Alcohol loss arising from microbial contamination of drivers' blood specimens

This paper describes the circumstances in which some drivers' blood specimens containing added sodium fluoride (1% w/v concentration) deteriorated as a result of microbial contamination, accompanied by a decrease of alcohol concentration. Strains of the bacteria Serratia marcescens and a Pseudomonas sp. were isolated from the specimens and proven capable of growing at ambient temperature in blood containing sodium fluoride at 1% w/v concentration. They were shown to be active in alcohol degradation in preservatised blood, the activity being dependent on sodium fluoride concentration and storage temperature. Blood diluters were assumed to be a source of microbial cross contamination from one blood specimen to the next. It is recommended that postmortem blood specimens be analysed in separate batches from drivers' specimens when automated blood diluters are used, that the content of fluoride ions be increased to an equivalent of 2% w/v sodium fluoride, and that storage of specimens at temperatures above 4 degrees C be minimised.     

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.     

Morphine-3-D glucuronide stability in postmortem specimens exposed to bacterial enzymatic hydrolysis

Medical examiners frequently rely on the finding of free morphine present in postmortem specimens to assist in certifying deaths associated with narcotics. In vitro hydrolysis of morphine-3-D glucuronide (M3DG) to free morphine was studied using variable specimen pH, initial degree of specimen putrefaction, storage temperature and time, and the effectiveness of sodium fluoride (NaF) preservation. Reagent M3DG was added to opiate-free fresh blood and urine and to autopsy-derived blood specimens. Reagent bovine glucuronidase was also added to certain specimens. Freshly collected and refrigerated NaF-preserved blood produced minimal free morphine, whereas four of five autopsy blood specimens produced free morphine from M3DG. Increased storage time, temperature, and initial degree of putrefaction resulted in greater free morphine generation despite the absence of viable bacteria. Hydrolysis occurring during specimen storage can generate free morphine from M3DG and may result in erroneous conclusions in certifying narcotic deaths.     

The effect of sodium fluoride on the stability of cyanide in postmortem blood samples from fire victims

Assigning a level of significance to cyanide concentrations found in the blood of fire victims is often hampered by the fact that cyanide is inherently unstable in cadavers and in stored blood samples. A few researchers have proposed that sodium fluoride can be used to minimize the instability of cyanide in blood samples; however, controlled studies have not been performed to support validation of this hypothesis. To test the sodium fluoride hypothesis, both treated and control blood samples from 14 autopsied fire victims were tested over a 25-30 day period. A 2% concentration of sodium fluoride was added to the blood samples at the start of testing and the samples were refrigerated between testing intervals. Cyanide concentrations in the treated and control samples were measured between 9 and 11 days post treatment and between 25 and 30 days post treatment. A statistically significant difference was not present between blood cyanide concentrations in treated and control samples between 9 and 11 days. During this time period, although there were small statistically significant increases in both treated and untreated samples the fluctuations were minor. Since the treated and control samples did not exhibit instability between 9 and 11 days, it is not surprising that the sodium fluoride appeared to have no effect. However, a statistically significant difference between blood cyanide concentrations in treated and control samples was observed between 25 and 30 days. Those samples treated with sodium fluoride showed a reduction in blood cyanide variability with virtually no overall change, over a 25-30 day period when compared to control samples, while unconditioned samples showed a significant, average increase of 35%. Based on the findings of this study, it is recommended that 2% sodium fluoride be added to blood samples obtained from fire victims to reduce cyanide instability due to bacteriological activity.     

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.     

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.     

大鼠口服敌敌畏和呋喃丹急性中毒后血液胆碱酯酶体外自动复能的动态观察

作者测定了中毒鼠血胆碱酯酶活性在体外室温下的动态变化,观察研究胆碱酯酶自动复能的特点。结果表明中毒鼠血胆碱酯酶活性体外自动复能在72小时内达最大值;敌敌畏中毒鼠血胆碱酯酶自动复能较呋喃丹组幅度大而下降也快;至第17天,与对照组相比,敌敌畏中毒鼠血胆碱酯酯活性仍有显著性差异。根据实验结果就中毒鼠血胆碱酯酶自动复能的法医学意义作了讨论。     

Stability of drugs in stored postmortem femoral blood and vitreous humor

The stability of 46 drugs in postmortem femoral blood stored for one year at -20 degrees C was investigated. The drugs included benzodiazepines, antidepressants, analgetics and hypnotics. For seven drugs we found a significant change in the concentration between the first and second analysis. Five substances; ethanol, desmethylmianserin, 7-amino-nitrazepam, THC and zopiclone showed a decrease in the concentration whereas the concentrations of two substances; ketobemidone and thioridazine increased. However, the changes observed were not of such an order that it would affect the interpretation in normal forensic casework. We also investigated the possible influence of potassium fluoride on the concentrations of the 46 drugs in vitreous humor after storage for one year. For two substances, ethanol and zopiclone, there were significantly lower concentrations in the samples without potassium fluoride. Furthermore, we also studied the correlation between the concentrations in femoral blood and vitreous humor. For 23 substances there was a significant difference between the concentrations in the vitreous humor and femoral blood. Significant correlations between the concentrations in these two specimens were found for 23 substances, indicating that vitreous humor can be an alternative specimen when blood samples are not available, provided that such correlation exists for the particular substance. Statistical analysis also revealed a correlation between the degree of protein binding of the different drugs and percentage of vitreous/femoral blood concentrations.     

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.     

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.     

Ethanol production by Candida albicans in postmortem human blood samples: effects of blood glucose level and dilution

We present two cases in which the ethanol concentration in blood samples taken after death continued to increase in the absence of any remarkable increase in n-propanol concentration. Species of bacteria and yeasts, including Candida albicans were isolated from these samples. We then examined whether C. albicans, the most common yeast in the general environment, was able to produce ethanol in human blood stored at room temperature. Ethanol production increased as the glucose concentration increased, indicating that C. albicans produced ethanol from the glucose. Our results also suggested that C. albicans produced ethanol more easily in blood diluted by intravenous infusions that included glucose than in undiluted blood. These findings are useful for the evaluation of postmortem ethanol production in subjects whose blood has been diluted by infusions with glucose. Furthermore, there was no quantitative relationship between the amount of n-propanol detected and the amount of ethanol production: n-propanol appears to be an unreliable index of putrefaction and postmortem ethanol production by C. albicans. It is possible for the blood ethanol level to be high and n-propanol not to be detected, even if the subject has not been drinking alcohol. We reconfirmed the necessity of immediately adding sodium fluoride to samples for ethanol analysis to prevent postmortem ethanol production.     

Interpreting results of ethanol analysis in postmortem specimens: a review of the literature

We searched the scientific literature for articles dealing with postmortem aspects of ethanol and problems associated with making a correct interpretation of the results. A person's blood-alcohol concentration (BAC) and state of inebriation at the time of death is not always easy to establish owing to various postmortem artifacts. The possibility of alcohol being produced in the body after death, e.g. via microbial contamination and fermentation is a recurring issue in routine casework. If ethanol remains unabsorbed in the stomach at the time of death, this raises the possibility of continued local diffusion into surrounding tissues and central blood after death. Skull trauma often renders a person unconscious for several hours before death, during which time the BAC continues to decrease owing to metabolism in the liver. Under these circumstances blood from an intracerebral or subdural clot is a useful specimen for determination of ethanol. Bodies recovered from water are particular problematic to deal with owing to possible dilution of body fluids, decomposition, and enhanced risk of microbial synthesis of ethanol. The relationship between blood and urine-ethanol concentrations has been extensively investigated in autopsy specimens and the urine/blood concentration ratio might give a clue about the stage of alcohol absorption and distribution at the time of death. Owing to extensive abdominal trauma in aviation disasters (e.g. rupture of the viscera), interpretation of BAC in autopsy specimens from the pilot and crew is highly contentious and great care is needed to reach valid conclusions. Vitreous humor is strongly recommended as a body fluid for determination of ethanol in postmortem toxicology to help establish whether the deceased had consumed ethanol before death. Less common autopsy specimens submitted for analysis include bile, bone marrow, brain, testicle, muscle tissue, liver, synovial and cerebrospinal fluids. Some investigators recommend measuring the water content of autopsy blood and if necessary correcting the concentration of ethanol to a mean value of 80% w/w, which corresponds to fresh whole blood. Alcoholics often die at home with zero or low BAC and nothing more remarkable at autopsy than a fatty liver. Increasing evidence suggests that such deaths might be caused by a pronounced ketoacidosis. Recent research has focused on developing various biochemical tests or markers of postmortem synthesis of ethanol. These include the urinary metabolites of serotonin and non-oxidative metabolites of ethanol, such as ethyl glucuronide, phosphatidylethanol and fatty acid ethyl esters. This literature review will hopefully be a good starting point for those who are contemplating a fresh investigation into some aspect of postmortem alcohol analysis and toxicology.     

The extent of postmortem drug redistribution in a rat model.

The aim of this study was to investigate the postmortem redistribution of several drugs in a rat model and to examine if any of the pharmacological properties was related to the extent of this phenomenon. One of the following drugs: phenobarbital (phenobarbitone), acetaminophen (paracetamol), carbamazepine, codeine, verapamil, amphetamine, mianserin, trimeprazine (alimemazine) or chloroquine was administered together with nortriptyline orally to rats 90 min prior to sacrifice. Heart blood was sampled immediately before sacrifice and after 2 h postmortem, as it has previously been shown that this is sufficient time for postmortem concentration changes to occur in heart blood. Blood was also sampled from the clamped abdominal inferior vena cava (representing peripheral blood) and tissue samples were taken from lungs, myocardium, liver, kidney, thigh muscle, forebrain, and vitreous humor together with a specimen from the minced carcass. Drugs were analyzed by high performance liquid or gas chromatography. For phenobarbital, acetaminophen and carbamazepine the postmortem to antemortem blood drug concentration ratios were close to 1.0 and tissue concentrations were low. The postmortem to antemortem heart blood drug concentration ratio for chloroquine (6.9 +/- 1.5) was higher than for nortriptyline (3.5 +/- 0.3), and the remaining drugs (codeine, verapamil, amphetamine, mianserin, and trimeprazine) showed ratios of the same magnitude as nortriptyline. The postmortem to antemortem blood drug concentration ratios for both heart blood and blood from the vena cava and also the lung to antemortem blood drug concentration ratio were closely related to the apparent volume of distribution for the drugs studied (p < 0.001). Accordingly, an apparent volume of distribution of more than 3-4 L/kg is a good predictor that a drug is liable to undergo postmortem redistribution with significant increments in blood levels. The postmortem drug concentration in blood from vena cava was closely related to the antemortem blood level, confirming that among the postmortem samples, the peripheral blood sample was the most representative for the antemortem blood concentration.     

Determination of a newly encountered designer drug "p-methoxyethylamphetamine" and its metabolites in human urine and blood

A newly synthesized designer drug, para-methoxyethylamphetamine (PMEA) was unexpectedly detected in the postmortem specimens of fatality involving drug intoxication in 2005, Japan. For unequivocal identification, the isomeric discrimination of PMEA and its positional-isomers was performed by GC/MS with the trifluoroacetylation. In order to prove the intake of PMEA, the characteristic metabolites of PMEA were also identified by GC/MS analysis of the urine specimen with trifluoroacetylation. As a result, para-methoxyamphetamine, para-hydroxyethylamphetamine (POHEA) and para-hydroxyamphetamine were identified as the major metabolites of PMEA. For the quantitative analyses of PMEA and its three metabolites in body fluids, an automated column-switching LC/MS procedure was developed, and applied to the postmortem blood and urine specimens. In this fatal case, blood concentration of PMEA was estimated to be 12.2 microg/mL and this level seemed extremely high in comparison with lethal blood-levels of its analogues, representing acute-intoxication of the victim. Based on the quantitative results, PMEA was found to be extensively metabolized to POHEA via O-demethylation, partly followed by its conjugation.     

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.     

Poisoning from oral ingestion of carbofuran (Furadan 4F), a cholinesterase-inhibiting carbamate insecticide, and its effects on cholinesterase activity in various biological fluids.

A case is presented of a fatal ingestion of Furadan (carbofuran), a cholinesterase-inhibiting carbamate insecticide. A 26-year-old white male was found dead with a partially filled 1-gal (3.8-L) container of Furadan 4F insecticide-nematocide (44.9% carbofuran). The individual had ingested approximately 345 mL of the mixture. Analysis of cholinesterase activity in various biological fluids was performed spectrophotometrically using propionylthiocholine and 5,5'-dithiobis-2-nitrobenzoic acid [Sigma Diagnostics, cholinesterase procedure No. 422 (PTC)] which was measured at 405 nm and 30 degrees C in a Gilford Stasar III Spectrophotometer. The cholinesterase activities were as follows: plasma, 245 units (U)/L (93% inhibition/7% normal activity); serum, 208 U/L (95.3% inhibition/4.7% normal activity); whole blood, 297 U/L (92.8% inhibition/7.2% normal activity); erythrocytes, 58 U/L (99% inhibition/1% normal activity); vitreous humor, 7 U/L; and bile, 148 U/L. Carbofuran was detected in the blood and gastric contents by thin-layer chromatography. No alcohol or other drugs were detected in the blood, urine, or gastric contents. Ingestion of the carbofuran produced acute visceral congestion and pulmonary edema. Death was caused by anoxia due to respiratory paralysis produced by cholinesterase inhibition from Furadan (carbofuran) ingestion.     

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.     

The influence of physical properties and lipid content of bile on the human blood/bile ethanol ratio

The relationship between ethanol levels in blood and bile was determined in human postmortem specimens. The influences of several physical properties--surface tension, specific gravity and viscosity--and bile lipid content on the blood/bile ethanol ratio were evaluated. A gas chromatographic direct injection technique was employed to determine the ethanol concentrations in postmortem blood and bile specimens. A positive correlation was established between the levels in the two fluids. No correlation could be found between the blood/bile ethanol ratios and the aforementioned physical properties of bile. Correction of the observed bile ethanol for lipid content had an insignificant effect on the ratio. The average blood/bile ethanol ratio was 1.03 +/- 0.29 (range: 0.32-2.91). The wide range observed makes it undesirable to use bile ethanol concentrations to predict specific blood ethanol concentrations. However, under certain conditions, bile ethanol levels may be used to estimate blood concentrations within a range of values.     

Timing and Appearance of Postmortem Root Banding in Nonhuman Mammals,

A study was undertaken using nonhuman mammal specimens to better understand environmental influences on postmortem hair root band (PMRB) formation and to see whether PMRBs would occur in nonhuman mammal hairs in a similar fashion to human hairs. Carcasses from surrounding roadways were the primary source of specimens for this study, augmented by donated deceased domestic pets. Sections of pelt from each specimen were placed in controlled environmental conditions while the remainder of the carcass was left in a secure outdoor setting. Hair samples were collected daily from outdoor and control specimens and examined for evidence of PMRBs. Several environmental factors were also recorded on a daily basis. Results demonstrate PMRBs can occur in nonhuman mammal hairs, and they have microscopic characteristics similar to human PMRBs. Factors found to correlate with PMRB formation include postmortem interval, temperature, pH, and the formation and subsequent volatilization of ammonia from the surrounding tissue.