Quantification of morphine, morphine 6-glucuronide, buprenorphine, and the enantiomers of methadone by enantioselective mass spectrometric chromatography in whole blood.

PURPOSE: Deaths among drug addicts are frequently caused by intoxication with methadone and/or morphine. These drugs are often used in combination with other drugs, such as buprenorphine. In addition, methadone is generally used as a mixture of R- and S-enantiomers. To date, a method for separation and quantitation of these specific drugs has not been developed. The aim of this study was to develop a sensitive enantioselective method for quantitation of morphine, its active metabolite morphine 6-glucuronide, buprenorphine, and R- and S-methadone, in a single analytical run. METHODS: Whole blood samples were diluted with 0.5 mol/L ammonium carbonate buffer and extracted on a Bond Elut C18 solid-phase extraction column with an automatic solid-phase extraction system. Chromatographic separation was performed on a chiral alpha-1-acid glycoprotein column with an acetonitrile/ammonium acetate buffer (10 mmol/L, pH 7.0, 22:78 v/v) mobile phase. The whole blood concentrations of the drugs were quantified by mass spectrometry using their stable isotope-labeled compounds as internal standards. RESULTS: The method was validated with respect to specificity, linearity, precision, limits of detection, and quantification and matrix effects. The precision (coefficient of variation) was below 15%, and the accuracy was between 90 and 115%. CONCLUSIONS: This method will be useful for routine analyses in forensic laboratories where blood samples are frequently analyzed for drugs of abuse. In some cases, sudden death from methadone overdose is caused by the enantiomeric form of the methadone, which makes the enantiomer separation capability of this method important.

Sudden unexpected death associated with lymphocytic thyroiditis.

A forensic autopsy study comprising 125 cases was carried out retrospectively in order to evaluate pathological changes in the thyroid gland in different groups of death. The five groups selected consecutively were: (i) opiate addicts who died from an overdose, (ii) alcoholics who died as a result of their alcohol abuse, (iii) cases of fatal poisoning other than opiate addicts, (iv) unknown cause of death and (v) controls without prior disease. Tissue samples from the thyroid gland were cut and stained with haematoxylin and eosin and van Gieson. Histology examinations were subsequently performed blind with semiquantitative assessment of the following six parameters: (a) height of the follicular epithelium, (b) the amount of lymphocytes, (c) the presence of plasma cells, (d) hyperplastic follicular changes, (e) oxyphilic changes, and (f) fibrosis. The most striking result was the finding of extensive lymphocytic infiltration of the thyroid parenchyma in five of the 124 cases, of which four belonged in the group of 'unknown cause of death'. This discovery leads to reflections regarding lymphocytic thyroiditis as a cause of death, either by itself or in combination with other disorders. Silent (painless) thyroiditis, especially, is easily overlooked at autopsy as there are no macroscopic changes and often no prior symptoms or history of thyroid disease pointing towards this condition. Analyses of thyroid hormones are unreliable in predicting endocrine status in life. Routine microscopy of the thyroid gland is therefore advocated in cases of sudden unexpected death in order to diagnose thyroid disease, in particular silent (painless) thyroiditis.

Ethical considerations for forensic scientists participating in humanitarian action: A personal reflection.

Humanitarian Forensic Action is aimed at relieving suffering by comforting and informing the relatives of disaster victims. It is not a priority to report human rights abuses to the authorities. It is thereby different from forensic human rights work with an inquisitive way of demonstrating culpability. The following tasks are relevant. The forensic scientist as an interviewer of the next of kin and witnesses. Emphasizing independency in armed conflicts. Assisting in man-made and natural disasters Assisting in the prevention and of the spread of epidemics.

Incidence, Frequency, and Clinical Characteristics of Type 3 Myocardial Infarction in Clinical Practice.

OBJECTIVES: Cardiac death in a patient with symptoms and electrocardiographic changes indicative of myocardial ischemia but without available measurements of cardiac biomarkers is designated a type 3 myocardial infarction. We wanted to investigate the incidence, the frequency, and the characteristics of patients diagnosed as type 3 myocardial infarction. METHODS: The occurrence of deaths in a well-defined geographic region was retrieved from the Danish Civil Registration System during a 1-year period from 2010 to 2011. Complementary data concerning causes of deaths were obtained from the Danish Register of Causes of Death, and ambulance and hospital patient files. Adjudication of the diagnosis was done by 2 local experts and one external senior cardiologist. RESULTS: A total of 2766 of the 246,723 adult residents in the region had died. A type 3 myocardial infarction was diagnosed in 18 individuals, corresponding to an annual incidence of 7.3/100,000 person-years. During the same 1-year period, 488 patients had other types of myocardial infarction implying a 3.6% frequency of type 3 myocardial infarction (18 of 506) among all myocardial infarctions. CONCLUSION: Type 3 myocardial infarction is a rare observation in clinical practice with an annual incidence below 10/100,000 person-years and a frequency of 3%-4% among all types of myocardial infarction. If autopsy data are included, the number of type 3 myocardial infarctions will increase.

Assessment of age at death by microscopy: unbiased quantification of secondary osteons in femoral cross sections.

The microscopic method of age at death determination was introduced by Kerley in 1965 [E.R. Kerley, The microscopic determination of age in human bone, Am. J. Phys. Anthropol, 23 (1965) 149-163.]. However, even though the method has been revised several times, there remain some fundamental issues concerning the reliability of the methods. This is because several basic histological features seen in a cross section of a bone have to be quantified according to the method, but the definition, and hence quantification, of these features leaves room for subjectivity. In a previous study we found that some of these features (osteon fragments and Haversian canals) could not be identified reliably. Only secondary osteons could be identified with a low inter and intra observer error. Furthermore, since the histological features are quantified in only parts of an entire bone cross section, the selection of these parts or areas is a potential source of bias. Finally, unless an unbiased method is used for addressing features on the borders of the selected areas, this will also introduce error. These issues have not been addressed specifically in previous studies. In this study, we used the methods of stereology to choose the regions of interest, as well as for dealing with border phenomena, and we only counted secondary osteons. Our results show a statistically significant increase in the median number of osteons per area unit with increasing age at death. However, this was after exclusion of one outlier. This result is probably due to the limited sample size (N = 24). As such, this study is preliminary, but does warrant applying the described techniques to a larger sample.

The ABCB1, rs9282564, AG and TT Genotypes and the COMT, rs4680, AA Genotype are Less Frequent in Deceased Patients with Opioid Addiction than in Living Patients with Opioid Addiction.

Sudden death due to acute intoxication occurs frequently in patients with opioid addiction (OA). To examine whether certain genotypes were associated with this, we examined the frequencies of 29 SNPs located in candidate genes related to opioid pharmacology: ABCB1, OPRM1, UGT2B7, CYP3A5, CYP2B6, CYP2C19, CYP2D6, COMT, KCNJ6 and SCN9A in 274 deceased patients with OA (DOA), 309 living patients with OA (LOA) and in 394 healthy volunteers (HV). The main hypothesis of the study was that subjects homozygous for the variant 3435T in ABCB1 (rs1045642) occur more frequently in DOA than in LOA and HV because morphine and methadone more readily cross the blood barrier in these subjects due to a lower efflux transporter activity of the ABCB1 (p-glycoprotein) transporter. Our results did not support this hypothesis, because no statistically significant difference (p = 0.506) in the frequency of the TT genotype of rs1045642 was observed between the DOA, LOA and HV cohorts. However, for another ABCB1 variant, rs9282564, we found that the frequencies of the AG and TT genotypes were 13, 21 and 25% in DOA, LOA and HV, respectively, and after correcting for age, sex and multiple testing, the differences between DOA and LOA were statistically significantly different (p = 0.027). The COMT rs4680 AA genotype frequencies were 25%, 35% and 31% in DOA, LOA and HV, respectively, and the difference between DOA and LOA was also statistically significant (p = 0.0028). In conclusion, this study generated two hypotheses suggesting possible associations of a reduced risk of death and carrying, respectively, the ABCB1 rs9282564 AG and TT genotypes and the COMT rs4680 AA genotype among patients with OA. These findings should be confirmed in independent cohorts, and if a causal relationship between these variants and fatal poisoning in OA is confirmed, then it may be possible at least in theory to personalize prevention of sudden death in this patient group.

Accumulation of triglycerides in the proximal tubule of the kidney in diabetic coma.

AIMS: The present study was initiated by a very recent histochemical observation of lipid accumulation in the renal cortex of a woman who died in a diabetic coma. Two older reports of lipid accumulation in the kidneys of patients who died, most likely in a state of non-regulated diabetes, supported this observation. We have examined whether lipid accumulation in the renal cortex is characteristic of diabetic coma and, if so, which type of lipid accumulates. METHODS: Three groups were studied. Ten subjects who died in a diabetic coma, eight diabetics who died of known causes unrelated to diabetes, and seven normal control subjects without any diagnosed diabetes who died of known causes. All were subjected to histological examination for lipid accumulation in the renal cortex. Detailed analysis of cortex lipids was performed for two of the subjects who died in a diabetic coma and all diabetic controls as well as non-diabetic control subjects. RESULTS: All subjects who died in a diabetic coma showed vacuolar lesions staining strongly for lipid in the proximal tubules. Neither normal controls nor non-coma diabetics showed these lesions. Compared with normal controls, renal cortex lipid was about tripled in the two analysed diabetic coma subjects due to 60-100-fold increases of triglycerides. The non-coma diabetics did not differ from the other controls with respect to triglycerides or other types of lipid, except that cholesteryl esters were elevated, though still a quantitatively minor component. CONCLUSION: Our findings strongly indicate that vacuolar lesions in the proximal tubules are characteristic of diabetic coma and that they are caused by accumulated triglycerides. Therefore, histological examination of renal cortex using a lipid stain may be a useful forensic tool in establishing diabetic coma as the cause of death.

The detection of phosphorus in the tissue of bomb victims in Gaza.

One of the authors went on a fact finding mission to Gaza immediately after the termination of the Israeli invasion. One of the allegations towards the invading Israeli army was the use of phosphorus bombs. The author was able to take samples of tissue from burn victims. Attempts were made to demonstrate the presence of phosphorus in the burn wounds. Using histology and Raman spectroscopy it was found with a high degree of probability that phosphorus was present, although it was not proven with absolute certainty.

Postmortem detection of hepatitis B, C, and human immunodeficiency virus genomes in blood samples from drug-related deaths in Denmark*.

Blood-borne viral infections are widespread among injecting drug users; however, it is difficult to include these patients in serological surveys. Therefore, we developed a national surveillance program based on postmortem testing of persons whose deaths were drug related. Blood collected at autopsy was tested for anti-HBc, anti-HBs, anti-hepatitis C virus (HCV), or anti-human immunodeficiency virus (HIV) antibodies using commercial kits. Subsets of seropositive samples were screened for viral genomes using sensitive in-house and commercial polymerase chain reaction (PCR) assays. Hepatitis B virus (HBV) DNA was detected in 20% (3/15) of anti-HBc-positive/anti-HBs-negative samples, HCV RNA was found in 64% (16/25) of anti-HCV-positive samples, and HIV RNA was detected in 40% (6/15) of anti-HIV-positive samples. The postmortem and antemortem prevalences of HBV DNA and HCV RNA were similar. Postmortem HIV RNA testing was less sensitive than antemortem testing. Thus, postmortem PCR analysis for HBV and HBC infection is feasible and relevant for demonstrating ongoing infections at death or for transmission analysis during outbreaks.

The postmortem distribution of ketone bodies between blood, vitreous humor, spinal fluid, and urine.

The distribution of the ketone bodies: acetone, acetoacetate, and D-beta-hydroxybutyrate, between blood, vitreous humor, spinal fluid, and urine was examined in 105 medico-legal autopsies. The ketone body concentration in the body fluids was determinated by head-space gas chromatography. The correlation between blood and the body fluids could be described with regression lines on the logarithmic-transformed results. The correlation is dependent on the ketone body concentration. The ketone bodies in spinal fluid show the best correlation to blood, followed by vitreous humor, and last urine. The concentration dependence in spinal fluid is mainly due to ketone bodies being metabolized in the brain. The human brain utilizes ketone bodies during normal nutritional state. In vitreous humor, the dependence is mainly due to protein bindings of acetoacetate and beta-hydroxybutyrate in blood and the difference in dry matter between blood and vitreous humor.

The detection of female DNA from the penis in sexual assault cases.

An investigation was performed with the aim of assessing the success rate of the examination of penile swabs or imprints for the detection of the female DNA profile. Two hundred twenty seven cases from a 3-year period were retrospectively reviewed. In 57% of the cases, no suitable material was available. Of the remaining 97 cases, 26 provided a DNA profile from the female victim. It is concluded that although the total success rate is low, it is worthwhile performing the examination, especially if there is a short interval between the alleged sexual assault and the examination.

The histological demonstration of lipids in the proximal renal tubules of patients with diabetic coma.

The Armanni-Ebstein phenomenon is a vacuolization of the cells of the proximal kidney tubules in diabetic coma. It has been believed to represent glycogen. In the present examination of 14 cases of diabetic coma, high contents of lipids, probably triglycerides, were demonstrated in the vacuoles by histological staining. Only weak positive reactions were found in diabetic controls. In both alcoholic and nonalcoholic-nondiabetics, no lipid was found. The sensivity was very high, but it was difficult from the present material to ascertain the level of specificity. The authors suggest the term fatty kidney in diabetics.

Surveillance of HIV and viral hepatitis by analysis of samples from drug related deaths.

OBJECTIVES: To determine the prevalence of antibodies against HIV, hepatitis B (HBV) and hepatitis C (HCV) in postmortem samples from drug related deaths (DRDs) in Denmark. DESIGN: Prospective cohort study. Postmortem samples tested for anti-HIV, anti-HCV anti-HBc and anti-HBs. Comparison to pre-mortem testing when possible. DRDs were searched for in the national register of drug treatment, national prison registers, and the national infectious disease register. SETTING: National level. PARTICIPANTS: Drug related deaths admitted to Danish Institutes of Forensic Medicine during 2004. MAIN OUTCOME MEASURES: Prevalence of antibodies, injection drug use, drug treatment experience and prevalence of cirrhosis. RESULTS: Samples for analysis were obtained from 78% (233/299) of DRDs. The prevalences of anti-HIV, anti-HCV and anti-HBc were 4% (9/214), 51% (110/215), and 35% (74/209), indicating a persisting low prevalence of HIV and a declining prevalence of HCV and HBV. Injecting ever was detected among 45% of DRDs and this was associated with a significantly higher prevalence of hepatitis B and C. Among the DRDs 56% received drug treatment and 12% had cirrhosis at autopsy. Evidence of vaccination against HBV was found among 16% (21/128). CONCLUSIONS: Monitoring of viral hepatitis and HIV among DRDs is feasible, and our survey indicates a falling prevalence among Danish drug users. Surveillance based on drug users in treatment may overestimate the true prevalence.