According to the World Health Organization and other studies, driving under the influence of psychoactive substances is a significant public health problem worldwide [
Forensic Sciences Research, Volume. 9, Issue 3, owae023(2024)
“Not everything that can be counted counts” in ethanol toxicological results: an antemortem and postmortem technical interpretation focusing on driving under the influence
Ethanol blood analysis is the most common request in forensic toxicology, and some studies point to positive results in approximately one-third of all unnatural deaths. However, distinguishing sober deaths from drunk deaths is not as simple as it may seem. This technical, clinical, and forensic interpretation is proposed to interpret the ethanol toxicological results, discussing several artefacts and pitfalls that must be considered, namely focusing on driving under the influence. This work is presented with a practical and objective approach, aiming to alleviate the complexities associated with clinical, physiological, pathophysiological, and toxicological aspects to enhance comprehension, practicality, and applicability of its content, especially to courts. Particularly the physical integrity of the body, the postmortem interval, putrefactive signs, anatomic place of blood collection, alternative samples such as vitreous humour and urine, the possibility of postmortem redistribution, the inclusion of preservatives in containers, and optimal temperature conditions of shipment are among some of the aspects to pay attention. Although several biomarkers related to postmortem microbial ethanol production have been proposed, their translation into forensic routine is slow to be implemented due to the uncertainties of their application and analytical difficulties. Specifically, in the interpretation of ethanol toxicological results, “not everything that can be counted counts and not everything that counts can be counted” (attributed to Albert Einstein).
Introduction
According to the World Health Organization and other studies, driving under the influence of psychoactive substances is a significant public health problem worldwide [
Methods
A thorough exploration was undertaken across various databases, encompassing PubMed (US National Library of Medicine), Web of Science, Embase, SCOPUS, and Google Scholar, to attain comprehensive cross-disciplinary coverage. No date limit was applied. This technical analysis was supplemented by referencing Portuguese legislation pertaining to this matter, including (i) Decree-Law No. 44/2005, dated 23 February—which regulates the Portuguese Road Code; (ii) Law No. 72/2013, dated 3 September—amends the Portuguese Road Code; (iii) Law No. 18/2007, dated 17 May—approves the regulation of driving under the influence of alcohol and other psychotropic substances; (iv) Ordinance No. 902-A/2007, dated 13 August—approved the fees applicable when driving under the influence of alcohol and other psychotropic substances; and (v) Ordinance No. 902-B/2007, dated 13 August—approves the materials for determining alcohol presence in expired air and for collecting and transporting biological samples to quantify blood alcohol concentration (BAC) or detect psychotropic substances in the blood.
Specific aspects of Portuguese legislation
When available, the laws that regulate driving under the influence of psychoactive substances vary among countries. One typical difference is the accepted limit of the BAC for someone to be charged. Thresholds ranged from the limit of detection (zero tolerance) to 0.8 g/L (i.e. 0.08%). The website
Consequences of ethanol on driving and variability of toxicological response
The effects of ethanol, which primarily acts as a central nervous system depressant, have been shown to lead to a decrease in essential capabilities for safe driving [
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Factors that can influence antemortem ethanol analytical results
It is important to highlight several aspects to pay attention, dependent on whether we are considering a breath or a blood sample.
Breath samples
Concerning breath samples, it is crucial to be mindful that the breath alcohol concentration (BrAC) relates more to the concentration of ethanol in arterial blood than to that in venous blood (typically collected from the median cubital vein) [
The quantification of BrAC is possible since the concentration of ethanol in exhaled air is in equilibrium with the concentration in the blood of the pulmonary capillaries [
The presence of residual alcohol in the oropharynx resulting from recent ingestion, vomiting or regurgitation of gastric contents, administration of cough medicines, mouthwash, or breath sprays may occasionally lead to higher values in exhaled air [
The breathing pattern immediately preceding a breath test can also impact the measured BrAC [
Blood samples
With respect to blood samples, collection is typically performed from antecubital fossa veins of the forearm [
Improper skin disinfection can serve as a potential source of contamination, introducing microorganisms from the skin microbiome [
The “after-drinking” scenario is another issue reported in the literature in cases related to driving under the influence of ethanol. These cases are referred to as the hip flask defence, in which following a motor vehicle accident, the driver evades law enforcement, and is later apprehended with a positive/illegal BAC [
The absorption of ethanol through the skin from antiseptics or hand sanitizers has not been demonstrated to elevate BAC [
Alcoholic fruit brandies and liqueurs included in chocolates were found to contain relevant concentrations of ethanol and congeners [
Finally, it is important to pay attention to the two methods and tubes commonly used for venous blood collection for forensic purposes. When blood is collected using a “syringe and a needle”, an additional step is required, i.e. transferring the collected blood from the syringe into a clean, dry tube for subsequent transport to the laboratory for analysis. On the other hand, “pre-evacuated grey-top tubes” for BAC analysis for forensic purposes provide a more convenient method for collecting venous blood. These tubes are sold with an expiration date, which ensures the integrity of the vacuum seal, and they typically contain a mixture of sodium fluoride (NaF, 100 mg) as the preservative and 20 mg of potassium oxalate as the anticoagulant for 10 mL of capacity [
Factors that can influence postmortem analytical results
The interpretation of the results of postmortem BAC is much more complex than that of antemortem blood, especially in cases of polytraumatized victims or those in a high state of decomposition. Moreover, in living individuals, blood is collected under sterile conditions, a fact that is not possible in postmortem samples since contamination most likely already occurred at the time of collection. Therefore, pragmatically considering two particularly important aspects, the risk of an artificial increase in BAC due to postmortem ethanol production or redistribution, is important.
The risk of postmortem ethanol production
In the first hours after death, intestinal bacteria migrate to the portal venous system and, after ∼6 h, contamination of systemic vessels already happen [
Endogenous ethanol production occurs by the anaerobic action of microorganisms on endogenous substrates, especially carbohydrates such as hexose glucose. The most commonly used pathway for glucose metabolism is the Embden–Meyerhof–Parnas glycolytic pathway, which generates two molecules of ethanol and two molecules of carbon dioxide from a glucose molecule. Therefore, and theoretically, if the glucose concentration in blood or urine is 1 g/L (i.e. (1g/L)/(180.156 g/L) = 0.00555 mol/L), simple calculations showed that glycolysis and fermentation would result in a BAC of 0.5 g/L (i.e. 0.005 55 mol/L glucose × 2 moles of ethanol×46.068 g/mol = 0.511 g/L of ethanol) [
Elevated environmental temperatures, duration of storage [
The recommended precautions to decrease (but most likely not to eliminate) microbial activity after sampling (and consequently decrease ethanol and other volatile formations) include blood collection for tubes with appropriate concentrations of an enzyme inhibitor preservative (e.g. 1%–2% w/v sodium or potassium fluoride) and storage under correct refrigerating conditions (4◦C) [
Under certain circumstances, BAC postmortem results should be compared with the analysis of other fluids, especially those that are more resistant to microbial contamination, such as the vitreous humour and urine [
The specific case of postmortem redistribution
Postmortem redistribution is a great challenge in postmortem toxicology since the corpse is not a static postmortem entity as far as the distribution of xenobiotics and endobiotics is concerned. This phenomenon depicts the movement of compounds between organs, between vascular compartments, and between vascular compartments and organs and vice versa and depends on several factors. Regarding ethanol, the passive diffusion from the gastric contents (i.e. not yet metabolized or absorbed), or from the respiratory tract as a result of aspiration of vomit, continues after death [
Finally, although the most frequent scenario is a postmortem increase in BAC, a decrease in BAC can also occur. Indeed, reanalysis of stored frozen samples (−20◦C) for up to 12 months is needed if a confirmatory analysis is requested, as these samples tend to produce <8% of the BAC even when preservatives containing fluoride are present [
Possibilities exist to differentiate exogenous ethanol sources from endogenous sources
In view of the various constraints described above, several studies have proposed strategies to differentiate exogenous/ingested ethanol from that resulting from endogenous production or postmortem redistribution processes. Among them, the following strategies are outlined:
Conclusions and future perspectives
The evaluation of states of influence by psychotropic substances while driving is a complex clinical and forensic issue [
Finally, the possibility of auto-brewery syndrome should not be disregarded; therefore, a clinical history is fundamental [
Acknowledgements
The author acknowledges the editorial support, namely, the constructive review of the manuscript and raised comments. A special thanks to Prof. Alan Wayne Jones for having created much of the current knowledge regarding forensic, clinical, pharmacokinetic, and pharmacodynamic aspects to ethanol abuse.
Compliance with ethical standards
Not applicable.
Conflict of interest
Ricardo Jorge Dinis-Oliveira holds the position of Editorial Board Member for Forensic Sciences Research and is blinded from reviewing or making decisions for the manuscript.
Disclosure statement
The author has no conflicts of interest to declare.
Funding
The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. The potential conflicts include employment, consultancies, honoraria, stock ownership or options, expert testimony, grants, or patents received or pending, and royalties.
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Ricardo Jorge Dinis-Oliveira. “Not everything that can be counted counts” in ethanol toxicological results: an antemortem and postmortem technical interpretation focusing on driving under the influence[J]. Forensic Sciences Research, 2024, 9(3): owae023
Category: Research Articles
Received: Jan. 25, 2024
Accepted: Mar. 29, 2024
Published Online: Sep. 22, 2025
The Author Email: Ricardo Jorge Dinis-Oliveira (ricardo.dinis@iucs.cespu.pt)