Postmortem Blood Samples Research Articles

Quantification of 2-aminothiazoline-4-carboxylic acid as a reliable marker of cyanide exposure using chemical derivatization followed by liquid chromatography–tandem mass spectrometry

In this research, we have developed a novel and simple liquid chromatography coupled with electrospray ionization–tandem mass spectrometry (LC/ESI–MS/MS) method for quantification of 2-aminothiazoline-4-carboxylic acid (ATCA), which is produced by the direct reaction of cyanide (CN) with endogenous cystine. In forensic science, detection of CN is important because CN is a poison that is often used for murder or suicide, in addition to being produced by the thermal decomposition of natural or synthetic materials. However, because CN disappears rapidly from body tissue, ATCA is thought to be a more reliable indicator of CN exposure. For the method reported herein, human blood samples (20 μL) were subjected to protein precipitation followed by derivatization with 4-bromoethyl-7-methoxycoumarin. Blood spiked with ATCA at concentrations ranging from 50 to 1500 ng/mL was used to prepare a calibration curve (lower limit of quantification; 50 ng/mL, lower limit of detection; 25 ng/mL). Our method uses chemical derivatization, so unlike previously reported methods, it does not require tedious pretreatment procedures, hydrophilic interaction liquid chromatography columns, or specialized equipment. In addition, our method allows for repeatable and accurate quantification of ATCA, with intra- and inter-assay coefficients of variation of below 5.0% and below 6.0%, respectively. We used the method to analyze ATCA in postmortem human blood samples, including samples from people who had intentionally ingested CN or were fire victims. Blood ATCA concentrations were higher among people who had ingested CN or were fire victims than among people in a control group (P < 0.0001). The data reported herein demonstrate that our LC/ESI–MS/MS method can be used to detect and quantify ATCA in postmortem blood samples and that CN exposure strongly affects ATCA concentration, providing a useful tool for detection of CN poisoning.

Effect of Temperature and Preservative on Neo-ethanol Formation in Postmortem Whole Blood Samples.

To determine the association of temperature, time duration, and preservatives with ethanol neo-genesis in postmortem whole blood samples. Observational cross-sectional study. Department of Chemical Pathology and Toxicology, Armed Forces Institute of Pathology, Rawalpindi, in collaboration with Combined Military Hospital, Rawalpindi from August 2020 to January 2021. Whole blood samples were obtained from cardiac chamber of an autopsy case with aseptic measures and were drawn into six tubes (3 x EDTA tubes and 3 x NaF tubes). These six tubes were stored at 4˚C, 25˚C, and 37˚C. All samples were analysed along with internal standard, on head space gas-chromatography on Day-0, Day-07, Day-14, Day-21, and Day-28 of sampling. A total of 60 samples out of 10 autopsies, were analysed. Blood alcohol concentrations of all samples were negative on 1st day (Day-0) of analysis. Samples stored at 4˚C did not produce any ethanol at any day of analysis, regardless of preservatives used. Neo-ethanol levels of blood samples stored in EDTA preservative were significantly raised (p <0.001) as compared to samples stored in NaF preservative. Samples stored at 37˚C showed significant increase in neo-ethanol concentration (p <0.001) in comparison with samples stored at 4˚C and 25˚C. Neo-ethanol levels increased with duration of storage till 14th day of analysis. Sodium fluoride (NaF) should be used as preservative to avoid ethanol neo-genesis during storage and transportation of blood samples for alcohol analysis. Cold chain maintenance is of pivotal importance to exclude false positive blood alcohol results. Key Words: Neo-ethanol, Neo-genesis, Ethylenediaminetetra acetic acid (EDTA), Sodium fluoride (NaF).

IgG4-specific responses in patients with Staphylococcus aureus bone infections are not predictive of postoperative complications.

The most prevalent pathogen in bone infections is Staphylococcus aureus; its incidence and severity are partially determined by host factors. Prior studies showed that anti-glucosaminidase (Gmd) antibodies are protective in animals, and 93.3 % of patients with culture-confirmed S. aureus osteomyelitis do not have anti-Gmd levels > 10 ng/mL in serum. Infection in patients with high anti-Gmd remains unexplained. Are anti-Gmd antibodies in osteomyelitis patients of the non-opsonising, non-complement-fixing IgG4 isotype? The relative amounts of IgG4 and total IgG against Gmd and 7 other S. aureus antigens: iron-surface determinants (Isd) IsdA, IsdB, and IsdH, amidase (Amd), α-haemolysin (Hla), chemotaxis inhibitory protein from S. aureus (CHIPS), and staphylococcal-complement inhibitor (SCIN) were determined in sera from healthy controls (Ctrl, n = 92), osteomyelitis patients whose surgical treatment resulted in infection control (IC, n = 95) or an adverse outcome (AD, n = 40), and post-mortem (PM, n = 7) blood samples from S. aureus septic-death patients. Anti-Gmd IgG4 levels were generally lower in infected patients compared to controls; however, levels among the infected were higher in AD than IC patients. Anti-IsdA, IsdB and IsdH IgG4 levels were increased in infected patients versus controls, and Jonckheere-Terpstra tests of levels revealed an increasing order of infection (Ctrl < IC < AD < PM) for anti-Isd IgG4 antibodies and a decreasing order of infection (Ctrl > IC > AD > PM) for anti-autolysin (Atl) IgG4 antibodies. Collectively, this does not support an immunosuppressive role of IgG4 in S. aureus osteomyelitis but is consistent with a paradigm of high anti-Isd and low anti-Atl responses in these patients.

Carbon monoxide related deaths in Jeddah, Saudi Arabia: A forensic carboxyhemoglobin autopsy-based study

Carbon monoxide (CO) poisoning is a prevalent global cause of death. This is the first epidemiological study investigating the relationship between CO poisoning (silent killer) and the cause of death in autopsy forensic samples received from 2009 to 2020 at the Poison Control and Forensic Medical Chemistry Center, Jeddah, Saudi Arabia. MethodsThe cause of death was classified based on the carboxyhemoglobin saturation level (COHb) in the postmortem blood samples. In 170 post-mortem cases, the level of COHb was positive. The double-wavelength spectrophotometric method was used to quantify the COHb and the samples were confirmed by another spectrophotometer techniques AVOXmeter 4000 system. ResultsJust more than half (58%) of the deaths were due to CO intoxication, and the median level of the COHb in the postmortem blood samples was 56%. The study confirmed that the cause of the death in 15% of the sample were classified as CO-related intoxication (median COHb 21%), and 27% had no relation with CO intoxication or undetermined cause of death (COHb level ≤ 10%). The manner of death was classified as accidental, suicidal, homicidal, natural or unknown cause of death. The majority (72%) of the causes of death was accidental death, 10% due to suicide, 4% to homicide and 5% to natural death with no relationship to CO poisoning. The manner of deaths were unknown in 15 cases due to a lack of information. ConclusionAlthough CO poisoning globally appears to decline, the rate in Jeddah remained stable through the study period. Safety and preventive educational interventions are required to reduce CO poisoning.

Assessment of chemical asphyxia caused by toxic gases generated from rigid polyurethane foam (RPUF) fires

Rigid polyurethane foam (RPUF) is widely used for thermal and sound insulation owing to their low thermal conductivity and light weight. However, they have serious disadvantages, including flammability and toxic gas generation, which can cause chemical asphyxia during a fire. Carbon monoxide (CO) and hydrogen cyanide (HCN) are representative toxic gases formed by incomplete combustion and HCN, in particular, is closely related to polyurethane product fires. In this study, the risk of inhalation of toxic gases such as CO, HCN and NO2 during RPUF fires was demonstrated convincingly through the analysis of carboxyhemoglobin (COHb), cyanide (CN-) and methemoglobin (MetHb) in the postmortem blood samples of 38 victims of RPUF fires. To better understand the toxic gas poisoning and chemical asphyxia, we classified all cases into two groups based on the extent of injuries and location where the victim was found. Mean concentrations of COHb and cyanide in group 1 without injuries were approximately two times higher than in group 2 with severe injuries, while concentrations of free MetHb showing possibility of NO2 inhalation were approximately six times lower than in group 2. Furthermore, we presumed concentrations of cyanide at the time of death and five cases showed the possibility of cyanide poisoning.

Application of Homogeneous Liquid-Liquid Microextraction With Switchable Hydrophilicity Solvents to the Determination of MDMA, MDA and NBOMes in Postmortem Blood Samples.

Synthetic drugs for recreational purposes are in constant evolution, and their consumption promotes a significant increase in intoxication cases, resulting in damaging public health. The development of analytical methodologies to confirm the consumption of illicit drugs in biological matrices is required for the control of these substances. This work exploited the development of an extraction method based on homogenous liquid-liquid microextraction with switchable hydrophilicity solvent (SHS) as extraction phase for the determination of the synthetic drugs 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine and N-methoxybenzyl-methoxyphenylethylamine derivates (25B, 25C and 25I) in postmortem blood, followed by liquid chromatography coupled to mass spectrometry in tandem. The optimized sample preparation conditions consisted of using 250 µL of ZnSO4 10% and 50 µL of NaOH 1 mol/L in the protein precipitation step; N,N-dimethylcyclohexylamine was used as SHS, 650 μL of a mixture of SHS:HCl 6 mol/L (1:1 v/v), 500 μL of whole blood, 500 μL of NaOH 10 mol/L and 1 min of extraction time. The proposed method was validated, providing determination coefficients higher than 0.99 for all analytes; limit of detection and limit of quantitation ranged from 0.1 to 10 ng/mL; intra-run precision from 2.16% to 9.19%; inter-run precision from 2.39% to 9.59%; bias from 93.57% to 115.71% and matrix effects from 28.94% to 51.54%. The developed method was successfully applied to four authentic postmortem blood samples from synthetic drugs users, and it was found to be reliable with good selectivity.

Tramadol-Related Deaths: Genetic Analysis in Relation to Metabolic Ratios.

Tramadol (TR) metabolism is mainly dependent on the enzymatic activity of CYP2D6, which is controlled by genetic polymorphisms. Individuals are classified as poor (PMs), intermediate (IMs), extensive (EMs) or ultrarapid metabolizers (UMs) according to their genotype or phenotype. The determination of the metabolic phenotype for CYP2D6 can be of utmost importance in forensic and clinical contexts that involve TR intake. The present study aimed to describe CYP2D6 genetic variants in cases of TR-related deaths and to assess which metabolic ratio(s) (MRs) would allow to determine CYP2D6 phenotype without having to perform genetic analyses. Forty-eight postmortem blood samples were selected from TR-related death cases previously analyzed in a forensic context in North of France between 2013 and 2019. Initial available data included blood concentrations of TR and its two main metabolites (M1 & M2) determined using an LC--MS-MS method. TR metabolism was expressed as various MRs comprising TR/M1, TR/M2 and M2/M1. After DNA extraction, sequencing was used for genetic variant detections that affect CYP2D6 activity/expression. In the present study, the allelic variants with the higher frequency were CYP2D6*1 (68%), followed by *4 (21%). The most frequent phenotype is EMs (59.6%), followed by IMs (23.4%), PMs (12.8%) and UMs (6.4%). There was no significant correlation between each calculated MR and the genotypically predicted phenotypes, except for M2/M1 which appears related to the PM phenotype. The observed distribution of CYP2D6 genetic variants in this TR-related death population was similar to that found in the general Caucasian population. The present study displayed that the blood M2/M1 ratio could be the best-correlated TR MR to the PM phenotype, and could thus be used in forensic contexts where genetic analyses are not possible or poorly informative. For the other phenotypes, especially the UM phenotype, genetic analysis appears to be the only reliable method to predict the CYP2D6 phenotype.

Direct coupling of MEPS to ESI-QqTOF-MS for the simultaneous analysis of tricyclic antidepressants and benzodiazepines in postmortem blood

The combination of chromatography and mass spectrometry is the golden standard in forensic sciences. Although this approach provides high sensitivity and selectivity levels, it is time-consuming. The circumvention of the chromatography can increase the sample throughput, but the selectivity must be compensated by using a selectivity enhanced instrumental technique, such as high-resolution mass spectrometry (HRMS), and designing an appropriate sample preparation step. Microextraction by packed sorbent (MEPS) has proven potential to treat complex biological samples in a fast and reproducible way. It allows processing low sample volumes, remaining a green procedure due to low solvent consumption. This article presents the direct coupling of a MEPS syringe to the electrospray ionization source (ESI) of a quadrupole time-of-flight mass spectrometer (QqTOF) using an infusion pump for sample introduction, as a proof of concept. The developed method was validated for the simultaneous analysis of benzodiazepines and tricyclic antidepressants in postmortem blood samples. Limits of quantification (LOQ) were between 25 and 50 ng mL−1 for all compounds. Accuracy and precision were evaluated at three concentration levels (50, 125 and 350 ng mL−1) and expressed in terms of relative error (%RE) and relative standard deviation (%RSD), respectively. Both parameters were better than 20% for the lower concentration evaluated and better than 15% for the other two concentration levels. The developed method was used to analyze authentic postmortem blood samples, to test the applicability in the forensic toxicological context. The results are in agreement with the data provided by the medico-legal services. Also, the application of HRMS has allowed the identification of unexpected targets in the samples.

Cell survival and DNA damage repair are promoted in the human blood thanatotranscriptome shortly after death

RNA analysis of post-mortem tissues, or thanatotranscriptomics, has become a topic of interest in forensic science due to the essential information it can provide in forensic investigations. Several studies have previously investigated the effect of death on gene transcription, but it has never been conducted with samples of the same individual. For the first time, a longitudinal mRNA expression analysis study was performed with post-mortem human blood samples from individuals with a known time of death. The results reveal that, after death, two clearly differentiated groups of up- and down-regulated genes can be detected. Pathway analysis suggests active processes that promote cell survival and DNA damage repair, rather than passive degradation, are the source of early post-mortem changes of gene expression in blood. In addition, a generalized linear model with an elastic net restriction predicted post-mortem interval with a root mean square error of 4.75 h. In conclusion, we demonstrate that post-mortem gene expression data can be used as biomarkers to estimate the post-mortem interval though further validation using independent sample sets is required before use in forensic casework.

Determination of Lidocaine in Postmortem Whole Blood Samples after Unsuccessful Cardiopulmonary Resuscitation

Forensic toxicologists often detect lidocaine in the biological fluids of the deceased, due to cardiopulmonary resuscitation (CPR) attempts prior to death. Here, we describe the development of a rapid, sensitive and robust method for the detection of lidocaine in postmortem whole blood using liquid−liquid extraction (LLE) followed by GC/MS analysis. The method showed a dynamic linear range of 100 to 6000 ng/mL with a linearity expressed by the regression coefficient (R2) and a value of 0.9947. The quantitation limit (LOQ) was found to be 0.03 ng/mL and the detection limit (LOD) 0.01 ng/mL. Recovery accuracy and repeatability were satisfactory. Finally, the method was applied to 23 real whole blood samples from cases where CPR was attempted. Blood concentrations ranged from 0.21–0.96 μg/mL.

Ethyl glucuronide and ethyl sulfate: a review of their roles in forensic toxicology analysis of alcohol postmortem.

This review presents the current methods used for determining ethyl glucuronide (EtG) and ethyl sulfate (EtS) concentrations in postmortem specimens, including sample preparation, analysis and the role of EtG and EtS in the postmortem assessment of the extent of alcohol abuse. Papers pertaining to postmortem investigation were collected from scientific databases and reviewed. The papers were published between January 2006 and October 2020. Most of the analyses involved postmortem blood and urine samples, with a few reports using other bodily specimens and tissues. The method validation was not conducted for all applications. These reports were mostly intended to present data rather than interpret them, and the lack of effort in relating these ethanol biomarkers with the cause of death and/or determination of the time of deaths due to ethanol intoxication might decrease the applicability of these makers after a promising start between 2006 and 2010. Nevertheless, by the beginning of 2020, papers investigating ethanol biomarkers were still increasing. A considerable number of methods used liquid chromatography coupled with mass spectrometry (LC-MS) techniques that require less sample preparation (e.g., protein precipitation extraction, dilution, filtration, and centrifugation). Although solid-phase extraction can be applied, only three applications were reported. Matrix effects can be a substantial challenge in analytical methods based on LC-MS because they directly affect the ionization of analytes. However, these problems can be avoided due to the high cutoff values used to identify positive results for these ethanol biomarkers, which are often above 0.1-1mg/L, and using internal standards. Research on using tissue specimens is recommended as most of the reported results on this type of specimen were promising.

Fatal methemoglobinemia: A case series highlighting a new trend in intentional sodium nitrite or sodium nitrate ingestion as a method of suicide

Unintentional exposure to nitrite- or nitrate-containing toxic salts is a recognized cause of acquired methemoglobinemia (MetHb). This systemic alteration of the blood can be fatal if not recognized and treated promptly. The intentional ingestion of sodium nitrite (NaNO2) or sodium nitrate (NaNO3), causing MetHb, is an uncommon and recently identified method of suicide, with the first reported case in the literature occurring in New Zealand in 2010. In this case series we present 28 cases of sudden death of individuals with evidence of MetHb and/or toxic salt ingestion, occurring in the Province of Ontario, Canada, between the years 1980 and 2020, inclusive. Of the 28 deaths in our case series, 25 showed evidence of intentional ingestion of sodium nitrite or sodium nitrate salts. Our year-over-year data demonstrated this is an increasingly used method of suicide in our provincial population, with the majority of cases occurring in the final two years of our study.Postmortem detection of MetHb is typically established via screening techniques such as scene evidence suggesting fatal consumption of a toxic salt in addition to the characteristic grey-purple lividity observed upon the body. The diagnosis can be established via postmortem blood testing demonstrating elevated methemoglobin saturation. Additionally, we have confirmed that postmortem MRI in cases of MetHb demonstrates a T1-bright (hyperintense) signal of the blood; both within intracardiac blood on chest MRIs and postmortem blood samples in tubes.

Screening algorithms for HBV, HCV, HIV and syphilis in an anatomical donation program

BackgroundUsers of anatomical donors are at risk of exposure to bloodborne pathogens. This study evaluated screening algorithms for hepatitis B and C virus, human immunodeficiency virus and Treponema pallidum during donor allocation and assessed the impact of postmortem time on hemolysis and how hemolysis affects test results and donor discard rate. MethodsFrom 2011 to 2018, demographic data of anatomical donors, time of postmortem blood sampling, presence of sample hemolysis, serological test results (negative; active infection; false reactive screening test; historic infection; inconclusive; technically impracticable) and the actual donor allocation were collected. ResultsDonors (n = 537) had a mean age of 77.53 ± 13.67 (24–103) year. Nine (1.68%) had laboratory test results indicative for active infection for hepatitis B (n = 1) and C virus (n = 2), human immunodeficiency virus (n = 5) and T. pallidum (n = 1). Negative screenings ranged from 74.67 to 97.58%, depending on the pathogen. According to the original screening algorithms, 479 (89.20%) donors should have been accepted. In practice, a donor acceptance rate of 91.20% was found. Analysis of potential donor allocation interpretation obstacles resulted in simplification of the in-house laboratory testing algorithms and addition of a nucleic acid test to increase the reliability for identification of active (acute) human immunodeficiency virus infection. Hemolysis was more common when sampling was performed more than 24 h after death (p < 0.001). Hemolytic samples more frequently showed a reactive or indeterminate human immunodeficiency virus test result (p < 0.001). Screening for human immunodeficiency virus and T. pallidum was technically more impracticable when hemolysis was present (p = 0.042 and p = 0.003, respectively). Donors with hemolytic blood samples were more often discarded (46.88%) compared to bodies with non-hemolytic samples (6.32%) (p < 0.001). ConclusionsDespite the implementation of donor screening algorithms, a significant number of bodies have an inconsistent allocation. New algorithms, to be evaluated in future research, were suggested. Early postmortem blood sampling is key as hemolysis can influence certain test results and donor allocation.

DNA methylation of decedent blood samples to estimate the chronological age of human remains.

Chronological age estimation may offer valuable investigative leads in human identification cases. Bisulfite pyrosequencing analysis of single CpG sites on five genes (KLF14, ELOVL2, C1orf132, TRIM59, and FHL2) was performed on 264 postmortem blood samples from individuals aged 3months to 93years. The goals were to develop age prediction models based on the correlation between the methylation profile and chronological age and to assess the accuracy of the prediction. Linear regression between methylation levels and age at each CpG site revealed that the five markers show a statistically significant correlation with age. The methylation data from a training set of 160 postmortem blood samples were used to develop an age prediction model with a correlation coefficient of 0.65, explaining 73.1% of age variation, with a mean absolute deviation from the chronological age of 7.60years. The accuracy of the model was evaluated with a test set of 72 samples producing a mean absolute deviation of 7.42years. The training and test sets were also categorized by specific age groups to assess accuracy and deviation from chronological age. The data for both sets revealed a lower prediction potential as an individual increases in age, particularly for the age categories above 50years.

Simple implementation of muscle tissue into routine workflow of blood analysis in forensic cases – A validated method for quantification of 29 drugs in postmortem blood and muscle samples by UHPLC–MS/MS

Whole blood is most often the matrix of choice for postmortem analysis but it is not always available. In these cases, muscle tissue can be used as an alternative matrix. Therefore, an ultra-high-performance liquid chromatography–tandem mass spectrometry method for the quantification of 29 drugs and metabolites of toxicological interest in postmortem muscle tissue was developed and validated. Additionally, a validation of whole blood was carried out to compare the results from the two matrices. Solid-phase extraction was performed by an automated robotic system to minimize manual labour and risk of human errors, and increase robustness, sample throughput and sample traceability. The method was validated in terms of selectivity, matrix effect, extraction recovery, process efficiency, measuring range, lower limit of quantification, carry-over, stability, precision and accuracy. To correct for any inter-individual variability in matrix effects on analyte accuracy and precision, deuterated analogues of each analyte were used as internal standards. The lower limit of quantification in both blood and muscle homogenate ranged between 0.002 and 0.005 mg/kg, while the upper limit of quantification spanned from 0.20 to 1.0 mg/kg. Corrected with the 4-fold dilution factor, the corresponding concentrations in muscle tissue were 0.008–0.02 mg/kg at the lower limit of quantification and 0.80–4.0 mg/kg at the upper limit of quantification. The method showed acceptable precision and accuracy, with precision below 12% and accuracies ranging from 87% to 115% at up to 6 levels for all analytes in both matrices. In addition, comparison between calibration standards in spiked muscle homogenate and spiked blood showed that analyte concentrations in muscle samples could be quantified by using spiked blood samples as calibration standards with acceptable precision and accuracy when using deuterated analogues as internal standards. The investigation of matrix effects showed no great difference between blood and homogenates of non-decomposed and decomposed muscle tissue for most analytes. In the samples where high ion suppression or enhancement was observed, the results were corrected by the internal standards. Statistical comparison of quality control samples in blood and muscle tissue showed no obvious differences, and therefore muscle tissue was included in the routine method for analysis of blood samples and used in autopsy cases where no blood was available. By adding a semi-automated homogenization step before the remaining automated sample preparation, muscle tissue samples were easily incorporated into the workflow of the existing routine method. The present method has been successfully implemented in routine analysis of blood and muscle tissue since 2019.

Methamphetamine use and the risk of diabetic ketoacidosis.

Diabetic ketoacidosis (DKA) is a life-threatening condition arising in individuals with insulin-dependent diabetes mellitus, associated with hyperglycaemia and hyperketonaemia. While drugs such as methadone, cocaine and certain prescription medications may precipitate DKA, the potential effect of methamphetamine is unclear. Analysis of autopsy and toxicology case files at Forensic Science SA, Adelaide, South Australia, was therefore undertaken from 1 January to 31 December 2019 of all cases where methamphetamine was detected in post-mortem blood samples. There were 94 cases with 11 diabetics (n = 6 type 1 and n = 5 type 2). Four of the six decedents with type 1 diabetes had lethal DKA (66.7%; age range 30-54 years; average age 44.6 ± 10.5 years; M:F ratio 1:1). This incidence of DKA was higher than that of the general insulin-dependent diabetic population (6%) and also significantly higher than in medico-legal cases (13%; p < 0.05). The clinical and autopsy assessment of insulin-dependent diabetics presenting with DKA should therefore include specific screening for methamphetamine. The increase in both insulin-dependent diabetes and methamphetamine abuse in the community may lead to increases in such cases in medico-legal and health contexts.

Pathology, bacteriology and molecular studies on caseous lymphadenitis in Camelus dromedarius in the Emirate of Abu Dhabi, UAE, 2015-2020.

Caseous lymphadenitis (CLA) or pseudotuberculosis is a chronic zoonotic bacterial disease caused by Corynebacterium pseudotuberculosis, which affects livestock and humans. This study aimed to describe the pathology, bacteriology and confirm the identity of the pathogen by 16S rRNA gene sequencing in Camelus dromedarius. A total of 12 camels with suspected CLA in three regions of Abu Dhabi Emirate (Abu Dhabi, Al Ain and Al Dhafra), United Arab Emirate (UAE) were subjected to clinical and postmortem examinations from January 2015 to December 2020. Clinically, camels were emaciated and showed the presence of external caseous abscesses suggestive of CLA. Postmortem examination showed multiple abscesses of variable sizes with caseous material encapsulated by fibrous tissue in the liver, lungs, muscle, and lymph nodes. Following clinical and postmortem examination, blood, pus and different tissue samples were collected for subsequent analysis. Histopathological examination of all organs stained with Hematoxylin and Eosin (H&E) indicated a central caseo-necrotic core that was admixed with bacterial colonies and infiltration of chronic inflammatory cells, surrounded by a pyogenic membrane, and an outer fibrous connective tissue capsule. Bacterial culture identified the isolates of Corynebacterium pseudotuberculosis biotype ovis strain, and these isolates were shown to be sensitive to all antibiotics tested (penicillin, ampicillin, Co-trimoxazole, enrofloxacin and tetracycline). Moreover, the identity of the isolates was confirmed by partial sequencing of the 16S rRNA gene which showed a 100% identity to Corynebacterium pseudotuberculosis. Phylogenetic analysis based on 16S rRNA gene sequence clearly differentiates Corynebacterium pseudotuberculosis from other species of Corynebacterium. Briefly, this study provided the basic information for infection of Corynebacterium pseudotuberculosis in Camels and will help in controlling of this pathogen in the region.

Determination of 19 Psychoactive Substances in Premortem and Postmortem Whole Blood Samples Using Ultra-High-Performance Liquid Chromatography–Tandem Mass Spectrometry

An ever-increasing need exists within the forensic laboratories to develop analytical processes for the qualitative and quantitative determination of a broad spectrum of new psychoactive substances. Phenylethylamine derivatives are among the major classes of psychoactive substances available on the global market and include both amphetamine analogues and synthetic cathinones. In this work, an ultra-high-performance liquid chromatography-positive ion electrospray ionization tandem mass spectrometric method (UHPLC-ESI-MS/MS) has been developed and fully validated for the determination of 19 psychoactive substances, including nine amphetamine-type stimulants and 10 synthetic cathinone derivatives, in premortem and postmortem whole blood. The assay was based on the use of 1 mL premortem or postmortem whole blood, following solid phase extraction prior to the analysis. The separation was achieved on a Poroshell 120 EC-C18 analytical column with a gradient mobile phase of 0.1% formic acid in acetonitrile and 0.1% formic acid in water in 9 min. The dynamic multiple reaction monitoring used in this work allowed for limit of detection (LOD) and lower limit of quantitation (LOQ) values of 0.5 and 2 ng mL−1, respectively, for all analytes both in premortem and postmortem whole blood samples. A quadratic calibration model was used for the 12 quantitative analytes over the concentration range of 20–2000 ng mL−1, and the method was shown to be precise and accurate both in premortem and postmortem whole blood. The method was applied to the analysis of real cases and proved to be a valuable tool in forensic and clinical toxicology.

Definite SUDEP population in Bexar County, Texas: A 36-year data registry

ObjectiveSudden unexplained death in epilepsy (SUDEP) is one of the leading causes of mortality in epilepsy. In this study, cases of definite SUDEP are reported for Bexar County from a 36-year period. MethodsDeath reports by Bexar County Medical Examiner (BCME) from 1983 to 2018 were reviewed to identify cases with definite SUDEP. The findings were based upon investigators’ reports, which included medical history, medication list, sleep position, time of death, and pathology reports. In addition to these potential risk factors, body mass indices (BMI), as well as heart, lung, and brain weights were compared between SUDEP victims with therapeutic vs subtherapeutic anti-seizure medication (ASM) levels. ResultsDefinite SUDEP was identified in 286 cases. The incidence of definite SUDEP was 0.51 ± 0.26/1000 person-years among PWE and 0.56 ± 0.29 per 100,000 person-years among the general population in Bexar County. The median age was 37 years old (interquartile range 27–48), and the majority were male (65%). While 171 (60%) people were prescribed at least one ASM, ASM levels were subtherapeutic in 239 cases (83.6%). Risk factors for SUDEP did not differ between SUDEP victims with therapeutic vs subtherapeutic ASM levels. While BMIs were only slightly increased in adherent vs nonadherent SUDEP victims, they were significantly associated with subtherapeutic ASM levels. Abnormal lung, heart, and brain weights were reported in 48 (16.8%), 67 (23.4%), and 43 (15.0%) SUDEP cases, respectively. SignificanceThis study is one of the largest autopsy-based registries of definite SUDEP. Subtherapeutic ASM levels measured in post-mortem blood samples suggest that nonadherence to ASM therapy was a leading risk factor for SUDEP. As BMI was elevated in this cohort, and obesity was significantly associated with subtherapeutic ASM levels, it may also be a risk factor for SUDEP. Case-controlled studies are needed to validate the specific role of obesity and related comorbidities in this population.

Method development for the identification of methoxpropamine, 2-fluoro-deschloroketamine and deschloroketamine and their main metabolites in blood and hair and forensic application

The constant increase of new psychoactive substances, often available on the illicit drug market as 'research chemicals', poses a concern for public health and a significant analytical and legislative challenge. Β-keto-arylcyclohexamines represent a class of dissociative anesthetics recently introduced on the market of New Psychoactive Substances (NPS). There is still a lack of information about the pharmacological activity of many of such substances, usually depending on the potential chemical modifications introduced to circumvent the law. Furthermore, their intake may not be fully intentional, since consumers do not always have knowledge of the content of online purchases. The present study describes the characterization by liquid chromatography-high resolution mass spectrometry (LC-HRMS), using a benchtop Orbitrap instrument, of the novel ketamine analogues methoxpropamine, 2-fluoro-deschloroketamine and deschloroketamine, found in the post-mortem blood and hair samples from a forensic case of suicide by fall from height, and of some of their metabolites. This allowed the development of analytical methods for the determination of both the β-keto-arylcyclohexamines and the metabolites in LC-HRMS and in LC-MS/MS, providing a starting point for studying their toxicokinetics.