A comparative study of the microbiological contamination of postmortem blood and vitreous humour samples taken for ethanol determination

Postmortem blood and vitreous humour samples were taken from each of 51 subjects. None of the vitreous humour samples contained large numbers of bacteria or fungi, whereas many micro-organisms were detected in 32 of the blood samples. The results of the microbiological examinations provided useful information for the interpretation of some ethanol levels that might otherwise have been misleading.     

Post-mortem hypoxanthine levels in the vitreous humour an introductory report

Post-mortem hypoxanthine levels in vitreous humour were determined in 86 consecutive legal autopsy cases. In cases of sudden death caused by trauma or by myocardial infarction, levels ranging from 0 to 540 μmol/l were found. The mean value was about ten times higher than normal in vivo plasma levels. The hypoxanthine levels seem to be independent of time post-mortem, at least during the first 48 hours.It is known that augmentation of the hypoxanthine plasma, cerebrospinal fluid, and urine levels reflects tissue hypoxia. In the present material no elevation of hypoxanthine levels in the vitreous humour was found in cases of strangulation or suspension, while statistically significant elevation was found in cases of drug intoxication. It is concluded that this may reflect the effect of drug-induced prolonged tissue hypoxia caused by respiratory depression.     

Analysis of carbohydrate-deficient transferrin (CDT) in vitreous humour as a forensic tool for detection of alcohol misuse

Analysis of carbohydrate-deficient transferrin concentration in vitreous humour (VH-CDT) has recently been demonstrated to be useful for diagnosis of pre-mortal alcohol misuse, but more knowledge considering possible methodological problems is warranted. In a forensic sample we examined the stability of VH-CDT during laboratory handling as well as the possible affection of time-dependent changes of total transferrin concentrations in vitreous humour (VH-Transferrin) in the dead body on VH-CDT as indicator of alcohol misuse. By use of a commercial assay designed for serum analysis (CDTect) it was possible to measure VH-CDT with high precision, and detectable amounts were found in 20 of 21 alcoholics and in two of seven controls. The compound was demonstrated to be stable in vitreous humour during laboratory handling, since the results of the first analysis were well reproducible after 4 months storage of the specimens (rs=0.86, P=0.0002). Transferrin concentrations in vitreous humour (VH-Transferrin) correlated to the estimated time since the individual last time had been alive before the forensic examination (rs=0.57, P<0.005). However, in this small sample the discriminating property of VH-CDT as indicator of alcohol misuse was not decisively affected, whether or not the variables "VH-Transferrin" and "estimated time since the individual last time had been alive" were considered in a multivariate logistic regression analysis (minor change in the beta-coefficient from 0.57 to 0.52, P=0.086). We conclude that CDT is stable in vitreous humour during proper laboratory handling and that detection of VH-CDT is primarily a marker of alcohol misuse before death. However, the results of this study do not exclude that time-dependent changes of VH-Transferrin, possibly affecting VH-CDT, may occur in the dead body. VH-CDT analysis should thus, at present, be restricted to cases with rather short post-mortal time interval.     

Direct analysis of anticonvulsant drugs in vitreous humour by HPLC using a column switching technique

A method is described for the rapid HPLC analysis of a number of anticonvulsant drugs in vitreous humour by injection directly onto a preconcentration column without the need for prior extraction. The conditions for optimum precision and accuracy were investigated and the method was subsequently applied to the analysis of phenytoin in a number of overdose cases. Sensitivity was 0.05 microgram/ml on a 0.5 ml vitreous sample. The mean blood/vitreous ratio for phenytoin was found to be 2.8 (range: 1.4-5.1). The relationship between blood and vitreous levels had a correlation coefficient of 0.85. Vitreous was found to be a very clean, stable sample, ideally suited to this technique.     

Magnesium, potassium, sodium and calcium in post-mortem vitreous humour from humans

Levels of magnesium, potassium, sodium and calcium in post-mortem vitreous humour from human controls, fire fatalities and drowning victims have been determined. The effects of time-related internal changes, external environmental parameters and different causes of death are evaluated. Despite the positive correlation and marked increase of potassium and, to a lesser extent, of magnesium and calcium with the length of the post-mortem interval, individual biological variability severely limits the usefulness of predictions of post-mortem interval based on electrolyte metal data. At best, there is only a 2/3 chance of a prediction being within 12 h of the true value. Vitreous humour metal concentrations are affected by external influences, such as the elevated temperatures of fires which increase the rate of release of intracellular magnesium and potassium. In cases where drowning is suspected, establishment or exclusion of this cause of death is not possible on the basis of vitreous humour electrolyte metal data because of possible post-immersion diffusion across the permeable membrane of the eyeball. It appears, however, that magnesium in salt-water cases and sodium in fresh-water cases are related, albeit erratically, to the length of the immersion period.     

Isotachophoretic determination of lactate ions in vitreous humour samples and its use in forensic toxicology

Isotachophoresis has been successfully used to determine the lactate ion concentrations in 40 vitreous humour samples taken from subjects representative of those normally encountered by the forensic pathologist. The method is described, and the use of the analysis in forensic toxicology is discussed.     

The biochemistry of vitreous humour. A comparative study of the potassium, sodium and urate concentrations in the eyes at identical time intervals after death

Analysis of the vitreous humour from an individual's eyes collected at the same time since death showed variation between the eyes in the concentration of potassium, sodium and/or urate. This previously unreported finding would limit the use of measuring these electrolytes in determining the time of death.     

On the phenomenology of lethal applications of insulin

This study deals with the postmortem findings in cases of lethal hypoglycaemia due to injections of insulin. In 12 cases (four female; eight male; mean age 42 years) the following aspects were evaluated retrospectively: circumstances of life, scene of death, pathomorphological findings and postmortem biochemistry on cerebrospinal fluid, vitreous humour, blood, and urine (levels of glucose, lactate, hemoglobin A_1c and insulin). Furthermore, analyses of ethanol in blood and urine as well as toxicological and histological examinations were performed. Unexpectedly, the dead persons rarely represented diabetics, relatives of diabetics, or medical personnel. It is concluded, that the diagnosis of fatal hypoglycaemia can only be established by a synopsis of postmortem biochemistry, pathomorphological alterations and anamnesis. Besides, this diagnosis must always be made “per exclusionem”.     

Suicide by injection of a veterinarian barbiturate euthanasia agent: report of a case and toxicological analysis

The history and toxicological findings of a suicidal case involving injection of a veterinarian barbiturate euthanasia agent (Vetanarcol containing pentobarbital are presented. Blood pentobarbital concentrations compatible with drug overdose were determined. Almost identical levels were found in blood, cerebrospinal fluid (CSF) and vitreous humour (VH). The highest concentration was measured in the bile. The present case is compared with similar rare cases in the literature.     

Postmortem concentrations of citalopram

The postmortem concentrations of citalopram in blood, bile, liver, and vitreous humour were investigated in 14 cases using a specially developed high performance liquid chromatography assay. Concentrations from drug and non-drug related deaths were categorized to determine a postmortem therapeutic and toxic range. Therapeutic citalopram concentrations for blood, bile, liver, and vitreous humour ranged to 0.4 mg/L, 2.1 mg/l, 6.6 mg/kg, and 0.2 mg/L, respectively. In one potentially fatal response to citalopram, concentrations were 0.8 mg/L, 6.0 mg/L, 0.3 mg/L for blood, bile and vitreous humour, respectively.     

“You must be joking”: the role of humour in the law classroom

There is a body of literature, including persuasive empirical evidence, linking the use of positive humour in tertiary classrooms with the creation of a relaxed learning environment, student motivation, attendance and engagement as well as positive student evaluations of teacher performance. However, the literature on the use of humour in teaching law is generally limited to anecdotal evidence. Drawing on the literature on using humour in teaching courses that students perceive as “difficult” in other disciplines, in this article we explore the benefits and pitfalls of using humour in the law classroom and provide illustrations of how humour might be used appropriately and effectively in teaching law.     

Blood, bone marrow and eye fluid ethanol concentrations in putrefied rabbits

The effect of putrefaction on postmortem blood, bone marrow and eye fluid ethanol levels was evaluated in rabbits. Control and dosed animals were sacrificed and stored at either room temperature (approx. 19 degrees C) or cold temperature (approx. 3.5 degrees C) for as long as 28 days. Control animals stored at room temperature showed ethanol levels in the bone marrow that peaked at 7 days after sacrifice, followed by decreases to a nondetectable level at 21 days. Overall decreases were demonstrated in bone marrow of dosed rabbits stored at room temperature for all postmortem intervals. The control animals stored at low temperature showed no ethanol in the bone marrow and blood until 21 days after sacrifice. Dosed rabbits stored at low temperature showed no significant changes in blood and marrow ethanol until 21 days after sacrifice.     

Estimation of the post-mortem interval: Effect of storage conditions on the determination of vitreous humour [K+]

Obtaining a reliable estimate of the post-mortem interval (PMI) has been a long-running challenge in forensic medicine. Several more or less successful techniques for making such estimates have been developed, but in recent years important advances have been made thanks to the detailed study of the relationship between the PMI and the analytes - in particular K⁺ - of the vitreous humour (VH). The extraction and pre-treatment of VH samples has been standardized, the influence of certain environmental factors on analytical results has been quantified, and some of the circumstances under which techniques become unreliable have been identified. The present work examines how the conditions to which VH samples are subject in routine practice may alter the results of their analysis. Exposure to light and ambient temperature was found to alter the values returned in determinations of VH [K⁺], [Na⁺] and [Cl^-], while exposure to several freezing/thawing cycles (even with final heating) led to no significant modifications in determinations of VH [K⁺] and [Na⁺]. It is recommended that if analysis has to be delayed, VH should be frozen for storage in a refrigerator before bringing to room temperature for processing. It is also recommended that samples not be exposed to ambient light and temperature.     

A fatal poisoning involving Bromo-Dragonfly

This paper reports a fatal overdose case involving the potent hallucinogenic drug Bromo-Dragonfly (1-(8-bromobenzo[1,2-b; 4,5-b′]difuran-4-yl)-2-aminopropane). In the present case, an 18-year-old woman was found dead after ingestion of a hallucinogenic liquid. A medico-legal autopsy was performed on the deceased, during which liver, blood, urine and vitreous humour were submitted for toxicological examination. Bromo-Dragonfly was identified in the liver blood using UPLC–TOFMS, and was subsequently quantified in femoral blood (0.0047 mg/kg), urine (0.033 mg/kg) and vitreous humour (0.0005 mg/kg) using LC–MS/MS. Calibration standards were prepared from Bromo-Dragonfly isolated from a bottle found next to the deceased. The structure and purity of the isolated compound were unambiguously determined from analysis of UPLC–TOFMS, GC–MS, HPLC–DAD, ¹H and ¹³C NMR data and by comparison to literature data.The autopsy findings were non-specific for acute poisoning. However, based on the toxicological findings, the cause of death was determined to be a fatal overdose of Bromo-Dragonfly, as no ethanol and no therapeutics or other drugs of abuse besides Bromo-Dragonfly were detected in the liver, blood or urine samples from the deceased. To our knowledge, this is the first report of quantification of Bromo-Dragonfly in a biological specimen from a deceased person. This case caused the drug to be classified as an illegal drug in Denmark on 5th December 2007.     

Comparative study of ethanol levels in blood versus bone marrow,vitreous humor,bile and urine

Post-mortem ethanol levels in blood were compared to corresponding levels in rib bone marrow, vitreous humor, urine and bile. In forensic toxicology, a good correlation between blood and a tissue or body fluid is needed to estimate a blood alcohol concentration when blood is unavailable or contaminated. In this study, direct injection and headspace gas-chromatographic techniques were employed to quantitate the ethanol concentrations. Comparable findings by these two techniques showed a reproducibility of results. When the determined bone marrow ethanol levels were corrected for the lipid fraction, a consistent correlation could be established between ethanol levels in blood and bone marrow. The relationship (linearity and ratio range) between ethanol levels in blood and corrected levels in bone marrow was better than that between blood and vitreous humor, bile or urine. This study showed that blood ethanol levels can be predicted by extrapolating the corrected rib bone marrow ethanol level.     

An oxcarbazepine-related fatality with an overview of 26 oxcarbazepine postmortem cases

We present an oxcarbazepine-related fatality together with an overview of 26 postmortem cases involving oxcarbazepine observed during the period 2001-2006. The fatality case concerned a 27-year-old woman with epilepsy, who was found dead in her bed. Oxcarbazepine and its active metabolite, 10-hydroxycarbazepine, were the only compounds detected. The concentrations of oxcarbazepine were as follows: femoral blood, 2.9mg/kg; muscle, 1.8mg/kg; liver, 0.9mg/kg; gastric content (300ml), 860mg/kg; and vitreous humour, not detected. The concentrations of 10-hydroxycarbazepine were as follows: femoral blood, 66mg/kg; muscle, 40mg/kg; liver, 62mg/kg; gastric content, 27mg/kg; and vitreous humour, 25mg/kg. The analyses were performed by HPLC-DAD after liquid-liquid extraction. Oxcarbazepine intoxication was regarded as a possible cause of death. For the other 26 cases, the 10-hydroxycarbazepine concentrations ranged from 2.2 to 48mg/kg with a median of 25mg/kg.     

Postmortem production of ethanol and n-propanol in the brain of drowned persons

We examined endogenous ethanol and n-propanol levels in the brain in 29 drowning cases in which ethanol consumption was excluded. Based on the stage of putrefaction of the brain, our cases were classified into 4 groups: pulpified brain (PB, n = 11), softened brain (SB, n = 6), discolored brain (DB, n = 2), and normal brain (NB, n = 10). The endogenous ethanol and n-propanol levels (mg/g), respectively, in the brains from these groups were 1.06 +/- 0.401 and 0.076 +/- 0.032 in PB, 0.195 +/- 0.136 and 0.012 +/- 0.009 in SB, and 0.053 +/- 0.032 and 0.001 +/- 0.001 in DB. Ethanol and n-propanol were not detected in NB. The concentration ratios of ethanol to n-propanol were 16.2 +/- 7.1 in specimens with ethanol levels > or = 0.50 mg/g (n = 10), and 17.6 +/- 13.5 in specimens with ethanol levels of 0.10 to 0.49 mg/g (n = 9). Drinking may strongly be suspected when (1) ethanol concentration in the brain is > or = 0.50 mg/g and cerebral ethanol to n-propanol ratio is > or = 40; and (2) the concentration of ethanol is 0.10 to 0.49 mg/g and the ethanol to n-propanol ratio is > or = 60.     

The influence of ethanol on the level of ketone bodies in hypothermia

Presently available possibilities of macro- and microscopic diagnosis of death from hypothermia are very limited as the changes observed are either weakly specific (ecchymoses in the mucous membrane of the stomach, histological features of haemorrhagic pancreatic necrosis, cardiomyocyte necrosis or decreased content of glycogen in hepatocytes) or represent only local action of low temperatures (frostbites, violet patches in the region of knees and elbows, red livores) and they may not be present in cases of death from cooling at environmental temperature close to zero or higher.The study evaluated the usefulness of acetoacetic acid (Ac-Ac), beta-hydroxybutyric acid (beta-HBA) and acetone determinations in blood, urine and vitreous humour for diagnosis of death from hypothermia. These three substances called ketone bodies, are easily assimilated energetic substrates that get oxidized preferentially before glucose and fatty acids. In hypoglycaemia (also hypothermia-induced one), the tissues dependent on glucose (e.g. the brain) cover most of their energetic needs from oxidation of these compounds.The analysis of 16 cases of death in circumstances suggesting hypothermia (mainly of the alcohol abusers) showed that the degree of ketosis was inversely proportional to the blood ethanol concentration. This relation may result from stimulation of insulin release and a decrease in the release of its antagonists by ethanol, as well as from inhibition of free fatty acid (FFA) beta-oxidation due to increase in the NADH/NAD ratio. So, the antiketonaemic effects of ethanol (together with its influence on the dilatation of the peripheral vessels and inhibition of shivering thermogenesis by muscle relaxation), explain increased sensitivity of intoxicated persons to low temperatures.     

The influence of physical properties and lipid content of bile on the human blood/bile ethanol ratio

The relationship between ethanol levels in blood and bile was determined in human postmortem specimens. The influences of several physical properties--surface tension, specific gravity and viscosity--and bile lipid content on the blood/bile ethanol ratio were evaluated. A gas chromatographic direct injection technique was employed to determine the ethanol concentrations in postmortem blood and bile specimens. A positive correlation was established between the levels in the two fluids. No correlation could be found between the blood/bile ethanol ratios and the aforementioned physical properties of bile. Correction of the observed bile ethanol for lipid content had an insignificant effect on the ratio. The average blood/bile ethanol ratio was 1.03 +/- 0.29 (range: 0.32-2.91). The wide range observed makes it undesirable to use bile ethanol concentrations to predict specific blood ethanol concentrations. However, under certain conditions, bile ethanol levels may be used to estimate blood concentrations within a range of values.     

A comparison of post-mortem ethanol levels obtained from blood and subdural specimens

Post-mortem subdural ethanol levels have been proposed as a useful test in certain forensic cases involving head trauma, particularly when the time interval from injury to death may have caused a lowering of the blood ethanol concentration to insignificant or undetectable levels. This study of 75 autopsied persons from whom both blood and subdural ethanol levels were obtained, shows the usefulness of the subdural ethanol level, especially where there is a prolonged or unknown post-traumatic time interval. Use of such a test is recommended in these situations.