks for one gene: successful species identification from compromised bone materials with the 12S rRNA.

The availability of a reliable molecular assay in species recognition in forensic cases is of paramount importance when visual inspection or morphological methods are not exhaustive, especially from challenging samples. Here, two different caseworks involving bone samples founded during medico-legal outdoor investigations are presented. In order to exclude the human nature of the specimens and to determine the exact species they belong to, we proceeded with the molecular approach trying to generate sequences from the classical mtDNA markers cyt b and COI. However, they both gave critical results. For this reason, a short amplicon of ~ 150 bp of the 12S rRNA gene was used as an alternative.This short fragment was sufficient to identify the biological origin of the bone specimens with a high degree of certainty leading to the exclusion of their human nature. This work highlights the utility of the 12S rRNA and underlines the importance of deepen the choice of alternative shorter markers with respect to the classical ones, in order to achieve species identification even from challenging and degraded material in forensic criminal and wildlife caseworks.

Seizures of illicit substances for personal use in two Italian provinces: analysis of trends by type and purity from 2008 to 2017.

BACKGROUND: The use of illicit substances represents one of the most difficult problems to confront in the health system. Drug use is a global problem but is not uniform throughout the world, within the same country and changes over time. Therefore, knowing the illicit substances that are used in a territory is essential to better organize health services in that specific geographical area. To this aim, we analysed 4200 samples confiscated from individuals who held them for personal use by police forces in the Italian provinces of Modena and Reggio Emilia from 2008 to 2017. METHODS: The suspected samples were screened by gas-chromatography-mass spectrometry (GC-MS) and by liquid chromatography-tandem mass spectrometry (LC-MS/MS); all samples were subsequently analysed by gas chromatography-flame ionization detector (GC-FID) for quantitative analyses. RESULTS: Cannabis was the most seized illicit substance (70.7%). Over the study period, the number of seizures of herb with a high content of Δ9-THC increased. The number of cocaine seizures remained stable (total 16.1%), but the median purity of seized cocaine increased to 75% in 2017. Heroin seizures decreased over time, but the median purity of seized heroin reached 16.8% in 2017. In almost all the years, heroin samples with a purity exceeding the 97.5 percentile were found. Especially from 2014, the range of seized substances increased and started to include synthetic cathinones, phenylethylamines, UR-144, LSD, psilocybe, prescription opioid and hypnotics. In two cases, tramadol together with tropicamide was seized. Most of the seizures involved male subjects and 82% of the seizures were from individuals younger than 35 years of age. CONCLUSIONS: The persistence of old illicit drugs and the rapid emergence of new psychoactive substances represented a serious challenge for public health in the studied Italian area. Some useful interventions might be: informing mainly young people about the possible complications of cannabis use; implementing standardized procedures to diagnose and treat cocaine-related emergencies in hospitals; increasing the distribution of naloxone to antagonize possible heroin overdoses; equipping laboratories to be able to identify the new psychoactive substances.

The importance of forensic storage support: DNA quality from 11-year-old saliva on FTA cards.

Storage conditions influence the integrity of the recoverable DNA from forensic evidence in terms of yield and quality. FTA cards are widely used in the forensic practice as their chemically treated matrix provides protection from the moment of collection to the point of analysis with current STR typing technology. In this study, we assess the recoverability and the integrity of DNA from 11-year-old saliva on FTA cards using a forensic quantitative real-time polymerase chain reaction (qPCR) commercial assay. The quality after long-term storage was investigated in order to evaluate if the FTA device could assure enough stability over time, applying some internally validated quality criteria of the STR profile. Furthermore, we used a 3D interpolation model to combine the quantitative and qualitative data from qPCR to calculate the minimum optimal DNA input (MODI) to add to the downstream PCR reaction based on the quantitative and qualitative data of a sample. According to our results, when saliva sample is properly transferred onto FTA cards and then correctly stored according to the manufacturer's instructions, it is possible to recover sufficient amounts of DNA for human identification even after more than a decade of storage at ambient temperature. Degradation affected the quality of results especially when the Degradation Index exceeds the value of 2.12, requiring modifications of the standard internal workflow to improve the genotyping quality. Above this value, the application of a "corrective factor" to the PCR normalization process was necessary in order to adjust the recommended manufacturer's PCR DNA input taking into account the degradation level. Our results demonstrated the importance to consider in predictive terms the parameters obtained with the real-time quantification assay, both in terms of quantity (DNA concentration) and of quality (DI, inhibition). Informatics predictive tools including qPCR data together with the variables of storage duration and conditions should be developed in order to optimize the DNA analysis process.

Appearance/Image- and Performance-Enhancing Drug Users: A Forensic Approach.

Image- and performance-enhancing drugs include a wide range of substances used to promote physical changes to enhance appearance. Anabolic androgen steroids are the most widely used image- and performance-enhancing drugs along with a wide variety of additional substances. The aim of this study was to identify the pathological changes pertaining to the cardiovascular system possibly involved in the death and characterize the substances associated with steroid use and their possible role in the death. A series of deaths involving image- and performance-enhancing drug users were selected. Nandrolone and testosterone were the most frequently identified steroids. The most commonly illegal drugs found were tetrahydrocannabinol, cocaine, and methadone. Antidepressants, opioids, benzodiazepines, and barbiturates were also found. These findings highlight that results obtained from postmortem investigations should be meticulously evaluated to understand the weight that each identified substance may have played in the death process and exclude any alternative causes of death.

The role of ethyl glucuronide in supporting medico-legal investigations: Analysis of this biomarker in different postmortem specimens from 21 selected autopsy cases.

Ethanol determination in postmortem blood is one of the most frequently requested analyses in legal medicine and forensic toxicology. Ethyl glucuronide is a non-oxidative ethanol metabolite. It is also a useful marker of ante-mortem alcohol ingestion when ethanol itself has been completely eliminated from the body and could be considered in autopsy cases to obtain more reliable indications. The aim of the present study was to investigate the ethyl glucuronide distribution in postmortem specimens from autopsy cases found to be positive for ethanol. We presented 21 autopsy cases in which central blood, peripheral blood and liver samples were available. Specimens were analyzed for ethyl glucuronide by liquid chromatography tandem mass spectrometry; we also recorded postmortem interval, case history, cause of death, use of drugs, metabolic disorders if present, putrefaction if present, history of ethanol abuse and information about ethanol intake before death. Our aim was to evaluate and to compare the ethyl glucuronide levels in different matrices taken from the same subject in order to provide a better understanding of the interpretation of postmortem ethyl glucuronide concentrations.

Monitoring of adherence to headache treatments by means of hair analysis.

PURPOSE: The aim of this study was to evaluate the potential of hair analysis to monitor medication adherence in headache patients undergoing chronic therapy. For this purpose, the following parameters were analyzed: the detection rate of 23 therapeutic drugs in headache patients' hair, the degree of agreement between the self-reported drug and the drug found in hair, and whether the levels found in hair reflected the drug intake reported by the patients. METHODS: The study included 93 patients suffering from primary headaches declaring their daily intake of at least one of the following drugs during the 3 months before the hair sampling: alprazolam, amitriptyline, citalopram, clomipramine, clonazepam, delorazepam, diazepam, duloxetine, fluoxetine, flurazepam, levomepromazine, levosulpiride, lorazepam, lormetazepam, mirtazapine, paroxetine, quetiapine, sertraline, topiramate, trazodone, triazolam, venlafaxine, and zolpidem. A detailed pharmacological history and a sample of hair were collected for each patient. Hair samples were analyzed by liquid chromatography-electrospray tandem mass spectrometry, using a previously developed method. RESULTS: All 23 drugs were detected in the examined hair samples. The agreement between the self-reported drug and the drug found in hair was excellent for most analytes (P < 0.001, Cohen's kappa); a statistically significant relationship (P < 0.05, linear regression analysis) between dose and hair level was found for amitriptyline, citalopram, delorazepam, duloxetine, lorazepam, and venlafaxine. CONCLUSIONS: Hair analysis proved to be a unique matrix to document chronic drug use in headache patients, and the level found for each individual drug can represent a reliable marker of adherence to pharmacological treatments.

Hair analysis for detection of triptans occasionally used or overused by migraine patients-a pilot study.

PURPOSE: The aim of this study is to evaluate the detection rate of almotriptan, eletriptan, frovatriptan, sumatriptan, rizatriptan, and zolmitriptan in the hair of migraineurs taking these drugs; the degree of agreement between type of self-reported triptan and triptan found in hair; if the concentrations in hair were related to the reported cumulative doses of triptans; and whether hair analysis was able to distinguish occasional use from the overuse of these drugs. METHODS: Out of 300 headache patients consecutively enrolled, we included 147 migraine patients who reported to have taken at least one dose of one triptan in the previous 3 months; 51 % of the patients overused triptans. A detailed pharmacological history and a sample of hair were collected for each patient. Hair samples were analyzed by liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) by a method that we developed. RESULTS: All the triptans could be detected in the hair of the patients. The agreement between type of self-reported triptan and type of triptan found in hair was from fair to good for frovatriptan and zolmitriptan and excellent for almotriptan, eletriptan, sumatriptan, and rizatriptan (P < 0.01, Cohen's kappa). The correlation between the reported quantities of triptan and hair concentrations was statistically significant for almotriptan, eletriptan, rizatriptan, and sumatriptan (P < 0.01, Spearman's rank correlation coefficient). The accuracy of hair analysis in distinguishing occasionally users from overusers was high for almotriptan (ROC AUC = 0.9092), eletriptan (ROC AUC = 0.8721), rizatriptan (ROC AUC = 0.9724), and sumatriptan (ROC AUC = 0.9583). CONCLUSIONS: Hair analysis can be a valuable system to discriminate occasional use from triptan overuse.

Hair testing in clinical setting: Simultaneous determination of 50 psychoactive drugs and metabolites in headache patients by LC tandem MS.

Headache patients suffering from recurrent attacks are a population at risk of overuse and abuse of analgesic medications. Associated with triptans, the first-line drugs recommended for the acute treatment, these patients usually take other medications such as opioids analgesics for the attack treatment, antidepressants and antiepileptics for prophylaxis treatment and benzodiazepines, non-benzodiazepine hypnotics and antipsychotics for the treatment of comorbidities. Regular and frequent use of triptans, like of any other symptomatic analgesic, can cause chronic headache and medication-overuse headache (MOH). In these circumstances, a detoxification treatment is necessary and therefore the monitoring and follow-up of the patients are crucial to the success of the treatment. In the present study, a LC tandem MS method has been developed for the identification of 50 psychoactive drugs in human hair, including triptans, benzodiazepines and metabolites, analgesics, antiepileptic, antidepressants and metabolites, a non-benzodiazepine hypnotic (z-drug), antipsychotics and metabolites. Hair samples were decontaminated, pulverized and incubated overnight in methanol; the extracts were then purified by a new and rapid QuEChERS procedure and analyzed by LC-MS/MS under gradient elution with positive ionization MRM mode. The procedure was fully validated in terms of selectivity, linearity, limit of detection and lower limit of quantitation, precision and accuracy, carry-over, matrix effect, recovery and dilution integrity. The validated procedure has been applied to 234 real hair samples collected from headache patients with known type and dosage of the taken drugs; the obtained data could be of interest to evaluate the xenobiotic concentrations in patients with known therapy.

Development and validation of a liquid chromatography-tandem mass spectrometric assay for quantitative analyses of triptans in hair.

Triptans are specific drugs widely used for acute treatment of migraine, being selective 5HT1B/1D receptor agonists. A proper assumption of triptans is very important for an effective treatment; nevertheless patients often underuse, misuse, overuse or use triptans inconsistently, i.e., not following the prescribed therapy. Drug analysis in hair can represent a powerful tool for monitoring the compliance of the patient to the therapy, since it can greatly increase the time-window of detection compared to analyses in biological fluids, such as plasma or urine. In the present study, a liquid chromatography-tandem mass spectrometric (LC-MS/MS) method has been developed and validated for the quantitative analysis in human hair of five triptans commonly prescribed in Italy: almotriptan (AL), eletriptan (EP), rizatriptan (RIZ), sumatriptan (SUM) and zolmitriptan (ZP). Hair samples were decontaminated and incubated overnight in diluted hydrochloric acid; the extracts were purified by mixed-mode SPE cartridges and analyzed by LC-MS/MS under gradient elution in positive multiple reaction monitoring (MRM) mode. The procedure was fully validated in terms of selectivity, linearity, limit of detection (LOD) and lower limit of quantitation (LLOQ), accuracy, precision, carry-over, recovery, matrix effect and dilution integrity. The method was linear in the range 10-1000pg/mg hair, with R(2) values of at least 0.990; the validated LLOQ values were in the range 5-7pg/mg hair. The method offered satisfactory precision (RSD <10%), accuracy (90-110%) and recovery (>85%) values. The validated procedure was applied on 147 authentic hair samples from subjects being treated in the Headache Centre of Modena University Hospital in order to verify the possibility of monitoring the corresponding hair levels for the taken triptans.

Therapeutic and recreational methadone cardiotoxicity.

Several classes of drugs have been associated with an increased risk of cardiovascular disease and occurrence of arrhythmias potentially involved in sudden deaths in chronic users even at therapeutic doses. The study presented herein focuses on pathological changes involving the heart possibly due to methadone use. 60 cases were included in the study in total and were divided into three groups (therapeutic methadone users: 20 cases, recreational methadone users: 20 cases, and sudden death group in subjects who had never taken methadone: 20 cases). Autopsies, histology, biochemistry and toxicology were performed in all cases. Macroscopic and microscopic investigation results in therapeutic methadone users were similar to those observed in sudden, unexpected deaths in non-methadone users. In recreational methadone consumers, macroscopic and microscopic examination of the heart failed to provide results consistent with acute or chronic myocardial or coronary damage, thereby corroborating the hypothesis of death most likely following respiratory depression.

Postmortem diagnosis of anaphylaxis in presence of decompositional changes.

Eosinophil and activated mast cell identification in the spleen combined with mast cell tryptase determination in postmortem serum may diagnose fatal anaphylaxis with a high degree of certainty. Mast cell tryptase measurement and significance in corpses with decompositional changes remains however an issue of controversy. Analogously, immunohistochemistry in corpses with decompositional changes may be influenced by several mechanisms, including protein alteration, antigen diffusion and unspecific antibody binding to disrupted protein structures. The authors present an autopsy case involving a 55-year-old woman who unintentionally received clarithromycin. Due to difficult in administrative procedures, the postmortem examination was performed 96 h after death. Mast cell tryptase was measured in postmortem serum from femoral, aortic and right heart blood. The obtained results were consistent with mast cell activation. Histochemistry (Pagoda Red) and immunohistochemistry (anti-tryptase antibodies) allowed splenic eosinophils and mast cells to be detected. Based on the results of all postmortem investigations, the hypothesis of anaphylaxis following accidental clarithromycin administration was formulated.

Cardiac Toxicity in Selective Serotonin Reuptake Inhibitor Users.

Several classes of recreational and prescription drugs have been associated with an increased risk of cardiovascular disease and the occurrence of arrhythmias, which may be involved in sudden deaths in chronic users even at therapeutic doses. The study presented herein focuses on pathological changes involving the heart, which may be caused by selective serotonin reuptake inhibitor use and their possible role in the occurrence of sudden cardiac death. A total of 40 cases were included in the study and were divided evenly into 2 groups: 20 cases of patients treated with selective serotonin reuptake inhibitors and 20 cases of sudden deaths involving patients void of any drug treatment. The first group included 16 patients treated with citalopram and 4 with sertraline. Autopsies, histology, biochemistry, and toxicology were performed in all cases. Pathological changes in selective serotonin reuptake inhibitor users consisted of various degrees of interstitial and perivascular fibrosis as well as a small degree of perineural fibrosis within the myocardium of the left ventricle. Within the limits of the small number of investigated cases, the results of this study seem to confirm former observations on this topic, suggesting that selective serotonin reuptake inhibitors may play a potential, causative role in the pathogenesis of sudden deaths in chronic users even at therapeutic concentrations.

Pathological changes in anabolic androgenic steroid users.

Several classes of recreational and prescription drugs have additional effects on the heart and vasculature, which may significantly contribute to morbidity and mortality in chronic users. The study presented herein focuses on pathological changes involving the heart possibly due to anabolic androgenic steroid use. The role these hormones may play in their occurrence of sudden cardiac death is also investigated. 98 medico-legal cases including 6 anabolic androgenic steroid users were retrospectively reviewed. Autopsies, histology, immunohistochemistry, biochemistry and toxicology were performed in all cases. Pathological changes consisted of various degrees of interstitial and perivascular fibrosis as well as fibroadipous metaplasia and perineural fibrosis within the myocardium of the left ventricle. Within the limits of the small number of investigated cases, our results appear to confirm former observations on this topic and suggest anabolic androgenic steroid's potential causative role in the pathogenesis of sudden cardiac deaths in chronic users.

Lateral saccular laryngeal cyst and unexpected asphyxial death.

We present the case of a 41-year-old man with a herniated saccular laryngeal cyst in the left cervical region who died unexpectedly at home from acute asphyxia. A few days before death the patient complained an obvious palpable swelling on the left side of the neck. The autopsy showed an oval-shaped mass originating on the wall of the larynx and herniating into the extralaryngeal region. The critical reduction of the laryngeal lumen originated from an intense oedema of the laryngeal mucosa above the cyst, concurrent with an inflammatory process of the cystic wall, with probable associated glottis laryngospasm mediated by the superior laryngeal nerve. The rapid evolution of oedema was favoured by the extreme relaxation of the connective tissue of the laryngeal mucosa in the supraglottic and aryepiglottic regions which explains the sudden death. The case is useful for delineating the problems related to the possible evolution of laryngeal cysts, the mechanisms of asphyxial complications, the pathological diagnosis and the medico-forensic aspects.

Unexpected intrauterine fetal death in parvovirus B19 fetal infection.

Parvovirus B19 infection during pregnancy can be transmitted to the fetus through the placenta. The consequences for the health of the fetus are very variable and can be very serious. They include intrauterine fetal death (IUFD) and miscarriage, which can lead to medico-forensic questions. For the most part, cases of IUFD take place during the second trimester of gestation and present an anatomopathologic picture characteristic of fetal infection with hydrops, placental edema, serous effusion, and erythroblastosis with nuclear inclusions. Endocardial fibroelastosis, medullar and thymic hypoplasia, and hepatic hemosiderosis are frequently present. In the third trimester, the cases are less frequent, not accompanied by hydrops, and can depend more on placental compromise than on direct infection of the fetus. We present 5 cases of IUFD resulting from parvovirus B19 and we discuss the pathogenetic and anatomopathologic aspects and obstetric liability. In 4 cases, the IUFD took place suddenly, in the absence of symptoms, in women who had not previously shown any symptom of the viral infection. In one case, the patient was hospitalized following an ultrasound diagnosis of fetal hydrops and IUFD took place 5 days after admission. Of these cases 3 were verified in the second trimester and 2 in the third trimester. Only the cases of the second trimester and one of the 2 cases of the third trimester presented the characteristic aspects of fetal infection. The other case of third trimester was characterized by placental involvement.

Spontaneous umbilical cord hematoma: an unusual cause of fetal mortality: a report of 3 cases and review of the literature.

Spontaneous hematoma of the umbilical cord represents a rare cause of fetal morbidity and mortality and the outcome is poor in half of the cases. There are many risk factors, such as infections, morphologic anomalies, alterations of the vessel walls, prolapses, twisting and traction of the cord, but in many cases the causes remain unknown. We present 3 cases of umbilical cord hematoma which took place at the end of the pregnancy and were followed by perinatal death of the fetus. In the 3 cases, which were autopsied including macroscopical and histopathologic examination of the placenta and the umbilical cord, a cordonal pathology was present; in all cases, there were alterations of the vascular wall, and in the third case inflammatory vasculopathy was found. In all 3 cases, the cause of death was attributed to acute anoxia due to the cordonal hematoma.

Sudden cardiac death during anabolic steroid abuse: morphologic and toxicologic findings in two fatal cases of bodybuilders.

We report two cases of sudden cardiac death (SCD) involving previously healthy bodybuilders who were chronic androgenic-anabolic steroids users. In both instances, autopsies, histology of the organs, and toxicologic screening were performed. Our findings support an emerging consensus that the effects of vigorous weight training, combined with anabolic steroid use and increased androgen sensitivity, may predispose these young men to myocardial injury and even SCD.

Sudden death from tubercular myocarditis.

Tuberculous myocarditis is a rare finding. We present the case of a 33-year-old woman who was in good health and who died suddenly at home. Autopsy and histopathologic examinations revealed granulamatous lesions in the myocardium, lungs, lymph nodes, liver, and spleen. No fast acid bacilli were demonstrated on histological examination. The presence of a Mycobacterium tuberculosis DNA complex was identified using a polymerase chain reaction (PCR) on formalin-fixed paraffin-embedded histological samples. An HIV test carried out on the blood obtained during the autopsy was negative according to the DNA amplification technique (PCR) and enzyme-linked immunosorbent assay serological test. We hypothesize that the mechanism of death was severe ventricular arrhythmia due to granulomatous proliferation in the structures of the interventricular septum.