The review, conducted by researchers at the Universidade Federal do Pará in Brazil, searched the Scopus, Web of Science, and PubMed databases for English-language articles published between 2014 and 2024, excluding reviews and meta-analyses. After removing duplicates from an initial pool of 414 articles and screening titles, abstracts, and full texts, 54 studies made the final cut. From these, the team extracted data on study objectives, main results, and recommendations for collection, storage, and biomarker detection. The overarching conclusion is clear: no single sample tells the whole story. Instead, laboratories should analyze multiple matrix types and multiple biomarkers, collect samples in duplicate, refrigerate or freeze them depending on storage duration, use preservatives, and ensure that staff are properly trained.

The central problem the review addresses is microbial fermentation. After death, bacteria and yeasts can convert glucose in the body into ethanol and carbon dioxide, a process that produces so-called post-mortem ethanol. This neoformation is not limited to visibly decomposing bodies; it can occur in specimens kept under inadequate conditions, such as high temperatures or the absence of preservatives like sodium fluoride. The result can be false-positive blood alcohol values that have nothing to do with what the deceased actually drank. Conversely, ethanol already present can be lost through oxidation into acetaldehyde, producing false negatives. Pathological conditions complicate matters further: individuals with diabetes, whose bodies carry excess glucose, can generate significant post-mortem blood ethanol even when they consumed no alcohol at all.

To disentangle drinking from fermentation, forensic toxicologists increasingly rely on non-oxidative metabolites of ethanol, chiefly ethyl glucuronide (EtG) and ethyl sulfate (EtS). Because these compounds are direct products of the body’s metabolism of alcohol, they cannot be manufactured by microbes fermenting glucose. Their presence in post-mortem samples is therefore directly tied to alcohol consumed before death. Studies cited in the review demonstrated a selective association between ante-mortem drinking and EtG and EtS detection: the biomarkers were absent in ethanol-negative samples from both deceased and living individuals, but appeared and then declined over time in ethanol-positive ones. Notably, EtG and EtS are not spontaneously formed even under post-mortem hyperglycemic conditions, so their detection supports metabolic activity before death, including in diabetic cases.

But the two biomarkers behave differently as bodies decompose. EtG is more sensitive yet less stable, and can be degraded during advanced decomposition, risking false negatives. EtS, by contrast, remains reliable even in severely putrefied blood, particularly at high temperatures, making it the more dependable marker for excluding pre-death drinking when results conflict. One strategy the review highlights is measuring EtG in vitreous humor, the clear fluid inside the eye, which is more sterile than blood and better protected from decomposition. Interpretation, the authors stress, must always account for the matrix, preservation conditions, and degree of decomposition.

Acetaldehyde, ethanol’s oxidative metabolite, is another piece of the puzzle, though a treacherous one. Its origin is difficult to pin down: it may arise from bacterial activity, yeast fermentation, ethanol oxidation, putrefaction, or poor blood preservation. The review notes that in well-preserved bodies and living individuals, acetaldehyde concentrations stayed within a stable range of 0.14 milligrams per milliliter or less, while highly putrefied bodies showed significantly higher values, indicating an association with putrefaction. Abnormally high acetaldehyde in the presence of ethanol and higher alcohols such as 1-propanol and 1-butanol may signal microbial neoformation. Conversely, acetaldehyde appearing without ethanol or 1-propanol may indicate that ethanol was oxidized and lost, particularly under extreme storage conditions. Higher alcohols themselves, produced when amino acids released by protein degradation ferment, appear at far lower concentrations than ethanol; a blood 1-propanol cutoff of 0.104 milligrams per deciliter has been proposed as a criterion for identifying post-mortem microbial ethanol production, though some researchers question its reliability.

When it comes to choosing samples, the review finds strong support for a multi-matrix approach. Peripheral blood, generally drawn from the femoral vein to avoid contamination and post-mortem redistribution, remains the primary specimen. Urine is closely associated with blood and, in healthy individuals, contains no glucose, reducing fermentation risk, though diabetic victims or those with urinary infections can confound this advantage. Vitreous humor emerges as a standout: anatomically isolated, minimally susceptible to decomposition and glycosuria, easy to collect from both eyes, and stable after death. Its inclusion as a routine matrix is strongly recommended, especially for putrefied bodies, outdoor deaths, and diabetic individuals. Cerebrospinal fluid, particularly from the spinal canal rather than the cerebral ventricles, offers another protected alternative. More exotic options are also on the table: dried blood spots for stable metabolites, costal cartilage ground in liquid nitrogen, brain tissue, hair for chronic abuse markers, and even necrophagous insect larvae, in which EtS proved a promising and stable indicator of alcohol ingestion.

Preservation practices emerge as the make-or-break factor in sample integrity. Refrigeration at 4 degrees Celsius is the minimum requirement, but samples stored longer than 48 hours should ideally be frozen, preferably at minus 80 degrees. Ethanol losses at room temperature can reach 15 to 16 percent, and even at minus 20 degrees, variations of 8 percent over 30 days have been recorded. Because oxidation is a primary cause of ethanol loss, samples should be collected in duplicate so that unopened tubes remain available for reanalysis; previously opened tubes showed greater losses in long-term studies. Tubes should be filled more than halfway to limit oxygen headspace, sealed tightly, made of sterile glass, and homogenized gently before storage. Fluoride preservatives, at concentrations of roughly 1 to 2 percent sodium or potassium fluoride, are essential: they inhibit both microbial growth and the enzyme enolase, curbing glycolysis and endogenous ethanol production. Anticoagulants such as EDTA or potassium oxalate may accompany them, while lithium heparin alone proved ineffective even at minus 80 degrees.

The stakes of all this extend well beyond the laboratory bench. Blood alcohol levels influence medicolegal interpretation, investigative conclusions, and judicial decisions, and the integrity of the evidence chain from collection to courtroom underpins due process itself. The review’s authors argue that protocols enabling clearer correlations between results and real conditions can prevent or detect alterations in ethanol levels, ensuring validity, traceability, and a fairer trial. They call for integrating ethanol metabolite analysis and multiple matrices into routine laboratory protocols, supported by standard operating procedures, investment in technology, and ongoing staff training. Emerging frontiers, from epigenetic markers of alcohol use disorder to ethyl esters revealing combined use of alcohol and synthetic cannabinoids, promise to widen the analytical toolkit further. For now, the message to forensic science is one of disciplined redundancy: sample widely, preserve rigorously, and never trust a single number to tell the story of how alcohol entered a body, or when.

Subject of Research: Post-mortem ethanol detection and the selection, collection, and storage of biological matrices in forensic toxicology

Article Title: Biological matrices for determining the origin of post-mortem ethanol: Recommendations for collection and storage

Article References: da Paz, A. P. S., Nunez, L. B. B., Jr, & de Mello, V. J. (2026). Biological matrices for determining the origin of post-mortem ethanol: Recommendations for collection and storage. International Journal of Legal Medicine. https://doi.org/10.1007/s00414-026-03994-9

Image Credits: AI Generated

DOI: 10.1007/s00414-026-03994-9

Keywords: forensic toxicology, post-mortem ethanol, ethyl glucuronide, ethyl sulfate, vitreous humor, microbial fermentation, sodium fluoride, blood alcohol, sample preservation, forensic autopsy, biomarkers, chain of custody

The core argument of the correspondence is that fitness-to-drive assessment has become fixated on the biomarker at the expense of context. Biomarkers such as carbohydrate-deficient transferrin in serum, ethyl glucuronide in hair, and phosphatidylethanol in dried blood spots are powerful indicators of alcohol consumption, and their technical performance has been extensively validated. Recent comparative work, cited in the correspondence, has examined whether serum carbohydrate-deficient transferrin or hair ethyl glucuronide offers the more reliable tool for diagnosing alcohol abuse, and protocols now exist for quantifying phosphatidylethanol in dried blood spots to monitor abstinence. Studies in driver’s license regranting cases have combined hair ethyl glucuronide, urinary ethyl glucuronide and ethyl sulfate, and capillary dried blood spot phosphatidylethanol to reconstruct a person’s drinking history. These methods are genuinely impressive pieces of analytical chemistry.

The problem, Palmiere argues, is not the chemistry but the interpretation. Biomarker concentrations do not exist in a social vacuum. Patterns of alcohol consumption vary enormously between cultures, and reference intervals and decision thresholds are typically derived from populations whose drinking habits, body compositions, and genetic backgrounds may differ from those of the person being tested. A threshold calibrated in one population may flag as abnormal a pattern of consumption that is unremarkable in another, or conversely may miss hazardous drinking that presents atypically. Without awareness of these population-level differences, a laboratory value that looks like a clean, objective answer can quietly encode the demographics of the population in which it was validated.

The same concern applies with even greater force to the screening questionnaires that sit alongside laboratory testing. The Alcohol Use Disorders Identification Test, developed under the auspices of the World Health Organization, is one of the most widely used instruments for detecting excessive drinking in primary care and in traffic medicine. Its diagnostic performance in detecting alcohol use disorders in the general population has been studied extensively, and validated translations exist, including a French version whose reliability and validity in primary care settings have been documented. But translation is not equivalence. Idioms about drinking, culturally shaped patterns of what counts as a drink, and differing willingness to disclose personal behavior to an authority figure can all shift how respondents answer. A questionnaire that performs well in its original cultural setting may systematically under- or overestimate alcohol problems in respondents from other backgrounds, and the correspondence suggests that this possibility is too rarely acknowledged in the medico-legal context.

Linguistic barriers compound the psychometric ones. Fitness-to-drive evaluations are high-stakes encounters in which the person being assessed has strong incentives to present themselves favorably, and the assessor must judge not only what is said but how it is said. When the assessed person and the assessor do not share a fluent common language, or when interpretation is improvised rather than professional, subtle cues of evasiveness or misunderstanding may reflect communication difficulty rather than deception or impairment. Screening instruments administered in a language the respondent does not command lose their validated properties entirely, yet the correspondence implies that such administrations still occur and that their results still carry weight in licensing decisions.

Structural factors add a third layer of inequity. Access to driving is not a luxury in many communities; it is a precondition of employment, caregiving, and basic participation in social life, particularly in rural and peri-urban areas poorly served by public transport. A person who loses a license after a positive screening result faces consequences that ripple through an entire household, and the ability to navigate the regranting process, including repeated testing, documentation requirements, and sometimes costly follow-up assessments, is unevenly distributed across social groups. People with precarious employment, limited health literacy, or insecure immigration status may struggle to comply with procedural demands that more advantaged drivers meet without difficulty, so identical medical findings can lead to very different practical outcomes depending on the driver’s circumstances.

The correspondence situates these concerns within a specific legal framework, drawing on the Swiss Federal Act on Road Traffic and the Swiss Ordinance on the Admission of Persons and Vehicles to Road Traffic. Swiss law, like the law of many European jurisdictions, ties license retention and regranting to medical assessments of fitness to drive, including in cases involving alcohol abuse and psychotropic substance use. Scholarly work on medicinal drugs and fitness to drive, and on psychotropic substance abuse and driving license eligibility in Italy, illustrates how widespread these assessment regimes have become. Where the law mandates a medical judgment, the fairness of the underlying instruments becomes a matter of legal as well as clinical significance, because an inequitable test translates directly into an inequitable sanction.

What would a more equitable assessment look like? The correspondence does not propose abandoning biomarkers or questionnaires, which would be neither feasible nor scientifically justified. Instead, it points toward a more contextualized practice: awareness of the population-specific limits of reference intervals and cut-offs, use of professionally translated and locally validated instruments wherever possible, professional interpretation rather than ad hoc language support, and a structural understanding of how compliance burdens fall unevenly. It also implies that assessors should treat convergent evidence, combining laboratory markers, validated questionnaires, clinical examination, and driving history, rather than elevating any single value into a verdict. The literature on combining carbohydrate-deficient transferrin with ethyl glucuronide in hair, for example, already reflects the logic that no single marker is definitive; the equity argument extends that logic from analytical uncertainty to social context.

The wider significance of the argument reaches beyond traffic medicine. Fitness-to-drive assessment is one of many medico-legal domains in which standardized instruments, developed and validated in particular populations, are applied to increasingly diverse societies. Forensic toxicology, psychiatric evaluation, and occupational medicine all face versions of the same problem: tools that are objective within their validation samples can become instruments of disparity when transplanted without scrutiny. The correspondence’s contribution is to name this problem explicitly in a domain where the stakes, both for road safety and for individual liberty and livelihood, are unusually concrete.

Published as a correspondence in the International Journal of Legal Medicine, the piece is deliberately concise, and it raises questions more than it settles them. How large are the cultural and linguistic effects on biomarker interpretation and questionnaire performance in real licensing populations? Which jurisdictions have already adapted their assessment protocols, and which continue to apply one-size-fits-all thresholds? Those empirical questions now await the field’s attention. What the correspondence makes clear is that the pursuit of road safety through medical assessment cannot be separated from the pursuit of fairness in how that assessment is designed, administered, and interpreted. A system that protects the public from genuinely impaired drivers while treating every driver, whatever their language or background, as equally credible and equally comprehensible, is the standard toward which the field is being asked to move.

Subject of Research: Equity and validity in medical fitness-to-drive assessment

Article Title: Beyond the biomarker: cultural, linguistic, and structural equity in fitness-to-drive medical assessment

Article References: Palmiere, C. (2026). Beyond the biomarker: cultural, linguistic, and structural equity in fitness-to-drive medical assessment. International Journal of Legal Medicine. https://doi.org/10.1007/s00414-026-03937-4

Image Credits: AI Generated

DOI: 10.1007/s00414-026-03937-4

Keywords: fitness to drive, legal medicine, biomarkers, alcohol abuse screening, AUDIT, carbohydrate-deficient transferrin, ethyl glucuronide, phosphatidylethanol, health equity, psychometrics, road traffic medicine, Swiss road traffic law