Effect of sodium fluoride on cholinesterase activity in postmortem blood

Thirty-two postmortem blood specimens, with and without sodium fluoride as preservative, were analyzed for cholinesterase activity by the Michel method. The fluoridated specimens, which contained from 0.7 to 31 mg/mL (average 6.3) of sodium fluoride, were found to exhibit cholinesterase activities that were 5 to 59% (average 25%) lower than the duplicate unfluoridated specimens. We concluded that, while this decrease is quite significant, a fluoridated postmortem blood specimen may be used for the measurement of cholinesterase activity when a non-fluoridated specimen is unavailable.     

Screening postmortem blood and tissues for nine classes [correction of cases] of drugs of abuse using automated microplate immunoassay

A commercially available enzyme-linked immunosorbant assay (ELISA) was evaluated as a screening procedure for the detection of nine classes of abused drugs in postmortem blood and tissue specimens. Specifically, postmortem blood, fluid and/or tissue homogenates were screened for amphetamine (AMP), methamphetamine (MET), barbiturates (BARB), benzodiazepines (BZD), cannabinoids (CNB), cocaine (benzoylecgonine; BE), morphine-specific (MOR), opiates (class; OPI), phencyclidine (PCP) and lysergic acid diethylamide (LSD) by ELISA and by coated tube radioimmunoassay (CTR) (BARB, BE, OPI, PCP, LSD) or double-antibody radioimmunoassay (DAR) (AMP/METH, BZD, CNB). Specimens that screened 'positive' by any method were confirmed and quantitated by gas chromatography/mass spectrometry (GC/MS). The only assay that appeared to perform less optimally than RIA was the MOR assay (five false negatives). However, this assay is very specific for free morphine while the GC/MS confirmation method provided a total morphine value. The OPI assay was more sensitive, producing fewer false negatives, and is recommended for broad class opiate screening. EIA is an adequate alternative to RIA for screening postmortem specimens, including blood and tissue, for nine major classes of drugs.     

Evaluation of the Abbott TDx-radiative energy attenuation (REA) ethanol assay in a study of 1105 forensic whole blood specimens

In a preliminary study to determine the applicability of the Abbott radiative energy attenuation (REA) method for the quantification of ethanol in whole blood specimens it was concluded that a larger number of samples was required to evaluate the method, particularly for use in forensic toxicology applications. In this study, 573 blood specimens from suspected driving while intoxicated individuals (DWI blood) and 532 postmortem blood specimens (PM blood) were analyzed by the REA method and a headspace gas chromatographic method (GC) currently used in this laboratory. "Negative" specimens (less than 10 mg/dL by GC) and "positive" specimens (greater than or equal to 10 mg/dL by GC) in each category were analyzed. Linear regression analysis comparing the REA values with the GC values was performed for each type of blood specimen. The equation obtained for DWI blood specimens was REA = 0.943 GC + 1.54; the equation for PM blood specimens was REA = 0.980 GC + 2.76. The correlation coefficient for each group was greater than 0.99. The data suggested that a limit of detection of 10 mg/dL could be applied for DWI blood specimens, while 20 mg/dL would be recommended as the limit of detection for PM blood specimens.     

Quantitation of atenolol, metoprolol, and propranolol in postmortem human fluid and tissue specimens via LC/APCI-MS

Hypertension is a growing medical concern in the United States. With the number of Americans suffering from hypertension increasing, the use of antihypertensives such as beta-blockers is increasing as well. In fact, three beta-blockers - atenolol, metoprolol and propranolol - were among the 200 most prescribed medications in the United States in 2003. Pilots that successfully manage their hypertension can remain certified to fly. The Federal Aviation Administration currently designates approximately 8% of active pilots as "hypertensive with medication". The Civil Aerospace Medical Institute (CAMI) performs toxicological evaluation on victims of fatal aviation accidents. At CAMI beta-blockers are analyzed using gas chromatography with mass spectrometric detection. We have, however, recently developed a liquid chromatography with mass spectrometric detection (LC/MS) method for the simultaneous quantitation of three commonly prescribed beta-blockers, atenolol, metoprolol and propranolol. One advantage of our LC/MS method is the specificity provided by an ion trap MS. Utilizing an ion trap MS, we were able to conduct MS/MS and MS/MS/MS on each analyte. This method also eliminates the time-consuming and costly derivitization step necessary during GC/MS analysis. Additionally, by utilizing this novel method, any concerns about beta-blocker metabolite and/or sample matrix interference are eliminated. The limits of detection for this method ranged from 0.39 to 0.78 ng/mL and the linear dynamic range was generally 1.6-3200 ng/mL. The extraction efficiencies for each analyte ranged from 58% to 82%. This method was successfully applied to postmortem fluid and tissue specimens obtained from victims of three separate aviation accidents.     

Ethanol formation in unadulterated postmortem tissues

During the investigation of aviation accidents, postmortem samples obtained from fatal accident victims are submitted to the FAA's Civil Aerospace Medical Institute (CAMI) for toxicological analysis. During toxicological evaluations, ethanol analysis is performed on all cases. Many species of bacteria, yeast, and fungi have the ability to produce ethanol and other volatile organic compounds in postmortem specimens. The potential for postmortem ethanol formation complicates the interpretation of ethanol-positive results from accident victims. Therefore, the prevention of ethanol formation at all steps following specimen collection is a priority. Sodium fluoride is the most commonly used preservative for postmortem specimens. Several studies have been published detailing the effectiveness of sodium fluoride for the prevention of ethanol formation in blood and urine specimens; however, our laboratory receives blood or urine in approximately 70% of cases. Thus, we frequently rely on tissue specimens for ethanol analysis. The postmortem tissue specimens received by our laboratory have generally been subjected to severe trauma and may have been exposed to numerous microbial species capable of ethanol production. With this in mind, we designed an experiment utilizing unadulterated tissue specimens obtained from aviation accident victims to determine the effectiveness of sodium fluoride at various storage temperatures for the prevention of microbial ethanol formation. We found that without preservative, specimens stored at 4 degrees C for 96 h showed an increase in ethanol concentration ranging from 22 to 75 mg/hg (average 42 +/- 15 mg/hg). At 25 degrees C, these same specimens showed an increase ranging from 19 to 84 mg/hg (average 45 +/- 22 mg/hg). With the addition of 1.00% sodium fluoride, there was no significant increase in ethanol concentration at either temperature.     

The relationship of methanol and formate concentrations in fatalities where methanol is detected

An automated headspace gas chromatography method was developed for the determination of formate (formic acid) in postmortem specimens, based on the in situ sulfuric acid-methanol methylation of formic acid to methyl formate. Diisopropyl ether was used as an internal standard. The method was applied to over 150 postmortem cases where methanol was detected. Of the 153 cases presented, 107 deaths were attributed to acute methanol toxicity. In the vast majority of the remaining 46 deaths, the methanol was determined to be present as a postmortem or perimortem artifact, or was otherwise incidental to the cause of death. Of the 76 victims who were found dead and blood was collected by the medical examiner, all but one had a postmortem blood formate concentration greater than 0.50 g/L (mean 0.85 g/L; n = 74). The sole exception involved suicidal ingestion of methanol where the blood methanol concentration was 7.9 g/L (790 mg/100 mL) and blood formate 0.12 g/L. In 97% (72/74) of the cases where blood was available, the blood formate was between 0.60 and 1.40 g/L. In 31 of the 153 cases, the victim was hospitalized and blood obtained on admission or soon after was analyzed for methanol and formate during the subsequent death investigation; the vast majority (27/30) had antemortem blood formate concentrations greater than 0.50 g/L. Cases with samples taken prior to death with blood formate concentrations less than 0.5 g/L can readily be explained by active treatment such as dialysis. The blood formate method has also been useful in confirming probable perimortem or postmortem contamination of one of more fluids or tissues with methanol (e.g., windshield washer fluid or embalming fluid), where methanol ingestion was unlikely.     

Distinguishing antemortem from postmortem injuries by LTB4 quantification

Leukotriene B₄ (LTB₄) in skin samples from seven forensic cases was detected by HPLC to distinguish their antemortem or postmortem origin. In total, there were thirteen antemortem and seven postmortem specimens. The results showed that LTB₄ was found in all antemortem wound specimens which were either fresh, or refrigerated or fixed in formalin for less than 10 days. In contrast, LTB₄ could not be detected in postmortem wound specimens. These results suggested that detecting the content of LTB₄ is a useful method for distinguishing antemortem from postmortem injuries.     

Quantitation of nucleotides,nucleosides and bases in antemortem and postmortem bloodstains by high-performance liquid chromatography

Ante- and post-mortem bloodstains prepared from the blood of volunteers and corpses were analysed for ATP and its related compounds by reversed-phase high-performance liquid chromatography (HPLC). The results showed that (1) ATP was present in a large amount in antemortem bloodstains but not in postmortem stains, (2) AMP, adenosine, inosine, hypoxanthine, xanthine and uracil either were not detected or were detected in smaller amounts in antemortem than in postmortem bloodstains, and (3) ADP was present in both ante- and postmortem bloodstains. These differences suggest that quantitation of these compounds may be useful in identifying whether bloodstains are ante- or post-mortem.     

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.     

Morbus Fahr--considerations on a case of sudden death

This paper presents 21 cases related to cyanide intoxication by oral ingestion. Cyanide concentrations in biological specimens are especially different from the type of postmortem specimens, and very important in interpreting the cause of death in postmortem forensic toxicology. Besides the detection of cyanide in autopsy specimens, the autopsy findings were unremarkable. Biological samples (0.2mL or equal to less than 10μg of cyanide) were analyzed colorimetrically for cyanide. In a series of 21 cyanide fatalities, the concentration ranges (mean±SD) of cyanide in heart blood, peripheral blood and gastric contents were 0.1-248.6mg/L (38.1±56.6mg/L), 0.3-212.4mg/L (17.1±45.1mg/L) and 2.0-6398.0mg/kg (859.0±1486.2mg/kg), respectively. The ranges of the heart/peripheral blood concentration ratio and gastric contents/peripheral blood concentration ratio were 0.3-10.6 (mean 3.4) and 3.4-402.4 (mean 86.0), respectively. From the difference of cyanide concentration and the concentration ratio of cyanide in different types of postmortem specimens, the possibility of the postmortem redistribution of cyanide and death by oral ingestion of cyanide could be confirmed. We reported cyanide fatal cases along with a review of literature.     

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.     

Phosphoenolpyruvate carboxykinase activity in human liver

The activity of phosphoenolpyruvate carboxykinase (EC 4.1.1.32) (PEPCK), a rate-limiting gluconeogenic enzyme, was found decreased by others in genetically determined disorders and in Sudden Infant Death Syndrome (SIDS). To understand these findings, we made a systematic study of normal human hepatic PEPCK activities in specimens obtained under various conditions from patients not suspected of having SIDS. PEPCK was assayed by the method of Ballard and Hanson [J. Biol. Chem., 244 (1969) 5625] and activity reported as units (1 mumol/min) per gram protein. Intra-assay precision was 4.1% (n = 1094); inter-assay precision using the same homogenate was 10.4% (n = 51); and inter-assay precision using different homogenates of the same tissue specimen was 16.3% (n = 17). The assay was linear with time and enzyme concentration for at least 60 min up to 1.3 mU/assay and for at least 5 min up to 20 mU/assay. Biopsy specimens had significantly (P = 0.015) higher PEPCK activity, 12.60 +/- 3.01 U/g (range 3.5-10.4, n = 9) compared to specimens obtained at autopsy, 3.20 +/- 0.45 U/g (range 0-8.6, n = 33). Specific activity was not significantly correlated with the patient's age, fresh vs. frozen tissue, postmortem intervals up to 68 h, or length of storage at -70 degrees C up to 21 years. One patient had activity at autopsy (tissue obtained less than 2 h postmortem) 26% less than was observed in his biopsy specimen. Autopsy samples separated by differential centrifugation into mitochondrial and cytosolic fractions and checked with marker enzymes ornithine transcarbamylase (mitochondrial) and arginase (cytosolic) had considerable cross-contamination between the two fractions in fresh and frozen specimens.     

Immunohistochemical detection of fibronectin and tenascin in incised human skin injuries

Immunohistochemical detection of molecules involved in inflammatory reaction can be useful for the diagnosis of vitality in skin wounds. We studied the expression of fibronectin (FN) and tenascin (TN) in 58 human skin wounds (48 vital and 10 postmortem). The age of vital injuries ranged from 3 min to 8 h and postmortem specimens were collected after a postinfliction interval of 15-180 min. One hundred thirty-seven formalin-fixed paraffin-embedded sections (mean: 2.3 sections per case) were stained with each of two monoclonal antibodies against FN and TN using the streptABC technique. A reticular staining for FN in wound edge and dermis was observed in 50% of vital specimens versus 0% in postmortem cases. Immunoreactivity was reduced in 10 autolysed cases. FN positivity exclusively at the injury margin was observed in 39.4% of vital wounds and 10% of postmortem cases. TN was negative in all specimens. Vital and postmortem hemorrhage areas showed positivity for FN and TN. Due to its low sensitivity, immunohistochemical analysis of FN is useful for determining vitality only in a minority of cases. Different factors in everyday practice, including autolysis and technical problems often produce false negative reactions with the result that FN cannot be regarded as a reliable parameter of vitality. Positive reactions (network staining) are more valuable than negativity but are not pathognomonic. Both vital and postmortem hemorrhages show an enhanced positivity for FN and TN, thus impeding the diagnosis.     

Analysis of N,N-diethyl-m-toluamide (DEET) in human postmortem specimens

N,N-diethyl-m-toluamide (DEET) levels in postmortem specimens of stomach and contents, blood, liver, and urine are reported following ingestion of the compound. DEET was analyzed by gas chromatography with an OV-101 column and a nitrogen phosphorus detector. The presence of the compound in the four postmortem specimens was confirmed by mass spectrometry.     

The forensic science implications of site and temporal influences on postmortem blood-drug concentrations

The dependence of postmortem blood-drug concentrations on the collection site and on the postmortem interval before specimen collection has been studied. These studies consisted of both sequential sampling from the same collection site at defined time intervals and a comparison of the drug concentrations of postmortem blood simultaneously collected from various sites. A site and time dependence was observed for postmortem blood-drug concentrations. The heart blood-drug concentrations were, in general, significantly higher than those of peripheral specimens. As a result of this phenomenon, the analysis of peripheral blood specimens and solid tissues is often necessary before a definitive interpretation of postmortem toxicological analyses is possible.     

Development and validation of a LC–MS/MS method for analysis of vortioxetine in postmortem specimens. First data from an authentic case

Vortioxetine is an antidepressant recently licensed in USA and EU for the treatment of major depressive disorder. Neither fatal case due to overdose nor data about postmortem concentrations on blood or other specimens have been reported. The aims of this study were the development and validation of a method for vortioxetine analysis by Liquid Chromatography Tandem Mass Spectrometry (LC–MS/MS) in postmortem samples and its application in an authentic case. The method was validated and applied on blood, vitreous humor, bile, brain, liver, kidney, and gastric content. After protein precipitation, the supernatant was directly injected into LC–MS/MS. Analysis was carried out by Multiple Reaction Monitoring (MRM) mode. The authentic case concerned a 38 years-old woman, affected by depression, who was found hanged at home. The method determined an acceptable sensitivity, selectivity, linearity, precision, and accuracy for all matrices. No interference was shown for all matrices. The matrices do not significantly reduce the peak intensity of vortioxetine. No carryover was shown. Toxicological analysis of the authentic case showed vortioxetine in blood (234 ng/ml), vitreous humor (10.5 ng/ml), brain (490 ng/g), lung (479 ng/g), liver (3751 ng/g), kidney (798 ng/g), bile (2267 ng/ml) and gastric content (253 ng/ml). Our case suggests that even at blood concentrations of vortioxetine equal to 234 ng/ml, the subject was able to stage and carry out the hanging. Vortioxetine concentrations found in the other cadaveric samples (biological fluids, organs, and gastric content) may be helpful to evaluate further similar comparable cases.     

Olanzapine concentrations in clinical serum and postmortem blood specimens--when does therapeutic become toxic?

The concentration of olanzapine (Zyprexa) was determined in 1653 clinical serum specimens during routine drug monitoring, and in 58 postmortem whole blood specimens as part of routine toxicological analysis. The analysis of olanzapine was performed by the solid-phase extraction of 1.0 mL of buffered serum or blood, followed by gas chromatography separation with nitrogen-phosphorus detection. The analysis of the clinical serum samples showed that 86% of positive serum values were within the range of 5 to 75 ng/mL, with a mean and median of 36 and 26 ng/mL, respectively. These data suggest that the concentrations of olanzapine expected during therapy may be higher than those previously reported. In 58 postmortem whole blood specimens the mean olanzapine concentration was 358 ng/mL with a range of 10 to 5200 ng/mL. Further, investigation of deaths involving olanzapine suggest that potential toxicity should be considered at concentrations above 100 ng/mL. Although the majority of the olanzapine-related deaths were associated with many other drugs, death primarily due to olanzapine toxicity was determined at concentrations in post-mortem blood as low as 160 ng/mL.     

GC/MS determination of zolpidem in postmortem specimens in a voluntary intoxication

An ingestion of an unknown quantity of Ivadal (zolpidem) tablets in a case of drug abuse is described. The authors report a new and fast method of analysing and determining the zolpidem concentration in postmortem specimens. Quantitation of zolpidem was performed by ethyl acetate extraction from alkalinized body fluids before GC/MS analysis. The analyses were performed without any complex sample clean-up steps and with little sample material. Postmortem concentrations of zolpidem in body fluids are given. The proposed method is a rapid procedure of analysis in cases of deliberate poisoning with the sedative-hypnotic drug, zolpidem.     

免疫印迹检测人胸大肌肌钙蛋白I推断较晚期死后经过时间

目的研究人死后骨骼肌肌钙蛋白I(Skeletal troponn in I,sTnI)的变化规律,探讨较晚期死后经过时间推断方法。方法以人胸大肌为研究对象,利用免疫印迹(W estern b lot)结合图象分析技术半定量检测不同离体时间内人胸大肌sTnI的含量,观察其与离体时间的关系。结果人胸大肌sTnI含量随离体时间延长而逐渐下降,与离体时间的对数值呈近似的线性关系:Y=11 972.5-4 761.9 lgX,相关系数r=0.989;离体5d的人胸大肌仍可检出sTnI。结论检验人死后骨骼肌(胸大肌)sTnI的含量有望成为用于推断人体较晚期死后经过时间的新技术。