The determination of drugs of abuse in whole blood by means of FPIA and EMIT-dau immunoassays--a comparative study

Six groups of common drugs of abuse (cannabinoids, benzoylecgonine, opiates, barbiturates, benzodiazepines and amphetamines) were determined in whole blood after acetone precipitation, using enzyme multiplied immunoassay (EMIT dau) and fluorescence polarisation immunoassay (FPIA--Abbott TDx and ADx) methods. Both methods, designed primarily for urine, allowed the determination of all above mentioned class of drugs but amphetamine. Only 1 ml of a pre- or postmortem blood sample was needed. The sensitivity of cannabinoids determination was higher by FPIA. The FPIA method gave more precise results, particularly in the case of autopsy blood. The method was applied for drug screening in autopsy and police blood samples. The results (both positive and negative) were in agreement with those obtained with chromatographic methods.     

The Toxicology of Methadone‐Related Death in Infants Under 1 Year: Three Case Series and Review of the Literature

Methadone‐related fatalities occur rarely in infants under 1 year, with five confirmed cases in the literature. The interpretation of pediatric postmortem toxicology relies on adult data; however, infants have crucial physiological differences that may impact interpretation of results. Retrospective case review included scene investigation, interviews, autopsy, and NIH/CDC Sudden Unexplained Infant Death Investigation Reporting Form. Methadone levels were confirmed by liquid chromatography/tandem mass spectrometry (LC‐MS/MS). The fatal blood methadone levels in this case series ranged from 69 to 700 ng/mL. This review of the literature adds toxicological findings of three methadone‐related infant fatalities and reviews the current literature on methadone‐related death in infants under 1 year. With opioid use on the rise in today's society, forensic experts should have a high index of suspicion for the possibility of intentional or unintentional poisoning in infants.     

A survey of buprenorphine related deaths in Singapore

Buprenorphine is available in Singapore as substitution treatment for opioid dependence since 2002. This study surveys buprenorphine related deaths in Singapore between September 2003 and December 2004. The aims are to establish the autopsy prevalence of buprenorphine related deaths and the demographical and toxicological profile of the cases. Toxicological screening was performed for all unnatural deaths, deaths involving known drug addicts, as well as when autopsy revealed no obvious cause of death. Twenty-one cases had buprenorphine detected in post-mortem blood and/or urine samples. Eighteen were sudden deaths. There were two fatal falls from height and one death by hanging. All subjects were male. The age range was 24-48 years. Fourteen subjects were between 30 and 39 years of age. The mean age was 35 years. The majority (62%) were Chinese. Eleven (52%) were known drug abusers. For sudden deaths, two groups were identified. Six cases died from natural causes. Blood buprenorphine levels ranged from undetected (detected in urine) to 3.2 ng/mL (mean 1.4 ng/mL). Twelve cases were attributed directly and indirectly to mixed drug poisoning. Blood buprenorphine levels ranged from undetected (detected in urine) to 17 ng/mL (mean 3.2 ng/mL). Nineteen cases showed concurrent abuse of buprenorphine and benzodiazepine, diazepam being the most frequently detected, followed by nitrazepam and midazolam. The availability of buprenorphine as substitution therapy is associated with an increase in buprenorphine related deaths. The danger of co-abuse of buprenorphine and benzodiazepines is highlighted.     

Teens and Spice: A Review of Adolescent Fatalities Associated with Synthetic Cannabinoid Use

Synthetic cannabinoids (SCs) are commonly abused by adolescents with reported past year (2013) use in high school students between 3 and 10%. Standard adolescent postmortem toxicology does not include routine SC analysis, and thus, the true burden of fatalities related to SCs is unknown. A retrospective case review of two cases included scene investigation, interviews, autopsy, and toxicology. SCs were confirmed by liquid chromatography–tandem mass spectrometry (LC?MS/MS). Review of the eight adolescent SC‐associated fatalities in the literature revealed five of eight cases had no other discernible cause of death on autopsy. Compounds detected included PB‐22 (1.1 ng/mL), JWH‐210 (12 ng/mL), XLR‐11 (1.3 ng/mL), JWH‐122, AB‐CHMINACA (8.2 ng/mL), UR‐144 (12.3 ng/mL), and JWH‐022 (3 ng/mL). With synthetic drug use on the rise, forensic experts should have a high index of suspicion for the possibility of SC intoxication in adolescent fatalities with no other discernible cause of death.     

Prevalence of psychoactive substances in truck drivers in the Nord-Pas-de-Calais region (France)

A previous study conducted in 1995 showed that psychoactive drug use by workers was higher in safety/security workstations than in the rest of the labour force. In order to verify this finding, we conducted a new study in 2003-2004 in the Nord-Pas-de-Calais region, restricted to truck drivers. The aim of this study was to allow harmonizing the professional practice of the occupational physicians, proposing drug prevention and drug testing policies, validating the analytical methods and the guidelines in case of positive testing results. One thousand truck drivers were studied. Urines were tested for amphetamines, cannabinoids, cocaine, opiates, benzodiazepines, buprenorphine and methadone by immunoassay. Urine ethanol determinations were performed by an ADH method. Positive urines for drugs of abuse, methadone or buprenorphine were then tested by gas chromatography or liquid chromatography coupled to mass spectrometry. Out of the 1000 drivers, cannabinoids were detected in 85 cases, opiates in 41 cases, amphetamines in 3 cases and cocaine in only one case. Buprenorphine was detected in 18 cases, methadone in 5 cases and benzodiazepines in 4 cases. Urine ethanol was positive in 50 cases. We found only one case with 6-monoacetylmorphine. Other positive opiates were metabolites of antitussives. The relatively low number of benzodiazepine positive urines could be explained by the lack of sensitivity of the test we used. All these results confirm those of the previous study for cannabinoids and ethanol in safety/security workstations. Positive results for methadone and buprenorphine are eight times higher than in the general population. In conclusion, the authors think that it will be of a great interest to test urine of truck drivers for other classes of psychoactive drugs, using a liquid chromatography-mass spectrometry method.     

Determination of Acetamiprid and IM‐1‐2 in PostMortem Human Blood,Liver, Stomach Contents by HPLC‐DAD

An HPLC‐DAD method was developed to detect and quantify a neonicotinoid insecticide acetamiprid (ATP) and its metabolite IM‐1‐2 in autopsy samples of a fatal intoxication case. The postmortem blood and tissue distribution of ATP and IM‐1‐2 was determined for the first time. The method showed acceptable precisions and recoveries with relative standard deviations of <10% for ATP level and 1.38 % for IM‐1‐2. The detection and quantification limits for ATP were 0.015 μg/mL and 0.030 μg/mL for blood and were 0.035 μg/g and 0.050 μg/g for liver samples, respectively. The mean contents of ATP were 0.79 μg/g in the liver, 47.35 μg/g in the stomach contents and 2.7 μg/mL in the blood. IM‐1‐2 content was 17.0 μg/g in the stomach contents. ATP and IM‐1‐2 were not detected in the urine. The presence of ATP and IM‐1‐2 in the samples was confirmed by GC‐MS. The method can be exploited in future forensic casework.     

Determining the Toxicological Significance of Pregabalin in Fatalities

Pregabalin has become more widely prescribed and abused in recent years but is still not always included in laboratory analysis. An LC‐MS‐MS method has been developed and applied to measure pregabalin in 93 postmortem cases, including drug‐related deaths, alternative causes of death, and fatalities where pregabalin was likely to have contributed to death. Other drugs or alcohol was detected, and the most common drug types (in decreasing frequency) were antidepressants, opioids, benzodiazepines, opiates, alcohol, antipsychotics, cocaine, cardiac drugs, amphetamines, cannabis, anticonvulsants, and antihistamines. New psychoactive substances (methoxphenidine and synthetic cannabinoids) were only found in two cases. The results provide further data to assist in evaluating the significance of postmortem pregabalin concentrations and a toxicologically significant concentration of 25 mg/L is proposed. Pregabalin, especially with concomitant use of other CNS depressant drugs, presents a significant toxicological risk and existing laboratory protocols should be reviewed for their suitability to detect pregabalin.     

An evaluation of the Cozart RapiScan system as an on-site screening tool for drugs of abuse in a non-conventional biological matrix: vitreous humor

The aim of this project was to evaluate the Cozart RapiScan Oral fluid Drug Testing System as an on-site screening tool for vitreous humor samples collected during post-mortem examinations. Vitreous humor is easy to collect and as it is contained within the eye it is almost completely unaffected by post-mortem redistribution. The ability to carry out an initial drug screen on vitreous humor at the earliest stage of the death investigation process could contribute significantly to the assessment of the role drugs may have played prior to confirmation with toxicological analyses at the laboratory. Vitreous humor (n = 146) was collected from autopsy examinations (111 males and 35 females) with a specific focus on cases where death occurred following a road traffic accident or where an overdose was suspected. All samples were screened using the five-panel methadone Cozart RapiScan Cartridge with an overall positive rate of 29%. Of the positive results, 43% screened positive for benzodiazepines, 17% for cocaine, 7% for methadone and 33% for opiates. Positive samples, with the exception of benzodiazepines, and 20% of negative samples were analysed by GC/MS. This is the first reported use of this system as an on-site forensic tool in death investigation and for screening for drugs of abuse in vitreous humor. The conclusions from this study show that the Cozart RapiScan System could play an important role in obtaining information on the toxicological state of the person at the time of death.     

Methadone‐Related Deaths – Epidemiological,Pathohistological, and Toxicological Traits in 10‐year Retrospective Study in Vojvodina,Serbia

The number of methadone‐related deaths (MRDs) during a 10‐year period (2002–2011) in the region of Vojvodina, Serbia, was increased. The cases were evaluated according to epidemiological parameters, pathohistological findings, and toxicological screening. The majority of victims were men, aged from 20 to 38. Pathohistologically, the signs of acute focal myocardial damage were present in the heart of victims with drug abuse history shorter than 2 years, while both signs of recent and chronic focal myocardial damage were developed among victims with longer drug abuse history (2–5 years). In postmortem blood samples of 54.84% of victims, methadone was detected in combination with diazepam, both in therapeutic range. Alcohol was absent in most cases. Other detected drugs were antipsychotics and antidepressants in therapeutic concentrations. These findings raise the attention to the concomitant use of methadone and benzodiazepines with the need for further studies to clarify the mechanism of death in such cases.     

Bilateral First Rib Anomalous Articulations with Pseudarthroses Mimicking Healing Fractures in an Infant with Abusive Head Injury

Bilateral symmetric bone nodules were observed in the anterolateral first ribs of an infant with shaking injuries at autopsy. The location prompted diagnostic considerations of healing fractures versus anomalous articulations with pseudarthroses. The forensic pathologist worked with forensic anthropologists and pediatric radiologists to evaluate autopsy findings and compare premortem and postmortem X‐rays. Gross examination of the bones by the pathologist and anthropologists confirmed bilateral, callus‐like bone nodules in first‐rib locations associated with pseudarthroses. Histologic examination of one of the bones further showed features most consistent with pseudarthrosis, not a healing fracture. Radiologists then compared multiple premortem and postmortem radiographs that showed no remodeling of the bone over a 2‐week interval between the time of injury and death, which would be unexpected for a healing fracture in an infant. This multidisciplinary approach resulted in the appropriate diagnosis of pseudarthroses due to anomalous articulations, an uncommon finding in forensic pathology.     

Postmortem Blood Concentrations of R‐ and S‐Enantiomers of Methadone and EDDP in Drug Users: Influence of Co‐Medication and P‐glycoprotein Genotype

Abstract: We investigated toxicological and pharmacogenetic factors that could influence methadone toxicity using postmortem samples. R‐ and S‐methadone were measured in femoral blood from 90 postmortem cases, mainly drug users. The R‐enantiomer concentrations significantly exceeded that of the S‐enantiomers (Wilcoxon’s test, p < 0.001). The samples were divided into four groups according to other drugs detected (methadone only, methadone and strong analgesics, methadone and benzodiazepines, or methadone and other drugs). There was no significant difference in any of the R‐methadone/total methadone ratios among the four groups. The median R/S ratio was 1.38, which tends to be higher than that reported for the plasma of living subjects. In addition, we investigated whether small nucleotide polymorphisms in the MDR1 gene that encode the drug transporter P‐glycoprotein were associated with the concentrations of R‐ and S‐methadone and its metabolite 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine. No significant association was detected.     

Characteristics of Medical Examiner/Coroner Offices Accredited by the National Association of Medical Examiners

The National Association of Medical Examiners accredits medical examiner and coroner offices. Approximately 60 offices were fully or provisionally accredited as of late 2011, and these offices serve one‐quarter of the U.S. population. The calculated average population served was 1.6M but ranged from 0.3 to 10.5M. The calculated mean death rate was 794 deaths/100K population, and the mean homicide rate was 7.2 homicides/100K population. The calculated mean budget was $4.35M, but budgets ranged from $0.67 to $26.8M. The calculated mean budget/capita was $3.02 but ranged from $0.62 to $10.22. The average size of the facility was under 30,000 sq. ft. The calculated average staffing was found to include five forensic pathologists, four and a half autopsy technicians, and nine investigators. The mean forensic pathologists/1M population was 3.7. Calculated workload indices included 222 autopsies/pathologist and an autopsy rate of 77.6/100K population. These results show that offices of every size can achieve accreditation.     

Direct ELISA kits as a sensitive and selective screening method for abstinence control in urine

In 2009 cutoff values of assessment criteria to testify abstinence control in order to estimate driving ability were standardized in Germany. The cutoff values are lower than required in existing guidelines like SAMHSA and there is critical discussion about detection of low concentrations by using immunoassay, especially concerning amphetamines in urine (50 ng/ml). In this study Direct ELISA kits were tested for their applicability to identify the absence of amphetamines, cannabinoids, opiates, cocaine, methadone and benzodiazepines in urine. Results were confirmed by LC/MS or GC/MS analyses. Sensitivity, specificity, predictive values (positive as well as negative) and overall misclassification rates were evaluated by contingency tables and were compared to ROC-analyses. Sensitivity results as well as specificity results were satisfying showing sensitivity values higher than 96% for each analyte. The amphetamine test we used showed sensitivity and specificity of 100% and 88%, respectively, even if amphetamine tests usually react with high cross-reactivity. Our study results include high discrimination at required cutoff values between positives and negatives for each drug group and demonstrate that immunological tests complying with requirements of current decreased urine cutoff values for assessment of driving ability do exist.     

Optimization and validation of CEDIA drugs of abuse immunoassay tests in serum on Hitachi 912

Due to sensitive limits of detection of chromatographic methods and low limit values regarding the screening of drugs under the terms of impairment in safe driving (§ 24a StVG, Street Traffic Law in Germany), preliminary immunoassay (IA) tests should be able to detect also low concentrations of legal and illegal drugs in serum in forensic cases. False-negatives should be avoided, the rate of false-positive samples should be low due to cost and time. An optimization of IA cutoff values and a validation of the assay is required for each laboratory. In a retrospective study results for serum samples containing amphetamine, methylenedioxy derivatives, cannabinoids, benzodiazepines, cocaine (metabolites), methadone and opiates obtained with CEDIA drugs of abuse reagents on a Hitachi 912 autoanalyzer were compared with quantitative results of chromatographic methods (gas or liquid chromatography coupled with mass spectrometry (GC/MS or LC/MS)). Firstly sensitivity, specificity, positive and negative predictive values and overall misclassification rates were evaluated by contingency tables and compared to ROC-analyses and Youden-Indices. Secondly ideal cutoffs were statistically calculated on the basis of sensitivity and specificity as decisive statistical criteria with focus on a high sensitivity (low rates of false-negatives), i.e. using the Youden-Index. Immunoassay (IA) and confirmatory results were available for 3014 blood samples. Sensitivity was 90% or more for nearly all analytes: amphetamines (IA cutoff 9.5 ng/ml), methylenedioxy derivatives (IA cutoff 5.5 ng/ml), cannabinoids (IA cutoff 14.5 ng/ml), benzodiazepines (IA cutoff >0 ng/ml). Test of opiates showed a sensitivity of 86% for a IA cutoff value of >0 ng/ml. Values for specificity ranged between 33% (methadone, IA cutoff 10 ng/ml) and 90% (cocaine, IA cutoff 20 ng/ml). Lower cutoff values as recommended by ROC analyses were chosen for most tests to decrease the rate of false-negatives. Analyses enabled the definition of cutoff values with good values for sensitivity. Small rates of false-positives can be accepted in forensic cases.     

Evaluation of the One-Step ELISA kit for the detection of buprenorphine in urine, blood, and hair specimens

A solid-phase enzyme immunoassay involving microtiter plates was recently proposed by International Diagnostic Systems corporation (IDS) to screen for buprenorphine in human serum. The performance of the kit led us to investigate its applicability in other biological matrices such as urine or blood, and also hair specimens. Low concentrations of buprenorphine were detected with the ELISA test and confirmed by HPLC/MS (buprenorphine concentrations measured by HPLC/MS: 0.3 ng/mL in urine, 0.2 ng/mL in blood, and 40 pg/mg in hair). The intra-assay precision values were 8.7% at 1 ng/mL of urine (n = 8), 11.5% at 2 ng/mL in serum (n = 8), and 11.5% at 250 pg/mg of hair (n = 8), respectively. The immunoassay had no cross-reactivity with dihydrocodeine, ethylmorphine, 6-monoacetylmorphine, pholcodine, propoxyphene, dextromoramide, dextrometorphan at 1 and 10 mg/L, or codeine, morphine, methadone, and its metabolite EDDP. A 1% cross-reactivity was measured for a norbuprenorphine concentration of 50 ng/mL. Finally, the immunoassay was validated by comparing authentic specimens results with those of a validated HPLC/MS method. From the 136 urine samples tested, 93 were positive (68.4%) after the ELISA screening test (cutoff: 0.5 ng/mL) and confirmed by HPLC/MS (buprenorphine concentrations: 0.3-2036 ng/mL). From the 108 blood or serum samples screened, 27 were positive (25%) after the ELISA test with a cutoff value of 0.5 ng/mL (buprenorphine concentrations: 0.2-13.3 ng/mL). Eighteen hair specimens were positive (72%) after the screening (cutoff: 10 pg/mg) and confirmed by LC/MS (buprenorphine concentrations: 40-360 pg/mg). The ELISA method produced false positive results in less than 21% of the cases, but no false negative results were observed with the immunological test. Four potential adulterants (hypochloride 50 mL/L, sodium nitrite 50 g/L, liquid soap 50 mL/L, and sodium chloride 50 g/L) that were added to 10 positive urine specimens (buprenorphine concentrations in the range 5.3-15.6 ng/mL), did not cause a false negative response by the immunoassay.     

Comparative study of postmortem barbiturates, methadone, and morphine in vitreous humor, blood, and tissue

With the introduction of radioimmunoassay (RIA) techniques, it has become toxicologically possible to determine drug concentrations in postmortem vitreous humor. This study demonstrates and confirms this toxicological feasibility. In 49 medical examiner's drug related cases, postmortem tissue levels of morphine, barbiturates, and methadone were compared to the vitreous humor.     

Postmortem changes mistaken for traumatic lesions: a highly prevalent reason for coroner's autopsy request

Postmortem changes are well known for their possible misinterpretation as traumatic lesions which can mislead to suspicion of violent death and therefore to a forensic autopsy request. As far as we know, a systematic review of the prevalence of such a reason for coroner's autopsy request has not been done yet. A retrospective study of 230 forensic autopsies requested by the Coroner's office from 2002 to 2004 in the province of Quebec, Canada, was conducted by the authors. Of the 230 reviewed cases, postmortem artifacts mistaken for traumatic lesions were found in 18 cases. These misinterpretation were based on 5 categories of portmortem changes: purge fluid drainage in 12 cases (66.7%), bluish discoloration by lividity in 5 cases (27.8%), parchment-like drying of the skin in 4 cases (22.2%), bloating from gas formation in 4 cases (22.2%), and skin slippage in 1 case (5.56%). Therefore, postmortem artifacts misinterpretation occurred in 7.83% (95% confidence interval 0.05-0.12) of all requested forensic autopsies and in 35.29% (95% confidence interval 0.23-0.50) of decomposed autopsy cases. This study clearly establishes the high prevalence of postmortem artifacts as main reason for forensic autopsy request. Hence, in a context of forensic pathologist shortage, the improvement of coroner continuous training may reduce the workload.     

Benzodiazepine findings in blood and urine by gas chromatography and immunoassay

Gas chromatography (GC) and immunoassay techniques applied to blood and urine specimens were compared for the screening of benzodiazepines in postmortem forensic toxicology. Five hundred and six such successive postmortem cases in which both urine and peripheral blood was sent for toxicological analysis by the medical examiners were selected. The urine specimens were tested by the Emit((R)) d.a.u. Benzodiazepine Assay, and in parallel, the blood and urine specimens were screened for benzodiazepine drugs and their metabolites by an established automated dual-column GC method. The lowest number of positives (153) was obtained when immunoassay was performed without enzyme hydrolysis. When urine samples were hydrolysed before immunoassay, the number of positives increased to 175. The highest number of positives (200) was obtained in urine by GC, and the screening of blood by GC yielded 185 quantitative results. Despite the urine GC screening produced the most positives, the quantitative screening of the blood by GC appears to be the most efficient approach in postmortem forensic toxicology, considering the fact that although urine findings confirm the presence of the drug, quantitative results in urine are irrelevant to acute toxicity.     

眼内窥镜检查视网膜在法医学中的应用价值

目前对于不明原因的死亡主要是通过法医尸体检验明确死亡原因,然而在尸体解剖中由于某些原因几乎很少对视网膜进行检查,视网膜改变在法医学中的价值并未引起重视。眼内窥镜的出现和在尸体检验中的应用,使视网膜检查在确定死亡原因和推测死亡间隔时间方面的作用逐渐被法医所重视。     

Postmortem angiography of catheter-induced pulmonary artery perforation.

A case of pulmonary artery perforation by the placement of a balloon-tipped, flow-directed (Swan-Ganz) catheter, as demonstrated postmortem by angiography and confirmed by conventional autopsy method, is reported. Angiography is an effective adjunctive modality in the postmortem diagnosis and localization of pulmonary artery perforation. In cases of suspected catheter-induced pulmonary artery perforation and death, postmortem angiography may prove useful to the forensic pathologist.