The importance of sample size with regard to the robustness of postmortem reference values.

Evaluating postmortem toxicological results is a challenging task due to multiple factors affecting blood concentrations after death. In order to improve the diagnostic accuracy in cases of suspected fatal intoxication different compilations of postmortem reference drug concentrations are often used. However, it is not clear what constitutes a reliable postmortem reference value. The current study presents reference concentrations for 13 substances from seven substance groups according to a standardized protocol. The reference concentrations were gathered from 3767 autopsy cases and subdivided into intoxications by one substance only (Group A, n=611), multi-substance intoxications (Group B, n=1355) and postmortem controls, in which incapacitation by drugs were excluded (Group C, n=1801). In particular, this study presents statistical information about the precision and conformity change with various sample sizes. Based on the present data >10 detections are usually needed, for the substances examined, to differentiate between intoxication cases and controls. Repeated samplings show that the median of small samples (N=≤5) has a high variation (normalized interquartile range 138-75%) and that a high number of detections (N=>20) in each group are needed to reduce the variation.

Postmortem blood sampling-Comparison of drug concentrations at different sample sites.

Drug concentrations in postmortem blood samples can differ considerably, depending on the sample site - a phenomenon named postmortem redistribution. In this study, blood samples from 48 cases of suspected intoxications were collected during autopsy at the Department of Forensic Medicine in Stockholm, Sweden. Samples were collected from the right and left heart, the carotid artery, jugular vein, external iliac artery and external iliac vein. The mean ratio of right heart/iliac vein was 1.3, which confirms the results from previous studies that drug concentrations in central blood are generally higher than in peripheral blood. The mean ratio of the ext iliac artery/ext iliac vein and the ratio of the carotid artery/jugular vein were 1.3 and 1.4, respectively, suggesting that drug concentrations are higher in arterial than in venous blood. Drugs with a low volume of distribution had a lower ratio of central/peripheral blood than drugs with a high volume of distribution (1.2 vs 1.4) and also a lower ratio of arterial/venous blood (1.3 vs 1.4). In cases with a short postmortem interval (PMI) there were no significant concentration differences in central and peripheral blood, but in cases with medium and long PMI, the ratios increased (1.2 and 1.4). Cases with a long PMI had an arterial/venous concentration ratio of 2.0. The results suggest that postmortem blood sampling should be performed as soon as possible after death and that peripheral venous blood, if available, should be used for analysis.

Interpretation of postmortem vitreous concentrations of sodium and chloride.

Vitreous fluid can be used to analyze sodium and chloride levels in deceased persons, but it remains unclear to what extent such results can be used to diagnose antemortem sodium or chloride imbalances. In this study we present vitreous sodium and chloride levels from more than 3000 cases. We show that vitreous sodium and chloride levels both decrease with approximately 2.2mmol/L per day after death. Since potassium is a well-established marker for postmortem interval (PMI) and easily can be analyzed along with sodium and chloride, we have correlated sodium and chloride levels with the potassium levels and present postmortem reference ranges relative the potassium levels. We found that virtually all cases outside the reference range show signs of antemortem hypo- or hypernatremia. Vitreous sodium or chloride levels can be the only means to diagnose cases of water or salt intoxication, beer potomania or dehydration. We further show that postmortem vitreous sodium and chloride strongly correlate and in practice can be used interchangeably if analysis of one of the ions fails. It has been suggested that vitreous sodium and chloride levels can be used to diagnose drowning or to distinguish saltwater from freshwater drowning. Our results show that in cases of freshwater drowning, vitreous sodium levels are decreased, but that this mainly is an effect of postmortem diffusion between the eye and surrounding water rather than due to the drowning process, since the decrease in sodium levels correlates with immersion time.

A new model for the estimation of time of death from vitreous potassium levels corrected for age and temperature.

Analysis of potassium concentration in the vitreous fluid of the eye is frequently used by forensic pathologists to estimate the postmortem interval (PMI), particularly when other methods commonly used in the early phase of an investigation can no longer be applied. The postmortem rise in vitreous potassium has been recognized for several decades and is readily explained by a diffusion of potassium from surrounding cells into the vitreous fluid. However, there is no consensus regarding the mathematical equation that best describes this increase. The existing models assume a linear increase, but different slopes and starting points have been proposed. In this study, vitreous potassium levels, and a number of factors that may influence these levels, were examined in 462 cases with known postmortem intervals that ranged from 2h to 17 days. We found that the postmortem rise in potassium followed a non-linear curve and that decedent age and ambient temperature influenced the variability by 16% and 5%, respectively. A long duration of agony and a high alcohol level at the time of death contributed less than 1% variability, and evaluation of additional possible factors revealed no detectable impact on the rise of vitreous potassium. Two equations were subsequently generated, one that represents the best fit of the potassium concentrations alone, and a second that represents potassium concentrations with correction for decedent age and/or ambient temperature. The former was associated with narrow confidence intervals in the early postmortem phase, but the intervals gradually increased with longer PMIs. For the latter equation, the confidence intervals were reduced at all PMIs. Therefore, the model that best describes the observed postmortem rise in vitreous potassium levels includes potassium concentration, decedent age, and ambient temperature. Furthermore, the precision of these equations, particularly for long PMIs, is expected to gradually improve by adjusting the constants as more reference data are added over time. A web application that facilitates this calculation process and allows for such future modifications has been developed.

Analysis of 14C and 13C in teeth provides precise birth dating and clues to geographical origin.

The identification of human bodies in situations when there are no clues as to the person's identity from circumstantial data, poses a difficult problem to the investigators. The determination of age and sex of the body can be crucial in order to limit the search to individuals that are a possible match. We analyzed the proportion of bomb pulse derived carbon-14 ((14)C) incorporated in the enamel of teeth from individuals from different geographical locations. The 'bomb pulse' refers to a significant increase in (14)C levels in the atmosphere caused by above ground test detonations of nuclear weapons during the cold war (1955-1963). By comparing (14)C levels in enamel with (14)C atmospheric levels systematically recorded over time, high precision birth dating of modern biological material is possible. Above ground nuclear bomb testing was largely restricted to a couple of locations in the northern hemisphere, producing differences in atmospheric (14)C levels at various geographical regions, particularly in the early phase. Therefore, we examined the precision of (14)C birth dating of enamel as a function of time of formation and geographical location. We also investigated the use of the stable isotope (13)C as an indicator of geographical origin of an individual. Dental enamel was isolated from 95 teeth extracted from 84 individuals to study the precision of the (14)C method along the bomb spike. For teeth formed before 1955 (N=17), all but one tooth showed negative Δ(14)C values. Analysis of enamel from teeth formed during the rising part of the bomb-spike (1955-1963, N=12) and after the peak (>1963, N=66) resulted in an average absolute date of birth estimation error of 1.9±1.4 and 1.3±1.0 years, respectively. Geographical location of an individual had no adverse effect on the precision of year of birth estimation using radiocarbon dating. In 46 teeth, measurement of (13)C was also performed. Scandinavian teeth showed a substantially greater depression in average δ(13)C (-14.8) than teeth from subjects raised in Japan (-13.5), Middle East and North Africa (-12.7) and South America (-10.9). In summary, isotopic analysis of carbon in enamel from a single tooth can give a good estimate of the year of birth of an individual and also provide information about the geographical origin of the individual. This strategy can assist police and forensic authorities when attempting to solve unidentified homicide cases and may facilitate the identification work associated with mass disasters.

Postmortem identification of hyperglycemia.

The detection of diabetic coma postmortem requires accurate biochemical analysis. Due to continuous consumption of glucose by surviving cells postmortem, blood glucose levels decrease rapidly. Therefore, vitreous fluid has been used as a substitute in forensic practice, since it has a very low cell count. It has been repeatedly reported that the sum value of vitreous glucose and lactate should be used to estimate the original antemortem blood glucose level, based on the assumption that pre-existing glucose is gradually converted to lactate under anaerobic conditions during agonal phase and the early postmortem period. In this study, we applied a strategy including consistent sampling of vitreous fluid from the centre of both eyes of deceased subjects as soon as possible after arrival at the morgue, and immediate bedside analysis using a blood gas instrument. In total, 3076 cases were included during 2004-2006. We found that, after an initial drop of vitreous glucose during the very early postmortem period, the levels stayed stable for appreciable time postmortem. Analysis of a second sample collected at autopsy 1-3 days later gave similar results (R(2)=0.90). In contrast, the vitreous lactate levels showed a steady increase. This implies that the sum value of glucose and lactate increases with postmortem time, as reflected by vitreous potassium level. In fact, a statistically significant difference in the sum value was seen between subjects with potassium below 10 mmol/L (n=1086) and above 20 mmol/L (n=531), p<.001. In addition, in this large material, we did not identify a single case with circumstantial indication of hyperglycemia that only showed high vitreous lactate. We therefore suggest that vitreous glucose alone should be used to diagnose hyperglycemia postmortem and that the limit of 10 mmol/L should have a good specificity for diabetic coma, which theoretically would equal an original blood glucose value of about 26 mmol/L. As to the methodology, we found that sonication, centrifugation and addition of fluoride to the samples are unnecessary procedures when using a blood gas instrument. The strategy resulted in a doubling of the number of diabetic coma identified at our department compared to preceding period when analysis only was performed on selected cases.

Reference concentrations of antidepressants. A compilation of postmortem and therapeutic levels.

In approximately 95% of all medicolegal autopsies performed in Sweden between 1992 and 2005, femoral blood samples were collected and screened for antidepressant drugs. A total of 8591 cases were identified and used for detailed analysis and interpretation. The present compilation provides information about 15 antidepressant drugs determined in femoral blood from certified fatal intoxications and in postmortem "control cases". The postmortem data were subjected to a previously proposed strategy, based on strictly standardized conditions regarding collection, handling and toxicological analysis of the samples. The postmortem data were compared with a therapeutic drug monitoring material (Group T; n = 16,809). The strict inclusion criteria meant that only 2737 postmortem cases were included in the survey. Accordingly, Group A (n = 330) were certified as deaths involving intoxication with a single antidepressant drug; Group B (n = 864) were deaths involving intoxication with more than one drug and/or with a significant concentration of ethanol; and Group C (n = 1800) were deaths under circumstances not involving incapacitation by drugs. In addition to providing reference levels for each drug, the results may also be used to assess risk of toxicity and supply supplementary information to the standard fatal toxicity index.

Flunitrazepam: an evaluation of use, abuse and toxicity.

The benzodiazepine flunitrazepam is extensively prescribed to patients with insomnia in many countries, but has also become popular among alcohol- and drug abusers. Several reports indicate that it is used as a date rape drug and suggest that it may precipitate violent behavior. Furthermore, flunitrazepam is involved in many fatal intoxications in Sweden. This study was designed and conducted to explore the negative consequences of flunitrazepam abuse in Sweden, and to assess the trends in its use and abuse. The occurrence of flunitrazepam in cases referred to the Department of Forensic Chemistry in Linköping, Sweden 1992-1998, was investigated in detail. The detections were studied separately for different groups; medicolegal death investigations, drug abuse cases, driving under influence cases, and other medicolegal cases. These data were further compared with the sales, and seizures by the Swedish Customs and the Swedish Police. During 1992-1998, 641 fatalities occurred, where the cause of death was attributed to intoxication with flunitrazepam solely (130) or in combination with other drugs, or concomitant conditions (511). In 78% of all driving under influence cases, where flunitrazepam was detected, the analyses also disclosed the presence of illicit drugs. A similar association was seen in drug abuse cases. The seizures reported by the Swedish Customs revealed a substantial and increasing illegal trade. Cases, where flunitrazepam seemingly induced violent behavior were identified, and one of these is described in some detail. It is concluded that the abuse pattern and the toxicity of flunitrazepam should be kept in mind by forensic investigators and that this panorama also should be considered when decisions about the registration and classification of flunitrazepam are made in different countries.

[GHB--dangerous, addictive and uncontrollable "party drug"]. / GHB--farlig, beroendeframkallande och svårkontrollerad "partydrog".

This report reviews the pharmacology, toxicity and abuse pattern of gamma-hydroxybutyrate (GHB). The legislative changes pertaining to this substance are also addressed. Examples of abuse, driving under the influence and fatal intoxication are given. It is concluded that GHB is widely abused, particularly among the younger generation, and that further cases of severe intoxication are likely to occur as long as the substance is easily available from countless sources, including via the Internet. Despite the classification of GHB as a narcotic in Sweden and several other countries, continued problems are expected since the precursors gamma-butyrolactone (GBL) and 1,4-butanediol (BD) are widely--and legally--available.

Postmortem measurements of thyroid hormones in blood and vitreous humor combined with histology.

The aim of this study was to investigate whether clinical reference premortem values can be used to assess postmortem concentrations of thyroxine, triiodothyronine, and thyroid stimulating hormone (TSH), to compare the postmortem concentrations in blood and vitreous humor, and to study the possibility of diagnosing hyperthyroidism by comparing thyroid histologic appearance and postmortem hormone values. Biochemical analyses of free thyroxine (FT4), free triiodothyronine (FT3), and TSH in femoral blood and vitreous humor were made in 38 cases. In 40 cases, the hormones and thyroid histologic appearance were studied; 22 had no significant pathologic changes, and 18 showed focal hyperplasia of the follicular epithelium. A positive correlation was seen between the femoral blood and vitreous humor concentrations of FT4 (R = 0.66) but not between the corresponding concentrations of FT3 and TSH. A positive correlation was also seen between FT3 and FT4 in femoral blood (R = 0.74). In cases with normal thyroid histologic appearance, 58% were found to have FT4 values >24 pmol/L (clinical reference interval 9-24 pmol/L), mean value 27.5 +/- 9.4 pmol/L), which did not differ from the FT4 values in the cases with hyperplasia, 31.6 +/- 15 pmol/L. Only 5% of the T3 measurements in the group with normal histologic appearance were >9 pmol/L (clinical reference interval 3-9 pmol/L). The mean value of FT3 in cases with normal histologic appearance was 3.4 +/- 1.3 pmol/L, and in the group with hyperplasia 8.6 +/- 6.1 pmol/L. The difference was statistically significant P < .005). It is concluded that postmortem values of FT3 and FT4 in femoral blood are fairly comparable to premortem clinical reference values, but the upper normal limit, especially for T4, has to be adjusted upward. Analysis of vitreous humor cannot be used post mortem to assess thyroid function. Histologically, hyperplastic changes correlate well with elevated FT3 in femoral blood.

Incomplete shored exit wounds: a report of three cases.

Typical and atypical exit wounds are well described in the forensic literature. Included in the descriptions of atypical exit wounds are perforating, "shored" exit wounds, in which the perforation of the skin is associated with an abrasion, whether or not the bullet fully exits the body. The authors describe an atypical, incomplete, shored exit wound in which the skin was abraded by supporting material at the site the bullet was recovered, but there was no associated perforation of the skin. Recognition of this injury pattern can be important in reconstruction of the crime scene in relation to the victim at the time of the shooting.

Effect of anticoagulation on renal function and protein excretion in experimental acute ischemic renal failure.

Female Sprague-Dawley rats underwent right nephrectomy and 40 min left renal artery occlusion (RAO). After 15 min of reflow, polyethylene glycol 1000 (PEG1000) was infused to induce osmotic diuresis and to enable glomerular filtration rate (GFR) measurements. Urine was collected during a 90 min period, and the concentrations of PEG1000, albumin, IgG, IgM and fibrin(ogen)/degradation products (FIB) were assessed both in plasma and urine by radial immuno diffusion technique Groups of rats were subjected to saline + RAO, warfarin + RAO or sham-operation. GFR as measured by PEG1000 clearance averaged 0.61, 0.036 and 0.094 mL/min/100g BW/kidney in sham-operated, saline + RAO and warfarin + RAO rats, respectively. Urinary excretion of albumin and IgG increased substantially in both ischemic groups. IgM was not detected in any of the urine samples. FIB excretion was lowest in the saline + RAO group, possibly due to retention of FIB-containing obstructions in the tubules. Rats subjected to warfarin + RAO had significantly higher excretion of FIB. This result suggests that warfarin does not prevent the glomerular sieving of macromolecules in the glomerular filter, but reduces tubular obstruction by preventing fibrin formation, which may explain its positive effect on GFR.

Psychotropics and suicide prevention. Implications from toxicological screening of 5281 suicides in Sweden 1992-1994.

BACKGROUND: Systematic clinical investigations of consecutive suicides have found psychiatric disorders in 90-95% of subjects (depressive disorder 30-87%). AIMS: To investigate use of psychotropics in men and women of different ages who commit suicide. METHOD: Results of toxicological screening in 5281 suicides in Sweden 1992-94 were studied. RESULTS: Psychotropics were detected in 45.3% of the suicides. Antidepressants were detected in 12.4% of the men and 26.2% of the women (7.2% and 14.2%, respectively, of those under 30 years of age). Neuroleptics or antiepileptics (in the absence of antidepressants) were detected in 8.3%, and anxiolytics/hypnotics alone in 20.5% of the subjects. Overdose by an antidepressant was the probable cause of death in 2.1% of the men and 7.9% of the women. CONCLUSIONS: The pattern of psychotropics detected in toxicology was incongruent with the pattern of diagnoses found in the clinical investigations of suicides mentioned above. Depression appears to be under treated in individuals committing suicide, especially in men and in subjects under 30 years of age.

Cytochrome P450 2D6 (CYP2D6) genotyping on postmortem blood as a supplementary tool for interpretation of forensic toxicological results.

Debrisoquine hydroxylase (CYP2D6) is involved in the metabolism of many toxicologically important drugs. The gene encoding for this enzyme displays a polymorphic distribution in all populations examined. We report a study on 46 cases, where analyses of the CYP2D6 gene were conducted on postmortem femoral blood in order to investigate the occurrence of poor metabolizers (PM). A polymerase chain reaction (PCR) method, designed and routinely used for therapeutic drug monitoring, was employed, only slightly modified. Samples from 22 cases, where the parent drug to metabolite ratio was unexpectedly high were analyzed as well as samples from 24 control cases. Genotyping could be carried out in all but one case. Previous freezing or addition of potassium fluoride as preservative did not prevent analysis. Only one PM (from the control group) was discovered, implying an occurrence of only 2.2% as compared to the reported frequency of approx. 7% in Sweden. Among the extensive metabolizers (EM), however, a number of individuals with mutated genes were identified. Although it seems reasonable to suspect a PM genotype in cases with a high concentration of a drug metabolized by CYP2D6, but without suspicion of acute overdose, our study does not support the opinion that this interpretation pitfall is particularly common. This study rather indicates that drug interactions in EMs constitute a more frequent and important problem.

Alzheimer changes are common in aged drivers killed in single car crashes and at intersections.

With increasing age, diseases affecting the cognitive functions are more frequent. These diseases may increase the risk for fatal car crashes. We analyzed the frequency of neuropathological alterations characteristic of Alzheimer's disease (i.e. neuritic and diffuse plaques, and neurofibrillary tangles) in two association areas of the brain, parietal and frontal cerebral cortex, from 98 fatally injured aged drivers. In the age groups of 65-75 and over 75 years of age, 50% and 72% of the drivers, respectively, had neuritic plaques in either parietal and/or frontal cortex. In 14% of all killed drivers the number of neuritic plaques reached the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) age-related histologic score C, which indicates the diagnosis of Alzheimer's disease (AD), and an additional 33% had score B, which suggests the diagnosis of AD. Neuropathological AD changes were most common in the brains of drivers killed in single vehicle crashes, followed by multivehicle crashes at intersections and least common in multivehicle crashes elsewhere, but the differences did not reach statistical significance. In a great majority (80-85%) of cases the killed aged driver was the guilty party of the crash. The results imply, that incipient AD may contribute to fatal crashes of aged drivers, and therefore the forensic autopsy of these victims should include neuropathological examination.

Effect of anticoagulation upon nephron obstruction in experimental acute ischaemic renal failure. A morphological study.

Ischaemic-reperfusion injury as a model of acute renal failure (ARF) results in increased macromolecular permeability, tubular obstruction, and renal oedema. To investigate the role for coagulation in this model, anticoagulated and saline-pretreated rats were subjected to 60 min unilateral renal artery occlusion (RAO). After 15 min of reflow, specimens were collected for electron and light microscopic examination. Morphometry was employed to study podocyte changes and Bowman's space dilatation as measures of increased permeability and tubular obstruction, respectively. After 15 min of reflow, Bowman's space increased significantly and the podocytes were markedly widened and flattened. Rats pretreated with heparin or warfarin showed less widening of Bowman's space than saline-treated rats, whereas no significant difference was seen regarding the podocyte changes. In saline-treated rats, fibrin-positive material was seen in the tubules but not in the urine sediments collected after 90 min of reflow, either due to fibrinolysis or poor urinary elimination. The results suggest that anticoagulation does not preclude the glomerular sieving of macromolecules, but seems to reduce tubular obstruction, probably by preventing conversion of filtered fibrinogen into fibrin.

Fatal injections of heroin. Interpretation of toxicological findings in multiple specimens.

We report two fatalities due to injection of heroin. The first case was witnessed but during the early phase of the police investigation the question was raised whether the injection was self-administered. Multiple samples were collected from different sites and analysed in order to establish drug distribution and to determine the site of injection. Fresh injection marks were found in both antecubital fossae but histological examination failed to settle which one was the last. However, toxicological analysis of the tissues at the injection sites indicated that the injection in the right arm was the last one. This was consistent with the suspicion that the victim was given the injection by another person although probably in agreement with the deceased. In the second case, a similar toxicological procedure was used. This fatality was not witnessed, however ample evidence indicated that it was an isolated event in a former intravenous heroin addict and there was only one fresh injection mark. Even in this case, the concentration of morphine was much higher in the tissue sample from the injection mark than in any of the blood samples.

A cluster of fentanyl-related deaths among drug addicts in Sweden.

During a 16-month period, nine fatalities occurred among white male drug-addicts, where fentanyl was detected at postmortem toxicological analysis. The street samples associated with these cases confirmed the presence of fentanyl as an additive in low-concentration amphetamine powders with caffeine, phenazone and sugar as cutting agents. In seven of the cases, an acute intoxication by fentanyl was considered to be the immediate cause of death, and in one case, it was likely, but no analysis of fentanyl was performed in blood, and in another case the death was suicide by hanging. This appears to be the first report of a cluster of fentanyl-related deaths outside the United States, and the occurrence of fentanyl in combination with amphetamine has not previously been reported. In addition, in all cases, femoral blood was collected, and samples were handled and analysed according to standardized, quality-controlled procedures. The previous history, circumstances surrounding the death, autopsy findings, histology and toxicology examination of each case are presented. The gas chromatographic-mass spectrometric method for fentanyl is also described. Fentanyl concentrations ranged from 0.5 to 17 ng g-1 blood, and from 5 to 160 ng ml-1 urine. Other drugs found were amphetamine (8 cases), ethanol (5 cases) and benzodiazepines (5 cases). Morphine was found in only one case. The average age of men was 33.9 years (range 22-44); six were found in their own of friend's apartment, two inside buildings (stairways) and one was found outdoors. We conclude that fentanyl is a dangerous substance that should be considered in drug-addict deaths even outside the United States, particularly when the remaining toxicology is unremarkable, and the cause of death cannot be ascertained

Site of entrance wound and direction of bullet path in firearm fatalities as indicators of homicide versus suicide.

In a retrospective study, 288 firearm deaths were analysed to identify factors indicating homicide. The cases were classified as suicides (213), homicides (54), accidents (14) and undetermined cases (7). A number of factors, related to the circumstances and the autopsy findings, were studied. Whereas 41% of the homicide victims were females, women only constituted 1.4% of the suicide victims. The mean age was 37 and 50 years among homicide and suicide victims, respectively. Most suicides were committed at or in the vicinity of the residence, whereas a majority of the homicides occurred elsewhere. The majority of the suicide victims had either left a suicide note or had medical records of previous psychiatric illness. In all groups, shotguns were the most frequent weapon used. Fifty-seven percent of the homicide victims, but only three percent of the suicide victims had sustained more than one gunshot wound. One-third of the homicide victims were shot at close range. Alcohol was present in one-third of both homicide and suicide victims. Whereas 38% of the homicidal gunshot wounds had their entrances at anatomical regions typical of suicide, the direction of the bullet path often differed from the directions recorded in suicides. Hence, in this material, an entrance wound in the right temple indicated suicide, but in combination with a direction back-to-front, the wound was more likely to be homicidal. This study suggests that an estimation of the direction of the internal bullet path should be conducted in firearm fatalities, and that this factor may assist in the determination of the manner of death.