Ethylglucuronide in head hair (HEtG) is nowadays the most accredited marker to prove chronic alcohol abuse, due to its undeniable performances in terms of discrimination between high-risk and low-risk alcohol drinkers. Among scientific societies, many efforts have been made to prepare consensus documents and protocols, so as to regulate the interpretation of analytical results and the consequent clinical/legal judgments. Nevertheless, warnings about the general applicability of the HEtG have been recently raised. In this study, we summarize the results of the HEtG determination across a large cohort of subjects (n=20,293), that have been obtained over 4 years of activity (2009-2013) in our lab in Orbassano (Piedmont, Italy). Our aim was to provide a general perspective of HEtG determination and investigate if age, gender, BMI, period of sampling, and site of hair collection could bias the analytical results. The experimental results were interpreted by statistical analysis, on the assumption that large population datasets will provide similarly-shaped distributions, leveling off the specific contribution of individual alcohol consumption.

Hair samples were collected from medical commissions for driving relicensing located in Piedmont, Italy. These medical commissions examine drivers who are called to prove low-risk intake of alcohol by means of HEtG analysis, using 30pg/mg as a cut-off value. HEtG was determined by HPLC-MS/MS, as previously published [V. Pirro et al. Forensic Sci. Int. (2011) 210:271–277].

The examined population consisted mainly of male subjects (93%). The majority of them (48%) was aged above 40 years (n=9731), while 29% was in the range 31–40 (n=5878) and 23% was in the age ranging 18–30 (n=4684). The Yate's Chi-square, Krushall- Wallis and Mann-Whitney tests were applied to compare HEtG level distributions for all samples showing a measurable concentration, 12% of whom presented HEtG concentrations over 30pg/mg. A significant distribution difference (P-values < 0.05) based on age was observed, possibly due to sociological reasons. The percentage of hair samples classified as positive ranged from 4% in the 18–30- year-old group to 18.0% in the group over 40 years old. On the other hand, no differences were found by considering gender and BMI factors. Axillary and pubic hair were confirmed not to represent viable alternative to scalp hair, since biased HEtG distributions were observed, whereas no significant differences were observed between chest and scalp hair: even applying various cut-offs (from 30 to 200pg/mg), the percentage of positive results between chest (n=1349) and head hair (n=15507) was constantly the same. Lastly, a “seasonality” factor was evaluated by comparing the period of collection (March, June, September and December), approximately representing the hair growth in winter, spring, summer and autumn. Across a population of 18917 subjects, the seasonal trend was observed over three cycles, showing the highest HEtG levels in winter and minimum levels in summer, suggesting that sources of bias may exist, likely related to sweating and frequency of hair washing. The average percentage of positive samples for head hair samples grown in winter, spring, summer and fall were 16.7%, 14.1%, 8.3% and 11.7% respectively.

The experimental HEtG distributions conclusively confirm that chest hair sampling can be trusted as an alternative to scalp. Furthermore, among biological and external factors, the season of sampling may significantly influence the measured HEtG concentration, and this potential source of bias should be taken into account when the results are interpreted.