What Is In Vitro Fermentation — And Can It Produce a False BAC Result?

One of the most significant and least understood challenges to blood test accuracy in DWI cases is in vitro fermentation. This is the process by which microorganisms inside the blood collection tube produce alcohol after the blood was drawn, artificially inflating the BAC result. When in vitro fermentation occurs, the laboratory is not measuring the alcohol that was in your bloodstream at the time of the draw. It is measuring that alcohol PLUS alcohol that was manufactured inside the tube. This is one failure point among many in a blood case, and the rest of the sequence from the needle to the report is on Can a DWI Blood Test Be Wrong?

How In Vitro Fermentation Happens

The human body harbors vast populations of microorganisms, including bacteria and yeasts. One of the most common is Candida albicans, a yeast that is present on the skin and in the gastrointestinal tract of most people. When blood is drawn and placed in a collection tube, some of these microorganisms may enter the tube with the blood.

If the conditions inside the tube allow it, these microorganisms (particularly Candida albicans) can metabolize the glucose (sugar) naturally present in the blood and produce ethanol as a byproduct. This is the same fermentation process that produces alcohol in beer and wine, except it happens inside a blood sample tube. The mechanism is documented in the forensic literature, including controlled work on ethanol production by Candida albicans showing that the effect depends heavily on glucose level and dilution, and research on ethanol formation in samples containing fluoride ions.

Conditions That Promote In Vitro Fermentation

• Insufficient sodium fluoride. Blood collection tubes for DWI testing are supposed to contain sodium fluoride, a preservative that inhibits microbial growth and enzyme activity. If the tube contains an insufficient amount of sodium fluoride, or if the blood volume is larger than the tube was designed for (diluting the preservative), microbial activity may not be fully suppressed.
• Elevated storage temperature. Microorganisms are more active at higher temperatures. If the blood sample is stored at room temperature or in a warm environment (such as the trunk of a patrol car in Texas summer heat) rather than being refrigerated promptly, fermentation is more likely to occur.
• Delay between collection and analysis. The longer the sample sits before being analyzed, the more time microorganisms have to produce ethanol. Delays of days or weeks between collection and laboratory analysis increase the risk of fermentation.
• Elevated blood glucose. Individuals with diabetes or pre-diabetes often have elevated blood glucose levels, which provides more substrate (food) for the microorganisms to ferment into ethanol. Diabetic individuals are particularly vulnerable to false BAC elevations from in vitro fermentation.
• Improper blood draw technique. If the phlebotomist does not follow proper aseptic technique, additional microorganisms from the skin surface may be introduced into the sample.

How Much Alcohol Can In Vitro Fermentation Produce?

Research has shown that in vitro fermentation can produce BAC increases ranging from 0.01 to over 0.20 g/dL depending on the conditions. In cases with significant delays, elevated glucose, and inadequate preservation, the amount of alcohol produced can be substantial — easily enough to push a result from below 0.08 to above 0.08, or from below 0.15 to above 0.15 (the threshold that raises a first DWI to a Class A misdemeanor, added to Texas Penal Code 49.04 by House Bill 1199 and effective September 1, 2011).

One caveat belongs here rather than in a footnote, because the State will raise it. Much of the published work on microbial ethanol production was done on postmortem specimens, where the conditions are far more permissive than they are in a properly preserved antemortem tube. Several studies of antemortem blood have found little or no fermentation, and have reported that the more common direction of change in a stored sample is a loss of ethanol rather than a gain. That does not defeat the argument, but it does define it: this is a challenge built on the specific conditions of a specific tube, not a general proposition that blood samples ferment.

How to Detect In Vitro Fermentation

In vitro fermentation can be detected through several analytical indicators:

• Presence of other fermentation byproducts. Microbial fermentation produces not only ethanol but also other compounds such as n-propanol, n-butanol, and acetaldehyde. If the chromatogram shows peaks for these compounds alongside ethanol, it suggests fermentation occurred in the tube.
• Elevated ethanol in the second sample. The Texas blood kit officers carry holds two tubes, so in most cases a second specimen exists even though no statute requires one. If that second tube, analyzed later, shows a higher BAC than the first, the difference is consistent with ongoing fermentation in the sample.
• Clotting or discoloration. Visual inspection of the sample may reveal clotting (which can indicate the anticoagulant failed) or discoloration (which can indicate microbial growth).
• Glucose depletion. If the glucose level in the sample is abnormally low compared to what would be expected for the patient, it suggests glucose was consumed by microbial fermentation.

Why Most Attorneys Miss This

In vitro fermentation is not something you learn in law school or at a CLE seminar. It requires understanding of microbiology, fermentation biochemistry, and the specific conditions under which blood samples are collected and stored in DWI cases. Our ACS-CHAL training covers this topic in depth and it can be an effective challenge available in blood test DWI cases.

Establishing it takes the underlying analytical data rather than the one-page report, because the fermentation byproducts that prove the case are visible on the chromatogram and nowhere else. What that data looks like and how it is read is on Understanding the GC-FID.

Your DWI Defense Should Be Built on Science

At Deandra Grant Law, our forensic credentials give us the foundation to challenge the prosecution’s evidence at a scientific level. If you are facing DWI charges in Texas, contact us for a free, confidential consultation.

Call (214) 225-7117 or schedule an appointment online at texasdwisite.com.