How Improper Blood Storage Techniques Employed By Utah Law Enforcement Leads To Increased Levels Of Alcohol In Sample

David Rosenbloom summerizing improper Blood Storage Techniques

Dave's Plain Language Summary: Blood draws are unquestionably the most accurate way to measure ethanol (alcohol) concentrations in humans and mammals, however, because the majority of samples are not refrigerated immediately to the proper temperature, and mailed via U.S. Postal Service to the forensic lab, those samples with the yeast Candida Albicans (present in about 20-30% of the adult population) or other bacterium eliminating ethanol as a waste product, will yield increased ethanol levels when finally tested forensically. Ethanol levels can increase as much as double the initial drawn level when samples are kept at room temperature and storage tubes containing less than 2% sodium fluoride (SF) are used (Utah law enforcement routinely use vacutainers using a 1% solution because officers are not trained in what the colored stoppers mean in terms of SF concentrations.

Collection and Storage of Specimens for Alcohol Analysis, Medical-Legal Aspects of Alcohol 237,  (J.C. Garriott, 4th ed. 2003), Anderson.

Dave's Plain Language Summary: Author summarized studies saying refrigeration and 2% Sodium Fluoride is required instead of 1%. Study falsely blames an automatic pipetting device for the anti law enforcement results. In other words, the results show increased levels of ethanol due to yeast produced ethanol levels

The Bacterial Production of Ethyl Alcohol, 8 Forensic Science Society Journal (1968), Blackmore.

The production of ethyl alcohol by a variety of bacteria from laboratory media and human tissue homogenates has been studied. Most bacteria commonly found at post-mortem can produce a significant amount of ethyl alcohol form glucose, sucrose, mannite or lactose. The same bacteria produced less than 20mg/100ml from ornithine, lysine, arginine and urea. Ethyl alcohol production can occur from tissue with gllucose concentrations of less than 20mg/ml. No ethyl alcohol was produced from carbohydrate and protein-fee urine. Sodium fluoride at a concentration of not less than 1% was needed to maintain sterility of stored blood. An analytical sequence is suggested for the determination of ethyl alcohol in samples taken at post-mortem in the effect of bacterial contamination is to be minimised.

Dave's Plain Language Summary: Sodium Fluoride at not less than 1% is required to maintain sterility of stored blood and avoid production of ethanol as a waste by-product of yeast and bacteria reproduction.

The Effect of Microbial Contamination of the Blood Sample on the Determination of Ethanol Levels in Serum, 60 Am. J. Clin. Path. 700, 701 (Nov. 1973), Blume & Lakatura.

Dave's Plain Language Summary: Sodium Fluoride is ineffective at inhibiting ethanol production by yeast and bacteria unless its use is combined with proper refrigeration techniques.

The Effect of Temperature on the Formation of Ethanol by Candida Albicans in Blood, 34(1) Journal of Forensic Sciences, 105-109(1989), Chang and Kollman.

The effect of temperature on microbial fermentation in blood was studied. Specimens of human blood from a blood bank were inoculated with Candida albicans, an organism capable of causing fermentation. A preservative was added to a portion of the inoculated specimens. These inoculated specimens, as well as uninoculated blood, were stored under various temperature conditions. Production of ethyl alcohol was monitored over a period of six months. Fermentation was found to be highly temperature dependent, with refrigeration proving to be most effective at inhibiting ethanol formation.

Abstract courtesy of www.pubmed.org - A service of the National Library of Medicine and the National Institutes of Health

Dave's Plain Language Summary: Sodium Fluoride of less than 2% volume is ineffective against the most common and dangerous pathogen of humans; Candida Albicans, without refrigeration of the sample below 5 degrees centigrade. Mailing blood results to the lab is thus unacceptable because a constant refrigerated temperature cannot be maintained.

Evaluation of Blood-Ethanol Profiles After Consumption of Alcohol Together with a Large Meal, 24(3) Canadian Society of Forensic Science Journal 165 (1991), Jones & Neri.

Storage of Specimens at +4°C or Addition of Sodium Fluoride (1%) Prevents Formation of Ethanol in Urine Inoculated with Candida Albicans, 23 Journal of Analytical Toxicology 333 (1999), Jones, Hylen, Svensson & Helander, 

Department of Forensic Toxicology, University Hospital, Linkoping, Sweden.

Dave's Plain Language Summary: Urine is the most inaccurate means of testing, however, urine must be both properly refrigerated and mixed with Sodium Fluoride (SF) 1% to insure against the growth of yeast and associated ethanol production as a yeast growth waste product.

The microbial synthesis of ethanol was investigated in urine specimens containing 0.5% or 1.0% (w/v) glucose and inoculated with the yeast Candida albicans (100 cfu/mL). Aliquots (10 mL) of urine were dispensed into plastic tubes containing enough sodium fluoride to give final concentrations of 0.1%, 0.25%, 0.5%, 0.75%, 1%, and 2% (w/v), and C. albicans was added. The tubes were tightly stoppered and allowed to stand either at room temperature (22 degrees C) or in a refrigerator (4 degrees C) for up to 34 days before concentrations of ethanol were determined by headspace gas chromatography. Urine samples stored at 22 degrees C without sodium fluoride produced 0.25 g/L ethanol after two days, and the concentration increased to 2.10 g/L and 4.50 g/L after eight days for specimens containing 0.5% (w/v) and 1% (w/v) glucose, respectively. The ratio of the serotonin metabolites 5-hydroxytryptophol/5-hydroxyindoleacetic acid (5HTOL/5HIAA) in urine remained within the reference range (< 15 pmol/nmol) despite high concentrations of ethanol being produced. Urine samples kept at 4 degrees C did not produce any ethanol (< 0.01 g/L) even without sodium fluoride present as a preservative. The production of ethanol by C. albicans was stopped completely by adding 1% or 2% (w/v) sodium fluoride but not by concentrations of 0.75% (w/v) or less. The microbial synthesis of ethanol in urine samples initially stored at room temperature without sodium fluoride was slowed down considerably by moving them into a refrigerator at 4 degrees C. In conclusion, the production of ethanol in urine by C. albicans can be prevented by storage of samples in a refrigerator at 4 degrees C or by adding sodium fluoride > or = 1% (w/v). Measuring the ratio of 5HTOL/5HIAA can help to distinguish postsampling production of ethanol from metabolism and excretion processes.

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 The Collection and Handling of the Blood Alcohol Specimen, 74 American Society of Clinical Pathologists 743 (1980), Kaye.

Proper collection, handling, and storage of the blood alcohol specimen are essential in medicolegal cases involving the question of sobriety. A standard operating procedure is necessary to ensure maximum reliability. Comments are offered on the advantages of using blood specimens in preference to urine or tissue specimens. The use of a conversion factor to obtain a calculated "presumed blood level" can be dangerous. Cautions and suggestions are offered regarding how and from where the blood should be obtained from a living person and during an autopsy. There are certain time limitations for storage of these blood-alcohol specimens. Each laboratory must establish its own limits for reliable storage, given the conditions in that laboratory. Unexpected and confusing results can lead to an erroneous interpretation if history, circumstances, type of injury, and survival time are not all carefully considered. Several possibilities for error in judgment are discussed.

Abstract courtesy of www.pubmed.org - A service of the National Library of Medicine and the National Institutes of Health

Dave's Plain Language Summary: Use of  1% or less of sodium fluoride solution in the storage module allows for only two (2) days storage (forty-eight (48) hours) without refrigeration, thus permitting acceptable analysis for on-site forensic testing (such as at a blood lab), but unacceptably inaccurate results when blood samples are shipped to off-site forensic labs via un-refrigerated mail or courier.

The Pharmacokinetics of Alcohol in Human Breath, Venous and Arterial Blood After Oral Ingestion, 26 European Journal of Clinical Pharmacology 619 (1984),  Martin, Moll, Schmid, Dettli.

The concentration-time profile of ethanol in breath air (AAC), arterial (ABAC) and venous blood (VBAC) of human volunteers was studied after four different oral doses of absolute alcohol--0.5, 0.75, 1.0, and 1.25 g/kg body weight. Seventy-eight single dose experiments were carried out in 42 subjects. In all 78 studies AAC was measured and VBAC was estimated simultaneously in blood collected from a cubital vein of 36 volunteers. Arterial blood, too, was collected from 8 subjects from a catheter in a brachial artery. All blood alcohol concentrations were analysed independently by gas chromatography (GLC) and an enzymatic (ADH) method. A one-compartment open model with first order absorption and pseudo-zero-order elimination was employed to calculate the pharmacokinetic parameters. The average values for the first order absorption rate constant (ka) ranged from 2.2 to 3.1, from 2.4 to 2.6 and from 1.0 to 1.7 h-1 for ACC, ABAC and VBAC, respectively. The pseudo-zero-order elimination rate constant (beta) was 0.17 to 0.18, 0.21 to 0.22 and 0.26 to 0.27 g X 1(-1) X h-1, respectively. During absorption ABAC tended to be higher than VBAC, peaking at a higher level (Cmax) and with a shorter time to peak (tmax) until an arterio-venous concentration equilibrium was reached, whereafter VBAC remained above ABAC. Although there was a close relationship between AAC, ABAC and VBAC during elimination, AAC closely followed the pattern of ABAC during absorption and tended to deviate from VBAC. AAC, therefore, is a much better predictor of ABAC during absorption than VBAC.

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 Factors Influencing the Significance of Alcohol Concentrations in Autopsy Blood Samples, The Medical Journal of Australia (1968);Plueckhahan and Ballard.

Dave's Plain Language Summary:  1% sodium Fluoride may function as an acceptable  preservative when testing times closely follow blood sample capture by less than forty-eight (48) hours. However, SF may effect the partitioning of ethanol, blood and water during gas chromatography (GC) testing, by changing the equilibrium temperature and time, resulting in a decreased cut off of GC peaks and troughs; requiring more careful analysis of the results.

The Effect of Biologic Specimen Type of the Gas Chromatographic Headspace Analysis of Ethanol and Other Volatile Compounds American Journal of Clinical Pathology 1987; 87: 79-85; Watts & McDonald, 

Gas chromatographic analyses of 37 degrees C headspace vapors above liquid phases saturated with sodium chloride demonstrated that the partitioning of isopropanol, n-propanol, and t-butanol from blood, plasma, or serum to headspace vapor was greatly reduced relative to that observed with the use of water as the liquid phase. The partitioning of ethanol and acetone was moderately reduced with these specimens relative to water, while no effect was seen with methanol or acetonitrile. Normal urine only slightly affected relative partitioning, and vitreous humor had no effect. The partitioning reductions observed with blood were not affected by changing the equilibration temperature to 25 degrees C, by lengthening the equilibration time to three days, nor by changing the concentration of the volatiles in the liquid phase. The use of saturated sodium sulfate enhanced the differences in partitioning observed between water and blood. Only with substantial dilution (5:1) of blood specimens was the effect abolished.

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