Dead or Alive? Identification of Postmortem Blood Through Detection of D-Dimer

Assessment of the role played by n-propanol in distinction of ethanol source in postmortem blood with the assistance of ethyl glucuronide and ethyl sulfate

Over the last 15 years, numerous deaths involving "Ecstasy" (3,4-methylenedioxymethamphetamine, MDMA) have been reported and described in the literature. In most cases, either antemortem or postmortem concentration data are available. Because of the wide range of results and potential idiosyncratic nature of MDMA toxicity, interpretation of both antemortem and postmortem concentrations is difficult. The possible influence of postmortem redistribution may be an overlooked factor, but existing data involve postmortem concentrations from varying anatomical sites. However, this paper describes for the first time an evaluation of the concentrations of MDMA and 3,4-methylenedioxyamphetamine (MDA) found in five fatalities admitted to hospital where both antemortem and postmortem blood samples were available. Admission MDMA and MDA concentrations ranged between 0.55 and 4.33 mg/L and 0 and 0.10 mg/L, respectively, in antemortem serum/plasma. Postmortem blood MDMA and MDA concentrations ranged between 0.47 and 28.39 mg/L and 0.02 and 1.33 mg/L, respectively. Postmortem concentrations were higher than corresponding antemortem concentrations in all 5 cases with postmortem/antemortem ratios between 1.1 and 6.6 for MDMA and 1.5 and 13.3 for MDA. Differences in concentrations were also observed between anatomical sites, with central sites (e.g., heart) having much higher concentrations than peripheral sites (e.g., femoral). Overall, MDMA and MDA appear to exhibit postmortem redistribution and concentrations measured in postmortem specimens (even from peripheral sites) are not directly comparable with antemortem findings close to or prior to death.

Ethanol stability in preserved antemortem blood has been widely studied since it is a common practice in cases involving suspected impaired driving to collect antemortem blood in evacuated blood tubes containing sodium fluoride. In some situations, antemortem blood is submitted to a forensic laboratory for ethanol analysis in evacuated blood tubes that contain only an anticoagulant. There has been limited research on ethanol stability in antemortem blood stored without a preservative. On two occasions, antemortem blood was collected from five ethanol-free individuals into 6-ml Vacutainer® tubes containing only 10.8mg potassium EDTA. The blood tubes were spiked with ethanol to approximately either 0.08 or 0.15 g/dl. Dual-FID headspace gas chromatography was used to analyze 58 blood tubes, 29 from each session, for ethanol 1 day after sample collection and again after 1 year of refrigerated storage (~4°C). Statistically significant decreases in ethanol were detected at the 0.05 level of significance. Mean decreases in ethanol after 1 year of storage for the 0.08 and 0.15 g/dl samples were 0.013 and 0.010 g/dl, respectively. The mean ethanol decrease across all tubes was 0.012 g/dl. The range of decreases for the 58 blood tubes was 0.003-0.018 g/dl. The mean ethanol decreases measured in this unpreserved antemortem blood are comparable in magnitude to those previously observed in antemortem blood containing sodium fluoride after 1 year of refrigerated storage. Ethanol did not increase in the antemortem blood samples despite the absence of sodium fluoride.

Tapentadol is a centrally acting synthetic analgesic which is prescribed for the treatment of a range of chronic pain conditions. Its use in treating various pain conditions is increasing and, as with other opioids, it has the potential to be abused. We describe a three-stage incorporation of tapentadol into validated screening and quantitative methods through: (i) addition of retention time/mass spectral data to a database, (ii) qualitative validation and (iii) quantitative validation. This represents an efficient and flexible approach to the incorporation of new compounds of interest to existing screening methods. Tapentadol was analyzed in blood and serum samples using alkaline liquid-liquid extraction with identification and quantitation by liquid chromatography/time-of-flight mass spectrometry. In a series of six post-mortem cases where tapentadol was detected but was not a primary causative factor in death, blood concentrations ranged from 0.01 to 1.0 mg/L. In two cases where tapentadol was a significant contributor to death, post-mortem blood concentrations were 1.7 and 3.9 mg/L. In one of these fatal cases, ante-mortem blood and serum were also analyzed. The tapentadol concentration in the post-mortem blood was 30% higher than in the ante-mortem blood after a post-mortem interval of 13 days, indicating some potential for post-mortem redistribution. The measured ante-mortem blood:serum ratio was 1.7, and is the first such ratio to be reported. Other drugs were detected in almost all cases, with the majority being prescription analgesics, sedatives and antidepressants. The number of cases in which tapentadol has been detected has increased in recent years, highlighting the importance of screening for this drug in forensic toxicological laboratories.

Postmortem blood drug concentrations change over time as a consequence of postmortem changes and therefore may not reflect the drug concentration at the time of death. In order to reduce the effect of postmortem redistribution on drug concentrations, early collection of samples for analysis is preferable. In addition, many other factors should be considered in the evaluation of drug concentrations in postmortem samples, such as stability of drugs during sample storage as this may be an additional source of variation. The aim of this study was to develop a method for quantifying drug concentrations in postmortem blood samples taken in drug-related deaths. A gas chromatography mass spectrometry (GC-MS) method with solid phase extraction (SPE) was developed and validated for the simultaneous determination of opioids and antidepressant drugs in whole blood. In addition, a liquid chromatography tandem mass spectrometry (LC-MS/MS) method with protein precipitation was developed and validated for simple and accurate analysis of gabapentin, morphine, morphine 3-glucuronide (M3G) and morphine 6-glucuronide (M6G) in whole blood samples. The methods were successfully verified using authentic postmortem blood samples. This study was designed to understand the stability of morphine and its glucuronides, in real postmortem blood samples, after storage in different conditions for short and long periods of time. The stability study revealed that, the concentrations of free and total morphine were stable during storage in the freezer for ~10 years and no significant losses were observed. The stability study of authentic case samples with and without preservative stored in the freezer for three years revealed that, sodium fluoride and potassium oxalate as a preservative for postmortem blood samples does not affect morphine, M3G and M6G stability under storage temperatures of -20°C for three years. This study also aimed to investigate morphine and morphine metabolites, together with their ratios, in order to achieve a comprehensive interpretation of time since death. The investigation of morphine to the respective glucuronide concentration ratio to estimate the survival times after administration of heroin revealed that, the lower ratios of these conjugates to total morphine (TM), are suggestive of a more rapid death, as there has been less time for the metabolism of morphine to occur. Similarly, the presence of 6MAM in the blood of a heroin toxicity death suggests a more rapid death. More specifically, when M3G/TM is less than 0.40 could show a quick death in the absence of 6MAM in the blood. The purpose of this study was to investigate changes in the concentration of specific drugs (gabapentin, morphine, M3G and M6G) in postmortem blood samples between death and autopsy, and to identify any patterns of these changes. The sampling technique and site of sampling (peripheral and central) were considered to further define the extent of PMR of drugs and identify a possible mechanism of PMR. Concerning sampling site, for all substances, femoral blood concentrations were significantly lower than those found in cardiac blood, indicating that femoral blood is probably less prone to PMR. In addition, the evaluation of drug concentrations in postmortem samples collected using different techniques of blood sampling revealed that, needle puncture in the upper thigh (blind stick) as opposed to dissection of the same vein, also appeared to have an effect on femoral drug concentrations, since femoral concentrations tended to be closer to cardiac concentrations with the dissection sampling than the blind stick sampling technique. Finally, the analytical results derived from sampling (blind stick) as soon as possible to assess whether it is necessary to obtain blood samples prior to autopsy and prevent contamination by PMR, suggest that PMR is a continuous phenomenon in central as well as in peripheral compartments, but also that femoral blood appears more resistant to it. Generally, the results conclude that PMR is an ongoing phenomenon in central as well as in peripheral compartments, but also that femoral blood seems more resistant to it. Therefore, to avoid the effect of the pre-autopsy interval on drug concentrations it is always preferable for early collection of samples for analysis, which are collected closer to the time of death, and would enable a better assessment of the likely contribution of drugs to the death.

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.

A simple, rapid and sensitive method is described for the determination of omethoate, dimethoate, diazinon, chlorpyrifos, parathion-ethyl, chlorfenvinphos, quinalphos and azinphos-ethyl in postmortem whole blood samples. The analytes and internal standard (ethion) were isolated from the matrix by solid-phase extraction, and were analysed by gas chromatography/mass spectrometry in the selected ion monitoring mode. The method has shown to be selective after analysis of postmortem samples of 40 different origins. Calibration curves were established between 0.05 (0.1 for omethoate) and 25 µg/mL, and the values obtained for intra- and interday precision and accuracy were within the criteria usually accepted for bioanalytical method validation. Lower limits of quantitation were 50 ng/mL for all compounds, except for omethoate (100 ng/mL); the limits of identification of the method were 25 ng/mL for all analytes, except for omethoate, for which 50 ng/mL was obtained. Absolute recovery was determined at three concentration levels, and ranged from 31 to 108%. The proposed method is simple and fast, and can be routinely applied in the determination of these compounds in postmortem whole blood samples within the scope of forensic toxicology. In addition, mass spectrometry has demonstrated to be a powerful and indispensable tool for the unequivocal identification of the analytes, since the acceptance criteria were accomplished even at very low levels, thus allowing obtaining forensically valid and sound results.

Development and validation of a multi-substance method for routine analysis of pesticides in post-mortem samples by Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry

Defined serological blood tests of deceased cornea donors are required to minimize the risk of viral infections of a transplant recipient as much as possible. Haemolysis, autolysis and bacterial contamination, may produce significant changes of post-mortem blood samples, which may lead to false serological test results. Pre- and post-mortem findings from the same cornea donors of the University Tissue Bank of the Charité in the years 2004-2009 (n = 487) were retrospectively analyzed and compared. The test results from pre-mortem blood samples were defined as the reference for the post-mortem blood test. Of 487 cornea donors, there were a total of 21 cases (4.3%) with discrepancies between serological test results from pre- and post-mortem blood samples. Of these, 7 values referred to the HBsAg-testing, 3 to the anti-HBs-, 1 to the anti-HBcIgG + IgM-, 1 to the anti-HCV-, 4 to the anti-HIV 1/2- and 5 to the TPLA-findings. False negative results within post-mortem serology occurred in 4 of 487 cases (0.8%). False positive results within the post-mortem blood samples occurred at a much more frequent rate, with 17 of 487 cases (3.5%). Discrepancies between serological pre- and post-mortem blood tests occur mainly due to the use of non-validated test systems. Therefore, it seems reasonable to test pre- and post-mortem blood samples serologically, whenever possible, at the same time, regardless of the sample age. Positive results, regardless of the sample type, should always be retested with validated confirmation tests (e.g. NAT), in order to differentiate between false and true positive results.

Benefits of simultaneous screening and quantitation of drugs and their metabolites in post-mortem samples by liquid chromatography-high resolution mass spectrometry

Plant toxin poisoning–a disguised diagnosis?

A common defense challenge when antemortem blood ethanol results are presented at trial is the assertion that ethanol was formed in the blood tube after the blood draw through fermentation of the blood glucose by Candida albicans (C.Albicans). In contrast, decades of research into the stability of ethanol in antemortem blood collected for forensic purposes have consistently shown that any analytically significant change in ethanol concentration is a decrease and initially, ethanol-negative blood remains ethanol-negative with storage. For there to be any possibility of fermentation to occur by C.Albicans in an antemortem blood sample there must be a plausible mechanism for introduction of C.Albicans into the blood. One mechanism proffered at trial is environmental contamination resulting from ambient air drawn into the evacuated blood collection tube. Blood was drawn from ethanol-free individuals into 6 and 10-ml gray-top Vacutainer® tubes containing sodium fluoride and 6-ml Vacutainer® tubes without a preservative. Following the blood draws, the tubes were stored unstoppered at room temperature for 24 or 48 h in various locations. Following unstoppered storage, the tubes were stoppered and stored refrigerated (~4°C), left at room temperature (~22°C), or placed in an oven (37°C). The refrigerated blood was analyzed for ethanol using headspace gas chromatography after both 5 days and 32 months. Unrefrigerated blood samples were analyzed after being stored at room temperature or in an oven for up to 30 days. Ethanol was not detected in any of the blood tubes after storage regardless of storage time, storage temperature, or preservative concentration.

Qualitative medetomidine detection in ante- and post-mortem samples via toxicology surveillance testing, Michigan 2024-2025.

Evaluation and usefulness of reverse dot blot DNA-PolyMarker typing in forensic case work

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).