A model of evaluative opinion to encourage greater transparency and justification of interpretation in postmortem forensic toxicology.

Over the past decades, the calls to improve the robustness of interpretation in forensic science have increased in magnitude. Forensic toxicology has seen limited progress in this regard. In this work, we propose a transparent interpretive pathway for use in postmortem forensic toxicology cases. This process allows the selection of the interpretive methodology based on the amount of previous information that is available for the drug(s) in question. One approach is an assessment of various pharmacological and circumstantial considerations resulting in a toxicological significance score (TSS), which is particularly useful in situations where limited information about a drug is available. When there is a robust amount of case data available, then a probabilistic approach, through the evaluation of likelihood ratios by the forensic toxicologist and of prior probabilities by the fact finder, is utilized. This methodology provides a transparent means of making an interpretive decision on the role of a drug in the cause of death. This will allow the field of forensic toxicology to take a step forward in using best practice in evaluative reporting, a tool already used by many other forensic science disciplines.

Designer Benzodiazepines Gidazepam and Desalkygidazepam (Bromonordiazepam): What Do We Know?

Designer benzodiazepines are one of the primary new psychoactive substance (NPS) threats around the world, being found in large numbers in postmortem, driving under the influence of drugs and drug-facilitated sexual assault cases. Even though when compared to many other NPS types, such as opioids and cathinones, there are relatively few designer benzodiazepines being monitored. Recently, a new NPS benzodiazepine has been reported in Europe, the USA and Canada, desalkygidazepam, also known as bromonordiazepam. This substance is a metabolite of the prodrug gidazepam, a drug licensed for use in Ukraine and Russia under the name Gidazepam IC®. In the paper, we review what is currently known about the use, pharmacology and analytical detection of gidazepam, its metabolite desalkygidazepam and their other possible metabolites.

The blood-to-plasma ratio and predicted GABAA-binding affinity of designer benzodiazepines.

PURPOSE: The number of benzodiazepines appearing as new psychoactive substances (NPS) is continually increasing. Information about the pharmacological parameters of these compounds is required to fully understand their potential effects and harms. One parameter that has yet to be described is the blood-to-plasma ratio. Knowledge of the pharmacodynamics of designer benzodiazepines is also important, and the use of quantitative structure-activity relationship (QSAR) modelling provides a fast and inexpensive method of predicting binding affinity to the GABAA receptor. METHODS: In this work, the blood-to-plasma ratios for six designer benzodiazepines (deschloroetizolam, diclazepam, etizolam, meclonazepam, phenazepam, and pyrazolam) were determined. A previously developed QSAR model was used to predict the binding affinity of nine designer benzodiazepines that have recently appeared. RESULTS: Blood-to-plasma values ranged from 0.57 for phenazepam to 1.18 to pyrazolam. Four designer benzodiazepines appearing since 2017 (fluclotizolam, difludiazepam, flualprazolam, and clobromazolam) had predicted binding affinities to the GABAA receptor that were greater than previously predicted binding affinities for other designer benzodiazepines. CONCLUSIONS: This work highlights the diverse nature of the designer benzodiazepines and adds to our understanding of their pharmacology. The greater predicted binding affinities are a potential indication of the increasing potency of designer benzodiazepines appearing on the illicit drugs market.

Forensic alcohol calculations in transgender individuals undergoing gender-affirming hormonal treatment.

There are an increasing number of individuals undergoing gender-affirming hormonal treatment (GAHT) to treat gender dysphoria. Current forensic alcohol calculations require knowledge of the sex of the individual, but this may disadvantage trans people as research has demonstrated that there are physiological changes in individuals who are undergoing GAHT. Using previously published studies on total body water (TBW) in cis individuals, and the known changes in lean body mass and hematocrit in trans individuals, it is possible to estimate TBW in trans individuals and compare them to those cis equation estimations. When using these revised rubrics, we determined that for trans women the use of the cis male anthropometric TBW equation only gives a small underestimation of TBW (0.9%) compared to the underestimation of TBW using the cis female TBW equation (-17.7%). For trans men, the use of the cis female TBW equation gives the largest disadvantage, underestimating TBW by -10.8% compared to the cis male TBW equation, that overestimates TBW by 6.6%. For this reason, we recommend that if the sex at birth of an individual is not known or disclosed, any forensic alcohol calculations in a forensic alcohol reports are made assuming that the gender declared by the individual is their sex at birth. Further research to develop validated anthropometric TBW equations are urgently needed as to not disadvantage trans people when forensic alcohol calculations are carried out.

Revised equations allowing the estimation of the uncertainty associated with the Total Body Water version of the Widmark equation.

Widmark calculations are the most commonly used alcohol calculations to estimate a) the amount of alcohol consumed based on blood alcohol concentration (BAC) and b) BACs at a set time after consumption of a known amount of alcohol. These calculations are vital in forensic casework. Previous work has demonstrated that using general error propagation-based equations the variability associated with alcohol calculations can be estimated, but these equations have only been determined for the volume of distribution version of the Widmark equation. However, recent investigations have shown that the total body water (TBW) version of the Widmark equation is more reliable than the version that utilizes the apparent volume of distribution of ethanol. To date, there is no general error propagation equation to determine the variability associated the TBW version of the Widmark equation. Using previously published studies of 185 individuals in which alcohol elimination rate (ß) and ethanol's volume of distribution were determined, we have shown that there is a negative correlation (-0.247) between the alcohol elimination rate (ß) and TBW. Using these data, we were able to produce equations allowing the estimation of the variability of the results calculated using the TBW version of the Widmark equation. This will allow forensic practitioners to give the best determination of the variability associated with Widmark calculations currently possible.

Just say no to postmortem drug dose calculations.

For years, a number of professional groups have warned forensic and clinical toxicologists against calculating an administered dose of a drug based on postmortem blood drug concentrations. But to date, there has been limited information as to how unreliable these dose calculations may actually be. Using amitriptyline as a model drug, this study used empirically determined pharmacokinetic variables for amitriptyline from clinical studies coupled with clinical overdoses (where the individual survived), and death case studies (ascribed to amitriptyline toxicity) in which the dose of amitriptyline was known. Using these data, standard pharmacokinetic equations, and general error propagation, it was possible to estimate the accuracy of calculated doses of amitriptyline, compared with the doses that were consumed. As was expected in postmortem cases, depending on the pharmacokinetic equation used, the accuracy (mean +128% to +2347%) and precision (SD ± 383% to 3698%) were too large to allow reliable estimations of the dose of amitriptyline consumed prior to death based on postmortem blood drug concentrations. This work again reinforces that dose calculations from postmortem blood drug concentrations are unreliable.

Femoral blood concentrations of the designer benzodiazepine etizolam in post-mortem cases.

Etizolam is a thienodiazepine that although licensed for clinical usage in Japan, India and South Korea is commonly abused and detected in post-mortem cases around the world. To date, there are limited data in the literature to allow for the interpretation of blood concentrations of etizolam in post-mortem cases. A liquid chromatography with tandem mass spectrometry method was used to quantitate etizolam concentrations in 28 post-mortem cases where etizolam was detected. The median concentration of etizolam in femoral blood was 8.5 ng/mL (range 1.0-172.0 ng/mL; n = 24); in antemortem plasma, the etizolam concentration range was 4-44 ng/mL (n = 4). The mean age of the individuals abusing etizolam was 38.5 ± 8.4 years (median 39 years), with the majority being male (86%). In all of the cases, multiple drugs were detected, with the most common being pregabalin (61%) followed by morphine/heroin (54%), diazepam (54%) and benzoylecgonine (21%), illustrating the increasing problem of poly-substance use in drug abusers. The cause of death in the cases in which etizolam was detected was multi-drug toxicity in 87.5% of the cases, with 12.5% unrelated to drug use (hangings and blunt-force trauma). These data will further help forensic practitioners with the interpretation of post-mortem etizolam concentrations.

Total body water is the preferred method to use in forensic blood-alcohol calculations rather than ethanol's volume of distribution.

During the prosecution and defence of drink-driving cases, forensic practitioners are often required to engage in various blood-alcohol calculations, such as whether or not the statutory limit was exceeded (e.g. 80mg/100mL, 0.08g/100mL or 0.80g/L). For this purpose, most forensic scientists utilize the Widmark equation, or some modification thereof, to calculate a person's blood alcohol concentration (BAC) based on information about the amount of ethanol consumed and the pattern of drinking. This equation comes in two main forms; one of which incorporates the apparent volume of distribution of ethanol (V) and the other a person's total body water (TBW). In this study, we utilised two independent data sets, one involving the determination of V for ethanol in 173 men and 63 women, and the other TBW determined for 582 men and 884 women. Those subjects included in the TBW group represented various racial groups (Caucasians, African Americans, Hispanics, Asians and Puerto Ricans), with body mass index (BMI) ranging from 17 to 80kg/m2. Both versions of the Widmark equation were evaluated in relation to their accuracy and precision in predicting TBW and/or V using the two most common anthropometric equations; those of Watson et al. and Forrest. Both anthropometric equations exhibited good accuracy (<4.3%) for the prediction of both TBW and V. However, the root mean square error was lower TBW was used for prediction (9.09-12.84%) rather than V (11.72-15.08%). Overall, this study has demonstrated (a) that blood-alcohol calculations are more reliable using TBW rather than V (b) that both equations (Watson et al. and Forrest) are applicable to ethnic groups other than Caucasians and (c) the Forrest equation predicts TBW in men and women with BMI from 17 to 35kg/m2 and that the Watson et al. equation works for those with more extreme BMI; females (17-80kg/m2) and males (17-67kg/m2).

The Contribution of Body Mass and Volume of Distribution to the Estimated Uncertainty Associated with the Widmark Equation.

The Widmark equation is used forensically for the determination of the amount of ethanol (alcohol) that may have been consumed and also to determine the blood alcohol concentration (BAC) of an individual at a specific time. It is important to be able to estimate the uncertainty associated with Widmark equations. To date, there has been no detailed determination of contribution to the final uncertainty of Widmark calculations of the volume of distribution of ethanol (Vd ), using anthropometric equations, or the contribution of an individual's body mass. Using published data, published literature, and freedom of information data, we determined that the variability (%CV) associated with Vd was ~10% (Watson et al. and Forrest anthropometric equations) and that the %CV associated with estimated body mass was ~15% compared to ~3% when body mass was directly measured. These data allow an estimation of the overall uncertainty of Widmark calculations using general error propagation. The estimated total uncertainty for BAC calculations increased from ~11% (volume consumed) and ~22% (BAC) to ~19% (volume consumed) and ~37% (BAC) when using measured body mass compared to estimated body mass. These results demonstrate that forensic practitioners should be mindful of the increase in estimated uncertainty in calculated Widmark equation results when estimated body mass is used rather than measured body mass. These data further improve the knowledge around the uncertainty of results calculated with the Widmark equation.

Using µX-Ray CT to observe postmortem diffusion from the stomach in a rat model.

The stomach has long been recognised as a depot for postmortem diffusion. A better understanding of the phenomena of postmortem diffusion would aid forensic practitioners in their interpretation of toxicological results. A limitation of previous stomach diffusion studies was the lack of ability to visualise postmortem diffusion in real time, the use of µX-ray Computed Tomography (CT) could overcome this problem. We utilised CT to track the diffusion of the contrast medium caesium ions (Cs+) (administered by oral gavage) from the rat stomach over 6 days. We investigated the influence of temperature (4°C and 20°C) and body position (horizontal and vertical). The results show that the a) diffusion of Cs+ from the stomach can be visualised over 6 days, over which a significant amount (∼50%) of the diffusion occurs in the first 24h following administration; b) storing the rat at 4°C reduces the distance of diffusion from the stomach by ∼66%; c) body position influences the route of diffusion and d) in 2 of the 16 rats studied Cs+ was found in the right lobe of the liver. Overall these results show that CT using Cs+ is a good model to visualise postmortem diffusion and that bodies show significant variation in postmortem diffusion. It is also clear that bodies should be refrigerated and postmortem samples should be taken as soon as possible to minimise the influences of postmortem diffusion from the stomach.

Application of a Bayesian network to aid the interpretation of blood alcohol (ethanol) concentrations in air crashes.

In the investigation of a fatal air crash, it is important to determine if the pilot, at the time of death, was contravening the regulations in relation to 1) the permitted concentration of ethanol (alcohol) in the blood and 2) whether the pilot had consumed alcohol within a specified period before flying. It is also important to assess whether any alcohol detected in the toxicological samples was present either because of consumption or because of post-mortem alcohol formation. We have developed a Bayesian Network that models the relationships between analytical results, circumstantial evidence and the concentration of alcohol at the time of death in cases of air crash. The model provides a rational, coherent approach to forensic interpretation, moving interpretation from a largely subjective, generalist approach to a more objective, case-specific methodology utilising available relevant data and accommodating the inevitable uncertainties within a case.

Post-mortem diagnosis of kidney impairment: An experimental study.

The determination of the role that drugs may have played in a death is an important part of the investigation into unexplained deaths. Renal impairment may lead to a reduction in drug excretion rate and therefore an accumulation of drugs or metabolites, leading to possible toxic or lethal effects. Creatinine levels are known to be stable in the post mortem period and in life can give an indication of kidney function. There are however widely reported limitations when using creatinine in isolation and so we investigated the usefulness of using estimated glomerular filtration rate (eGFR) for scoring an individual as having renal impairment using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula. We analysed unpreserved vitreous for creatinine in 812 individuals using an isotope dilution mass spectrometry (ID-MS) traceable enzymatic. We found that the biochemical analysis of post mortem vitreous creatinine and subsequent calculation of eGFR is a useful adjunct to the standard testing that takes place during a post-mortem examination and can assist in death investigation. Using an eGFR of <60 mL/min/1.73 m2 gave a sensitivity of 94.3% and specificity of 97.3% when scoring an individual as having renal impairment. We therefore recommend the calculation of eGFR for the determination of possible renal impairment in post mortem investigations. It is, of course, always pertinent to interpret any results using a wealth of case information. Extreme caution should be exercised in cases where insufficient clinical information/history is available, particularly in cases in which there is suspected diabetic ketoacidosis, dehydration or hospitalisation prior to death.

Uncertainty in Widmark calculations: ABV variation in packaged versions of the most popular beers in the UK.

Forensic practitioners regularly use the Widmark equation to determine theoretical blood alcohol concentrations for use in cases involving alcohol. It is important with these calculations to determine the uncertainty associated with any result. Previous work has investigated the uncertainty in percent alcohol by volume (%ABV) from beers produced by small independent breweries in the UK but did not study the top selling beers in the UK. The top selling lagers and ales/bitters in the UK were identified by sales volume and the %ABV determined. These data was then used to determine the percent coefficient of variation (%CV) that should be used by forensic practitioners when constructing alcohol technical defence reports for use in forensic cases. These samples, from what may be described as 'big' brewers, were determined to have a smaller root mean square error (RMSE) (±0.1%v/v, n = 35), and %CV than those previously reported for beers produced by small, independent breweries in the UK. The results from this study shows that different RMSE's should be used for %ABV when determining the uncertainty of results from Widmark calculations depending if the drinks consumed have been from either 'big' brewers or small, independent breweries.

Postmortem tissue distribution of morphine and its metabolites in a series of heroin-related deaths.

The abuse of heroin (diamorphine) and heroin-related deaths are increasing around the world. The interpretation of the toxicological results from suspected heroin-related deaths is notoriously difficult, especially in cases where there may be limited samples. To help forensic practitioners with heroin interpretation, we determined the concentration of morphine (M), morphine-3-glucuronide (M3G), and morphine-6-glucuronide (M6G) in blood (femoral and cardiac), brain (thalamus), liver (deep right lobe), bone marrow (sternum), skeletal muscle (psoas), and vitreous humor in 44 heroin-related deaths. The presence of 6-monoacetylmorphine (6-MAM) in any of the postmortem samples was used as confirmation of heroin use. Quantitation was carried out using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with solid-phase extraction. We also determined the presence of papaverine, noscapine and codeine in the samples, substances often found in illicit heroin and that may help determine illicit heroin use. The results of this study show that vitreous is the best sample to detect 6-MAM (100% of cases), and thus heroin use. The results of the M, M3G, and M6G quantitation in this study allow a degree of interpretation when samples are limited. However in some cases it may not be possible to determine heroin/morphine use as in four cases in muscle (three cases in bone marrow) no morphine, M3G, or M6G were detected, even though they were detected in other case samples. As always, postmortem cases of suspected morphine/heroin intoxication should be interpreted with care and with as much case knowledge as possible.

Evidence based survey of the distribution volume of ethanol: Comparison of empirically determined values with anthropometric measures.

The Widmark equation is commonly used when blood alcohol calculations are required in forensic and legal medicine, such as in road-traffic cases and alcohol-related deaths. An important biological variable in this connection is the volume of distribution (Vd) of ethanol, which is commonly referred to as the rho-factor. Although a person's Vd can be determined empirically through controlled drinking experiments, this approach is not very practical in reality. For this reason, a number of anthropometric equations have been developed that utilize sex, age, height and weight to estimate the person's total body water (TBW) and hence Vd of ethanol. To date, there are not any studies that compare Vd derived from anthropometric data with robust values measured empirically. From the literature we compiled information about the Vd of ethanol from drinking studies with 173 Caucasian males and 63 Caucasian females from Western Europe. These empirically derived values of Vd were then compared with estimates derived from various anthropometric equations. In males the Watson, Watson and Batt regression equation involving age, height and weight gave the most accurate results (bias was 0.00L/kg) and 95% range ±0.13L/kg. The equation derived by Forrest, which took into consideration a person's body mass index (BMI), gave the best estimates of Vd for females; mean bias -0.01L/kg and range ±0.15L/kg.

Use of the Randox Evidence Investigator immunoassay system for near-body drug screening during post-mortem examination in 261 forensic cases.

BACKGROUND: This paper describes the performance of four Randox drug arrays, designed for whole blood, for the near-body analysis of drugs in a range of post-mortem body specimens. METHODS: Liver, psoas muscle, femoral blood, vitreous humor and urine from 261 post-mortem cases were screened in the mortuary and results were obtained within the time taken to complete a post-mortem. Specimens were screened for the presence of amfetamine, barbiturates, benzodiazepines, benzoylecgonine, buprenorphine, cannabinoids, dextropropoxyphene, fentanyl, ketamine, lysergide, methadone, metamfetamine, methaqualone, 3,4-methylenedioxymetamfetamine, opioids, paracetamol, phencyclidine, salicylate, salicylic acid, zaleplon, zopiclone and zolpidem using the DOA I, DOA I+, DOA II and Custom arrays. RESULTS: Liver and muscle specimens were obtained from each of the 261 post-mortem cases; femoral blood, vitreous humor and urine were available in 98%, 92% and 72% of the cases, respectively. As such, the equivalent of 12,978 individual drug-specific, or drug-group, immunoassay tests were undertaken. Overall >98% of the 12,978 screening tests undertaken agreed with laboratory confirmatory tests performed on femoral blood. CONCLUSIONS: There is growing interest in the development of non-invasive procedures for determining the cause of death using MRI and CT scanning however these procedures are, in most cases, unable to determine whether death may have been associated with drug use. The Randox arrays can provide qualitative and semi-quantitative results in a mortuary environment enabling pathologists to decide whether to remove specimens from the body and submit them for laboratory analysis. Analysis can be undertaken on a range of autopsy specimens which is particularly useful when conventional specimens such as blood are unavailable.

The influence of alcohol content variation in UK packaged beers on the uncertainty of calculations using the Widmark equation.

It is common for forensic practitioners to calculate an individual's likely blood alcohol concentration following the consumption of alcoholic beverage(s) for legal purposes, such as in driving under the influence (DUI) cases. It is important in these cases to be able to give the uncertainty of measurement on any calculated result, for this reason uncertainty data for the variables used for any calculation are required. In order to determine the uncertainty associated with the alcohol concentration of beer in the UK the alcohol concentration (%v/v) of 218 packaged beers (112 with an alcohol concentration of ≤5.5%v/v and 106 with an alcohol concentration of >5.5%v/v) were tested using an industry standard near infra-red (NIR) analyser. The range of labelled beer alcohol by volume (ABV's) tested was 3.4%v/v - 14%v/v. The beers were obtained from a range of outlets throughout the UK over a period of 12 months. The root mean square error (RMSE) was found to be ±0.43%v/v (beers with declared %ABV of ≤5.5%v/v) and ±0.53%v/v (beers with declared %ABV of >5.5%v/v) the RMSE for all beers was ±0.48%v/v. The standard deviation from the declared %ABV is larger than those previously utilised for uncertainty calculations and illustrates the importance of appropriate experimental data for use in the determination of uncertainty in forensic calculations.