Performance of immunoassays in screening for opiates,cannabinoids and amphetamines in post-mortem blood

Several immunoassay methods for screening of abused drugs in whole blood were evaluated in post-mortem forensic toxicology. Blood samples known to be positive or negative for opiates, cannabinoids or amphetamines by gas chromatography-mass spectrometry (GC-MS) were analysed by EMIT II Plus and EMIT d.a.u., Syva RapidTest and Triage 8 after acetone precipitation. In these experiments, the EMIT immunoassay method was modified by using the Dade Behring VIVA analyser to detect substances more sensitively. Low concentrations of abused drugs were detected in blood samples. The sensitivities of the modified EMIT method for opiates, cannabinoids and amphetamines were 100, 86 and 98%, respectively, whereas the values were below 86% with the other methods. The specificities of all immunoassay methods for opiates and cannabinoids were 83% or above but 51-85% for amphetamines. Sample rejection occurred in a few cases with the EMIT amphetamine assays. The modified EMIT immunoassay system presented here seems to be useful for screening of drugs of abuse in post-mortem blood samples, especially when urine is not available.     

Elevated morphine concentrations determined during infant death investigations: artifacts of withdrawal of care

Abstract:  Three cases are reported of elevated postmortem blood morphine concentrations (189–3036 ng/mL) that were observed during the course of death investigations involving three children ranging in age from 1 week to 2 years, all of whom underwent withdrawal of life support. In all three cases, the presence of opiates in postmortem blood was indicated by immunoassay (ELISA) and quantitative confirmatory analysis of free morphine concentrations in postmortem blood was performed by solid-phase extraction followed by gas chromatography/mass spectrometry (GC/MS) in the selected ion monitoring mode. While the practice of withdrawing life support from terminally ill patients, with the accompanying administration of narcotics/analgesics has been reported in the medical literature, it has not been adequately described in the forensic literature. The implications of this practice on the forensic toxicological interpretation of morphine findings are discussed. To our knowledge, this is the first report of postmortem morphine concentrations arising directly from administration in conjunction with withdrawal of care in pediatric patients.     

The role of ethanol abuse in the etiology of heroin-related death

Toxicology analyses and other forensic science data were used to examine the mechanisms through which ethanol increased the risk for death caused by injected street preparations of heroin. The authors studied 505 victims of fatal heroin overdose and compared subjects who had concentrations of blood ethanol greater than 1000 mg/L (n = 306) with those who had concentrations less than, or equal to 1000 mg/L (n = 199). We found significant negative correlations between concentrations of ethanol and morphine (a heroin metabolite) in blood (R2 = 0.11, P = 0.0001 for log10-transformed variables) as well as between concentrations of blood ethanol and bile morphine (R2 = 0.16, P = 0.0001 for log10 bile morphine versus blood morphine). Toxicologic evidence of infrequent heroin use was more common in decedents with blood ethanol concentrations greater than 1000 mg/L than in those with lower concentrations. Our data suggest that ethanol enhances the acute toxicity of heroin, and that ethanol use indirectly influences fatal overdose through its association with infrequent (nonaddictive) heroin use and thus with reduced tolerance to the acute toxic effects of heroin.     

Comparison of the MTP immunoassay with EMIT in blood screening for drugs

We compared the MTP immunoassay with EMIT for the screening of drugs of abuse (opiates, cannabinoids, cocaine metabolites and amphetamines) in whole blood samples. These blood samples were obtained from the German police, when driving under the influence of drugs of abuse was suspected. For screening with the MTP immunoassay 25 microliters of serum or blood (without any pretreatment) was pipetted into the wells of the microtiter plates and the procedure was followed as described. Prior to screening with a Cobas Mira and EMIT reagents, the samples were treated with acetone to precipitate serum proteins. The cutoff for all drugs of abuse was set at 10 ng per ml of serum or blood. In most cases there was a good agreement between the negative and positive results of the two screening assays. The agreement between the two assays in the detection of opiates and cocaine was 91% and 93%, respectively, and for cannabinoids and amphetamines approximately 80%. The MTP immunoassay was more sensitive than EMIT for the detection of cannabinoids--but at the same time the MTP immunoassay was less specific. Both screening assays have a sensitivity of 100% for the detection of opiates and cocaine, but the specificity of the EMIT--also for opiates--was substantially lower. The MTP immunoassay has in respect to amphetamines a very high sensitivity, whereas the sensitivity of EMIT for amphetamines is inacceptable due to losses during sample preparation. The specificity of MTP immunoassay for amphetamines is not optimal, because a relatively large amount of samples tested false-positive for amphetamines at the cutoff of 10 ng/ml. In summary the MTP immunoassay, although not automated, performs well in comparison with EMIT, especially if the sample preparation for EMIT testing ist considered.     

Immunoglobulins, binding opioid peptides, biogenic amines, and opiates, in substance abusers

Immunoglobulins binding morphine, biogenic amines, and an opioid peptide dermorphine were measured by solid-phase enzyme immunoassay in patients abusing narcotics. The patients' ages varied from 20 to 40, with the duration of narcotic use 1-17 years. Narcotic dependence was found to involve increased production of immunoglobulins binding opioid and monoamine neuromediators. Enzyme immunoassay of antibodies to opiates detects latent forms of narcomania in cases when the narcotic is not present in the body. These results serve the basis for creating a new method for analysis of narcotic abuse and evaluation of the duration of their use, which can be used in practical medicine and in forensic medical expert evaluations.     

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.     

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.     

Heroin body packing: three fatal cases of intestinal perforation

Death from heroin body packing has been well described in the forensic literature. Most fatalities are due to drug leakage and consequent acute heroin toxicity. Recently, drug traffickers have become more sophisticated in their packaging, and the risk of rupture of drug packets is more remote. Though intestinal obstruction is a recognized risk of body packing, rarely has this resulted in death. We describe four cases of heroin body packing presenting to the Regional Medical Examiner Office in New Jersey. Death in three of these cases was due to intestinal obstruction, with resultant intestinal rupture and peritonitis. Toxicologic evaluation in these three cases was negative for opiates or other drugs of abuse. In one case, death was due to acute heroin toxicity, validated by toxicologic analysis. We briefly discuss the differing drug packaging found in these four cases and the ramifications of packaging as it relates to intestinal obstruction.     

Digoxin-like immunoreactive substance in postmortem blood of infants and children

A digoxin-like immunoreactive substance (DLIS) has been reported in the serum of infants not receiving digoxin. This study was undertaken to determine if DLIS is present in the postmortem blood and tissues of infants or children and whether the endogenous substance could interfere with forensic toxicological analysis in suspected overdose. Ninety blood specimens taken from the heart at autopsy of children or infants were screened for DLIS using commercial radioimmunoassay kits. The average age at death in these cases was 8.6 months, the median age was 2 months. DLIS equivalent to 0.25 to 2.0 ng/mL digoxin was found in one third of the cases. The incidence of positive findings was 5/6 stillborns, 10/45 Sudden Infant Death Syndrome (SIDS), 10/15 deaths as a result of infection, 4/7 homicides, 1/8 deaths caused by congenital defects, and 0/9 accidental deaths. The body distribution of DLIS was investigated and highest levels were found in the liver. Findings of DLIS in blood were correlated with renal failure, (elevated vitreous urea nitrogen), electrolyte imbalance, and liver trauma. Apparent concentrations were in the equivalent therapeutic range of digoxin and would not be confused with accidental or intentional overdose with digoxin.     

域外毒品鉴定错案成因分析与防范借鉴

近年来,域外毒品鉴定错案高发,深刻暴露出毒品鉴定管理的内源性问题,值得我国相关领域工作人员借鉴。本文首先概述了毒品鉴定的特点;其次,根据近年来美国、加拿大、英国等地出现的典型毒品鉴定错案,对相关成因做系统梳理,为提升毒品鉴定管理质量、降低毒品鉴定错案的负面影响提供参考依据。本文将鉴定错案分为"错误"和"失误"两大类,并提出了通过提升实验室内部管理、加强实验室外部监督以及落实鉴定意见的质证认证等环节,预防毒品鉴定错案的系统方法。对于防范毒物毒品错案的发生具有重要的参照意义。     

Dependence of acute opiate poisoning lethal outcome on the gender, age, and duration of addiction

The article presents toxicological characteristics of 198 cases of acute parenteral poisoning with morphine and heroin in the range of concentrations of their metabolites in the blood and urine which occur in practice. Risk of death is quantified in the range of possible morphine concentrations in the blood in acute opiates intoxication with reference to gender and age. Assessment of the criteria of quantitative opiates toxicity was made according to the method of logit regression and dose-effect curve suitable for analysis of correlations between probability of death and blood concentrations of opiates metabolites. Conditional lethal doses in respect to gender and age are calculated.     

Screening for cocaine metabolite fails to detect an intoxication

Testing for the presence of cocaine (COC) is common in postmortem and clinical laboratories. COC use may be detected by screening urine specimens for COC metabolite. In the forensic arena, screening positive results are confirmed by a more specific and sensitive technique, such as gas chromatography-mass spectrometry. This article reports the case of an individual who died of COC intoxication but whose immunoassay screen (EMIT) for COC metabolite was negative. Gas chromatography-mass spectrometry analysis of the urine detected benzoylecgonine (BE) at a concentration of 75 ng/mL and COC at 55 ng/mL. These concentrations explain the negative screening result since the cutoff concentration of the assay was 300 ng/mL for BE. The reported cross reactivity with COC was 25,000 ng/mL. However, heart blood concentrations of COC and BE were 18,330 and 8640 ng/mL, respectively. The results from this case provide evidence that an EMIT test alone may fail to detect COC use. Individuals utilizing results of drug screening by immunoassay must be aware of the limitations of this testing methodology.     

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.     

ELISA-based determination of opiates in purulent transformation of cadaveric material

Abuse of drugs has been a topical issue up to the present-day. Opiates like morphine and heroin are still prevalent. The routine methods of determination of drugs in blood made within the forensic medical expertise are time-consuming and samples must be specially prepared for them. ELISA, when used for the determination of opiates in blood and urine, significantly cuts the test time. According to our research results, the diagnosis of opiates by ELISA is advisable at the preliminary stage of the forensic expertise of cadaver; the method is informative, which is of extra importance in cases of purulent transformation of cadaveric material.     

Screening for drugs in forensic blood samples using EMIT urine assays

A screening method for the detection of drugs in haemolysed whole blood has been evaluated. Methanolic extracts of 300 forensic blood samples known to be positive or negative for drugs were analysed with EMIT d.a.u. assay kits for amphetamine, cannabinoids, opiates and benzodiazepines (the latter to analyse for diazepam and the main metabolite N-desmethyldiazepam). There were very few false positive results, except for the amphetamine assay in postmortem blood samples, where 9% were false positive. For amphetamine and cannabinoids a few false negatives were found, these were from samples with very low drug concentrations. No false negatives were found for opiates and diazepam. The present modification of the EMIT d.a.u. method seems to be a good method for screening of drugs in forensic blood samples, except for amphetamine in postmortem samples. The method is simple and requires only 0.5 ml blood.     

Importance and Approach to Manner of Death Opinions in Opioid-Related Deaths

The majority of opioid-related deaths are accidental. However, the number of opioid-related suicidal deaths is likely under recognized. Presented here is a case of suicide by heroin overdose. The manner of death would have likely been deemed accidental if not for critical information shared by the decedent’s family during follow-up telephone interviews between the forensic pathologist and the decedent’s family, which included text messages that were sent by the decedent just before his death that were not known at the time of the initial medicolegal death scene investigation. This case highlights that when a forensic pathologist establishes an engaged relationship with the decedent’s family, the information elucidated can prove to be invaluable in reaching an informed opinion about the manner of death. For overdose cases, identifying an accurate manner of death allows the design of public health efforts that adequately address the health risks in the community. For aid in the determination of the manner of death for overdose cases, we propose a five-step checklist that may assist forensic pathologists and medicolegal death investigators when approaching similar cases.     

Possibility of the species identification using blood stains located on the material evidences and bone fragments with the method of solid phase enzyme immunoassay with "IgG general-EIA-BEST" kit and human immunoglobulin G

The authors give the comparative analysis of Russian and foreign forensic medical methods of species character identification of the blood from the stains on the material evidences and bone fragments. It is shown that for this purpose it is feasible to apply human immunoglobulin G (IgG) and solid phase enzyme immunoassay (EIA) with the kit "IgG general-EIA-BEST". In comparison with the methods used in Russia this method is more sensitive, convenient for objective registration and computer processing. The results of experiments shown that it is possible to use the kit "IgG general-EIA-BEST" in forensic medicine for the species character identification of the blood from the stains on the material evidences and bone fragments.     

Frequency of positive diazepam-screening in post-mortem examinations (author's transl)]

The post-mortem blood specimens of 389 forensic autopsies were analyzed for diazepam. The age of the cases investigated was above 10 years and the survival time was less than 12 hours. Eighteen samples corresponding to 4.6% were found to be diazepam-positive. These 18 samples were distributed equally between men and women. The proportion of diazepam-positive samples was increased in the groups of suicide and poisoning (alcohol and opiates). The association between diazepam intake and poisoning was statistically highly significant. No correlation was found between diazepam intake and age. Alcohol was found to occur significantly more often in the group of the diazepam positive cases as compared to the diazepam negative group.     

Screening postmortem whole blood for oxycodone by ELISA response ratios

The objective of this study was to investigate the accuracy of screening postmortem whole blood for oxycodone using the ratio of the oxycodone immunoassay response to the response for the specimen obtained with a general opiate-class immunoassay. Fifty eight specimens which were negative for opiates and 158 postmortem whole blood specimens positive for opiates including 66 specimens known to contain oxycodone were assayed. Specimens were diluted 1:5 with assay buffer and analyzed by both the Neogen Oxymorphone/Oxycodone ELISA and the Neogen Opiate Group ELISA (Neogen Corporation, Lexington KY). The oxycodone equivalents in ng/mL from the Oxymorphone/Oxycodone ELISA were divided by the morphine equivalents in ng/mL from the Opiates ELISA to obtain an Oxycodone/Opiates Response Ratio. This ratio was compared with the GC/MS data for all specimens and for opiate positive specimens. Receiver Operating Characteristic (ROC) analysis suggested that optimum relative response ratio was 2.0. The sensitivity of the ELISA response ratio for the presence of oxycodone at a response ratio cutoff of 2.0 was 89.4% +/- 3.8% and the specificity was 88.1% +/- 3.2%. Specimens with a ratio of 2.0 or higher had a greater than 50% probability (positive predictive value) of containing oxycodone in a population with a greater than 15% prevalence of oxycodone.     

胰岛素中毒法医学研究进展

胰岛素作为一种临床上常见的降血糖药物,可以有效控制糖尿病患者血糖,使之维持在正常范围。但过量胰岛素可导致死亡,其中以意外中毒最多见,其次为自杀,胰岛素他杀虽极为罕见,但值得高度重视。胰岛素中毒致死的法医学鉴定极为困难,虽然相关研究已取得一定的进展,但在法医学实践中,这类案件仍然是一大难题。本文从法医病理学的角度,对胰岛素中毒的死亡机制、病理学改变、检测手段、注意要点及诊断要点进行综述,为法医学实践提供帮助。