Lack of fermentation in antemortem blood samples stored unstoppered in various locations.

A common defense challenge when antemortem blood ethanol results are presented at trial is the assertion that ethanol was formed in the blood tube after the blood draw through fermentation of the blood glucose by Candida albicans (C. Albicans). In contrast, decades of research into the stability of ethanol in antemortem blood collected for forensic purposes have consistently shown that any analytically significant change in ethanol concentration is a decrease and initially, ethanol-negative blood remains ethanol-negative with storage. For there to be any possibility of fermentation to occur by C. Albicans in an antemortem blood sample there must be a plausible mechanism for introduction of C. Albicans into the blood. One mechanism proffered at trial is environmental contamination resulting from ambient air drawn into the evacuated blood collection tube. Blood was drawn from ethanol-free individuals into 6 and 10-ml gray-top Vacutainer® tubes containing sodium fluoride and 6-ml Vacutainer® tubes without a preservative. Following the blood draws, the tubes were stored unstoppered at room temperature for 24 or 48 h in various locations. Following unstoppered storage, the tubes were stoppered and stored refrigerated (~4°C), left at room temperature (~22°C), or placed in an oven (37°C). The refrigerated blood was analyzed for ethanol using headspace gas chromatography after both 5 days and 32 months. Unrefrigerated blood samples were analyzed after being stored at room temperature or in an oven for up to 30 days. Ethanol was not detected in any of the blood tubes after storage regardless of storage time, storage temperature, or preservative concentration.

Testing antemortem blood samples for ethanol after four to seven years of refrigerated storage.

The previous studies on ethanol stability in antemortem blood samples stored under various conditions have shown that ethanol concentration decreases with storage. The feasibility of measuring a forensically meaningful blood ethanol concentration in antemortem blood samples stored refrigerated (~4°C) from 4-7 years after the blood draw was evaluated in this research. All blood samples were collected into two 10-ml gray top Vacutainer® tubes as part of police driving under the influence investigations. In 29 cases, blood in the tube originally analyzed was retested after 5-7 years of refrigerated storage. Blood in 41 cases was analyzed in a previously unopened blood tube from the case after 4-7 years of refrigerated storage. The first analysis of blood in each case occurred within 35 days of the blood draw. Initial blood ethanol concentrations ranged from 0.094 g/dl to 0.301 g/dl. No samples showed an increase in ethanol concentration with storage that exceeded the uncertainty of the initial measurement. All decreases in ethanol concentration were less than 0.020 g/dl. The mean differences in ethanol concentration in previously opened and unopened tubes were -0.014 g/dl and -0.010 g/dl, respectively. The results of this research support that antemortem blood in previously opened and unopened refrigerated blood tubes can be analyzed for ethanol content more than 4 years and as much as 7 years after the blood draw and provide a result consistent with the amount of ethanol loss expected from a test done within 1-3 years of the blood draw.

Large-scale reanalysis of refrigerated antemortem blood samples for ethanol content at random intervals.

Ethanol stability in antemortem blood stored under various conditions has been widely studied. Most such studies have somewhat limited sample size (<50) and limited variation in the length of time between the blood draw and the first analysis and between the first analysis and the reanalysis. In the work presented here, the antemortem blood drawn for forensic purposes and stored refrigerated (~4°C) in 371 cases was analyzed for ethanol concentration using headspace gas chromatography at various times after the blood draw based on routine case flow and then also analyzed at various times within approximately 1 year after the first analysis. This methodology is intended to provide insight into the range of differences expected when cases are analyzed in the normal flow of casework and then reanalyzed at random times afterwards as occurs when reanalysis is performed by the defense or by the laboratory if the original analyst is unavailable to testify. In 22 cases, the same blood tube from the case was reanalyzed. The previously unopened blood tube from the case was analyzed in 349 cases. The 25 cases in which the blood was ethanol-negative based on the first analysis remained ethanol-negative when reanalyzed. The average difference in ethanol concentration between tests for the ethanol-positive cases was -0.004 g/dL. This decrease was statistically significant at the 0.05 level of significance. The range of differences was -0.0197 to 0.0103 g/dL. The difference measured in 85% of the ethanol-positive cases was in in the range of -0.008 to -0.001 g/dL.