Preanalytical factors influencing the results of ethanol analysis in postmortem specimens.

Excessive drinking and drunkenness are underlying factors in many fatal accidents, which make the quantitative determination of ethanol in postmortem (PM) specimens an essential part of all unnatural death investigations. The same analytical methods are used to determine ethanol in blood taken from living and deceased persons although the interpretation of the results is more complicated in medical examiner cases owing to various preanalytical factors. The biggest problem is that under anaerobic conditions ethanol can be produced naturally in decomposed bodies by microbial activity and fermentation of blood glucose. Ways are needed to differentiate antemortem ingestion of ethanol from PM synthesis. One approach involves the determination of ethanol in alternative specimens, such as bile, cerebrospinal fluid, vitreous humor and/or urine, and comparison of results with blood alcohol concentration (BAC). Another approach involves the analysis of various alcohol biomarkers, such as ethyl glucuronide, ethyl sulfate and/or phosphatidylethanol or the urinary metabolites of serotonin 5-hydroxytryptophol/5-hydroxyindoleacetic acid (5-HTOL/5-HIAA). If ethanol had been produced in the body by microbial activity, the blood samples should also contain other low-molecular volatiles, such as acetaldehyde, n-propanol and/or n-butanol. The inclusion of 1-2% w/v sodium or potassium fluoride, as an enzyme inhibitor, in all PM specimens is essential to diminish the risk of ethanol being generated after sampling, such as during shipment and storage prior to analysis. Furthermore, much might be gained if the analytical cut-off for reporting positive BAC was raised from 0.01 to 0.02 g% when PM blood is analyzed. During putrefaction low BACs are more often produced after death than high BACs. Therefore, when the cadaver is obviously decomposed, a pragmatic approach would be to subtract 0.05 g% from the mean analytical result. Any remaining BAC is expected to give a more reliable indication of whether alcohol had been consumed before death.

Comparisons of blood alcohol concentrations between initial testing and reanalysis from unopened tubes preserved with 0.25% NaF following refrigerated storage up to 3.93 years.

Few studies support the usage of <1% nominal sodium fluoride (NaF) to preserve ethanol in antemortem blood. Of these studies, several are limited by short study durations of 90 days or less, and there is limited research of authentic samples preserved with <1% NaF. In this retrospective analysis, data from initial and reanalysis testing of blood alcohol concentration (BAC) in antemortem blood collected in 6 mL gray-top tubes (providing 0.25% nominal NaF) has been compiled, to evaluate changes in ethanol concentration that occurred after periods of refrigerated storage. The time between initial and reanalysis was determined by court request(s), and ranged 0.30-3.93 years. In each case, a previously unopened tube was selected for reanalysis testing. Comparable BAC values were found from initial testing, with BACs ranging from 0.025 to 0.394 g/100 mL, and reanalysis testing, with BACs ranging from 0.021 to 0.393 g/100 mL. Ethanol changes ranged from -0.013 g/100 mL (decrease) to 0.008 g/100 mL (increase). BAC reanalysis values that increased (n = 6) were within the uncertainty of measurement (UM) from the initial BAC test, that is, were not statistically or analytically significant. For BAC decreases (n = 29), four test values exceeded the UM of the original test, with BAC losses ranging from 0.004 to 0.011 g/100 mL (relative percent changes 6.5-16.0% loss). The average ethanol change was -0.004 g/100 mL, which is comparable to or less than ethanol losses from studies using 1% NaF preservative of varying temperature and storage duration.

Preanalytical Factors Influencing the Stability of Ethanol in Antemortem Blood and Urine Samples.

The quantitative analysis of ethanol in blood and other biological specimens is a commonly requested service from forensic science and toxicology laboratories worldwide. The measured blood alcohol concentration (BAC) constitutes important evidence when alcohol-related crimes are investigated, such as drunken driving or drug-related sexual assault. This review article considers the importance of various preanalytical factors that might influence changes in the ethanol concentration in blood after collection and before analysis or reanalysis after various periods of storage. When blood samples were collected by venipuncture from living subjects in evacuated tubes containing sodium fluoride (NaF) preservative, there was no evidence that the BAC increased after collection. Most studies found that the BAC decreased after collection depending on storage conditions, such as time and temperature, and the amount of NaF preservative. After the storage of blood specimens in a refrigerator (4oC) for up to 1-4 weeks, the changes in the BAC were not analytically significant. After storage for up to 12 months at 4oC, under the same conditions, the BAC decreased on average by 0.01-0.02 g%. The loss of ethanol does not appear to depend on the type of evacuated tubes used (glass or plastic), nominal volume (5 mL or 10 mL) or the amount of NaF preservative. Urine alcohol concentrations were also stable after various periods of storage, although in cases of glycosuria and urinary tract and/or Candida infections, the addition of NaF (1% w/v) was essential to prevent post-sampling synthesis of ethanol.

Statistical comparisons of 0.08 g/100 ml fortified blood alcohol samples from 6-ml and 10-ml gray-top tubes.

This experiment supplements the study, "Statistical comparisons of blood alcohol samples from 6-ml and 10-ml grey-top tubes". The initial study analyzed fortified samples for blood alcohol concentration (BAC) using two sizes of gray-top tubes: a 10-ml tube containing a nominal 1% sodium fluoride (NaF), a preservative, and a 6-ml tube containing 0.25% NaF, using the variables of time, storage temperature, fill volume, and concentration. From that study, paired t-tests determined no difference in BAC from data sets of the two tube sizes, however, Analysis of Variance, or ANOVA, testing determined the two tube sizes yielded different results at 0.08 g/100 ml. To investigate this potential disparity, this study fortified new samples at 0.08 g/100 and focused on two variables: fill volume and storage duration, while increasing the sample population (to n = 8). For this study, the two tube types yielded equivalent concentrations under the majority of conditions studied. Differences between the two tube types were found using paired t-test for the high-volume samples on Days 7 and 30; ANOVA yielded the same results but also determined one additional statistical difference for the Day 30 low-volume samples. However, the differences observed between tube sizes fall within standard deviation ranges established for the analytical method precision profile, indicating statistical differences are not analytically significant. Further studies are needed to investigate the comparability of the tubes under real-world conditions, under which oxygen and/or additives are not added during sample preparation steps or by usage of blood bank products.

Statistical comparisons of blood alcohol samples from 6-mL and 10-mL grey-top tubes.

Historically, blood alcohol concentration (BAC) studies utilized a 1% concentration of the preservative sodium fluoride (NaF), leaving an information gap supporting usage of lower concentrations of NaF to preserve ethanol. As many forensic laboratories utilize Becton, Dickinson and Company 6-mL gray-top tubes (0.25% NaF), statistical comparisons were conducted to determine whether significant differences exist between BAC values obtained from 6-mL tubes versus 10-mL tubes (1% NaF). Whole blood was spiked at three concentrations, (0.04, 0.08, and 0.15 g/100 mL) and aliquoted into tubes at "low," "medium," and "high" fill volumes. Tubes were split into refrigerated or ambient storage and analyzed after 1, 3, 5, 7, and 30 days, using headspace gas chromatography. Each 6-mL and 10-mL tube pair, prepared, stored, and analyzed under identical conditions, was compared by t-test (95% confidence level). For refrigerated tubes, 32 of 45-tube pairs did not reject the null hypothesis (that 6-mL and 10-mL tubes yield equivalent BACs), and 31 of 45 ambient stored tube pairs did not reject the null hypothesis. Analysis of variance (ANOVA) found no significant differences between 6-mL and 10-mL gray-top tubes for 0.04 and 0.15 g/100 mL concentrations over 30 days; significant differences were observed for 0.08 g/100 mL concentration tubes, which warrants further study. Paired t-tests of grouped samples found no significant differences between 6-mL and 10-mL tubes at any concentration.