Thomas M. Maenhout

Non-oxidative ethanol metabolites as a measure of alcohol intake

Recent alcohol intake can be monitored by the measurement of indirect biomarkers. Elevated levels of liver enzymes (i.e. gamma-glutamyl transferase (GGT), alanine amino transferase (ALT) and aspartate amino transferase (AST)) in blood are commonly used in clinical practice as an indicator of alcohol-induced liver damage. With the exception of carbohydrate-deficient transferrin (CDT), the specificity of indirect markers is only moderate because many cases of elevated levels are unrelated to alcohol consumption. Because of their intermediate half-life and tendency to accumulate in hair, non-oxidative ethanol metabolites can be used as markers with an intermediate timeframe between ethanol measurements and GGT and CDT with regard to recent alcohol consumption occurring between hours to 1 week. Additionally, these biomarkers offer a high ethanol-specificity in combination with approximately a two-fold higher sensitivity in comparison with indirect alcohol markers. In case of forensic use of direct ethanol metabolites, caution has to be taken in interpretation and pre-analytical pitfalls should be considered.

Carbohydrate Deficient Transferrin in a Driver's License Regranting Program

AIMS Carbohydrate deficient transferrin (CDT) is a common diagnostic marker for detecting chronic alcohol abuse. For over 2.5 years, it has been used in traffic medicine among subjects applying for drivers license renewal or regranting in Belgium. We report on data collected during the program and provide an estimation of an applicable cut-off point in forensic situations. Using this cut-off, the success of the drivers license regranting program is evaluated. METHODS CDT was assayed at Ghent University Hospital by capillary zone electrophoresis, measured on the Capillarys 2™ system, in 3977 subjects applying for drivers license regranting. Determination of a cut-off was done by using Bhattacharya statistics and by adding a measurement uncertainty interval. The outcome of the program was evaluated by monitoring CDT values for 163 subjects during one entire year. RESULTS In 3977 subjects (3481 males and 496 females), CDT values were significantly higher in men compared with women, but there is no need for a gender-specific cut-off value. Drunk drivers under the age of 30 have significantly lower CDT values than older subjects, and a separate cut-off could be calculated. A general cut-off of 2.3% CDT was calculated for the entire study population. Using this cut-off value for evaluating the outcome of the program for 163 subjects, the percentage offenders at the beginning (29%) decreased to 8% after 1 year. CONCLUSION Applying a marker for chronic alcohol abuse such as CDT for drivers license renewal or regranting is a powerful tool. Analysis of data collected over 2.5 years reveals a favorable outcome of the program and a useful cut-off point could be determined.

Capillary electrophoresis of urinary prostate glycoproteins assists in the diagnosis of prostate cancer.

Prostate marker assays are widely used for detection of prostate cancer (PCa) but are associated with considerable sensitivity and specificity problems. Therefore, we investigated prostatic protein glycosylation profiles as a potential biomarker. We determined the urinary asparagine‐linked glycan (N‐glycan) profile of prostatic proteins of healthy volunteers (n = 25), patients with benign prostate hyperplasia (BPH; n = 62) and newly diagnosed PCa patients (n = 42) using DNA‐sequencer‐assisted fluorophore‐assisted carbohydrate electrophoresis. Through squeezing of the prostate, a sufficient amount of prostatic proteins was obtained for direct structural analyses of N‐glycan structures. N‐glycans of PCa compared to BPH were characterized by a significant decrease in triantennary structures (p = 0.047) and overall fucosylation (p = 0.026). Prostate‐specific antigen (PSA) and the urinary glycoprofile marker showed comparable overall receiver operating characteristic curve analysis as well as in the diagnostic gray zone with serum PSA values between 4 and 10 μg/L. However, when combining PSA and the urinary glycoprofile marker, the latter gave an additive diagnostic value to serum PSA (p ≤ 0.001). In conclusion, N‐glycosylation profiling demonstrated differences between BPH and PCa. These changes could lead to the discovery of a new biomarker for PCa.

Automated indirect immunofluorescence microscopy enables the implementation of a quantitative internal quality control system for anti-nuclear antibody (ANA) analysis.

Abstract Background: Screening for anti-nuclear antibodies by indirect immunofluorescence (ANA-IIF) remains mandatory in the serological work-up of connective tissue diseases. Recently, automated approaches were introduced that may improve harmonization. Here, we investigated whether the introduction of automated ANA-IIF and more specifically the use of its quantitative measure, could improve ANA-IIF internal quality control (IQC) management. Methods: We retrospectively reviewed results of two cohorts of routine samples and parallel IQC data collected from January 2010 to February 2013 and from February to mid October 2013. For the first cohort, data were collected using conventional microscopy. The second cohort was analyzed by an automated ANA-IIF microscope (Zenit G sight, A. Menarini). Retrospectively, we evaluated the applicability of the probability index (PI) of control material measurements and patient results for IQC management based on Westgard multirules. This approach was also compared with monthly monitoring of the %ANA-IIF positive samples. Results: In our historical data set, we showed that monitoring of %ANA positives identified systematic errors that were not detected by monitoring control material results. Data resulting from automated microscopy showed that PI measurements on control material remained stable within the observed period and that Westgard multirules can be used for IQC follow-up. Parallel monitoring of the daily median patient PI and the monthly %ANA positives, showed that the daily median was a sensitive and fast tool for detecting systematic errors. Conclusions: The introduction of the automated ANA-IIF microscope could enable objective IQC procedures and should be considered an important step forward in ANA-IIF harmonization.

Immature granulocyte count in peripheral blood by the Sysmex haematology XN series compared to microscopic differentiation

Recent advances in automation of haematology analysers have significantly shortened turn-around-times for reporting leukocyte differential and count in the peripheral blood. Due to the introduction of improved flagging rates, the amount of microscopic peripheral blood slide reviews can be minimised.1 Having access to an enormous amount of data, one has to find the balance between productivity and clinical quality. Reliable automated blood cell characterisation and quantification remain challenging in pathological samples, whereas slide reviews due to unnecessary flagging should be avoided in normal samples. An important feature of the automated haematology cell counters is their ability to identify and quantify immature granulocytes (IG) in a peripheral blood sample. Recently, the Sysmex XN series was introduced and its performance was evaluated.2 It provides the possibility to automatically count the IG in the peripheral blood. After applying a specific lysing agent (Lysercell WDF), the IG are measured in the WDF channel and differentiation is made based on granularity (side scatter) and nucleic acid content (side fluorescence by the Fluorocell WDF reagent). The IG cluster is found right above the neutrophil cluster in the biparametric histogram side scatter/fluorescence. The IG fraction includes promyelocytes, myelocytes and metamyelocytes (blasts and band cells are not included). In the present study, …

Usefulness of Indirect Alcohol Biomarkers for Predicting Recidivism of Drunk-driving among Previously Convicted Drunk-driving Offenders: Results from the Recidivism Of Alcohol-impaired Driving (ROAD) Study

AIM In several European countries, drivers under the influence (DUI), suspected of chronic alcohol abuse are referred for medical and psychological examination. This study (the ROAD study, or Recidivism Of Alcohol-impaired Driving) investigated the usefulness of indirect alcohol biomarkers for predicting drunk-driving recidivism in previously convicted drunk-driving offenders. DESIGN, SETTING, PARTICIPANTS AND MEASUREMENTS The ROAD study is a prospective study (2009-13) that was performed on 517 randomly selected drivers in Belgium. They were convicted for drunk-driving for which their licence was confiscated. The initial post-arrest blood samples were collected and analysed for percentage carbohydrate-deficient transferrin (%CDT), transaminsase activities [alanine amino transferase (ALT), aspartate amino transferase (AST)], gamma-glutamyltransferase (γGT) and red cell mean corpuscular volume (MCV). The observation time for each driver was 3 years and dynamic. FINDINGS A logistic regression analysis revealed that ln(%CDT) (P < 0.001), ln(γGT) (P < 0.01) and ln(ALT) (P < 0.05) were the best biochemical predictors of recidivism of drunk-driving. The ROAD index (which includes ln(%CDT), ln(γGT), -ln(ALT) and the sex of the driver) was calculated and had a significantly higher area under the receiver operator characteristic curve (0.71) than the individual biomarkers for drunk-driving recidivism. Drivers with a high risk of recidivating (ROAD index ≥ 25%; third tertile) could be distinguished from drivers with an intermediate risk (16% ≤ ROAD index < 25%; second tertile; P < 0.001) and a low recidivism risk (ROAD index < 16%; first tertile; P < 0.05). CONCLUSIONS Of all routinely used indirect alcohol markers, percentage of carbohydrate-deficient transferrin is the major predictor of recidivism of drunk-driving. The association with gamma-glutamyltransferase, alanine amino transferase and the sex of the driver could have additional value for identifying drunk-drivers at intermediate risk of recidivism. Non-specific indirect alcohol markers, such as alanine amino transferase, gamma-glutamyltransferase, aspartate amino transferase and red cell mean corpuscular volume have minimal added value to % carbohydrate-deficient transferrin for distinguishing drunk drivers with a low or high risk of recidivism.

Immunonephelometric Carbohydrate-Deficient Transferrin Results and Transferrin Variants

To the Editor: Carbohydrate-deficient transferrin (CDT)1 is a biomarker of growing importance in the assessment of alcohol abuse after conviction for drunk driving. CDT is a more specific indicator for alcohol than traditional liver function tests and is used for identification and follow-up of chronic high alcohol consumption (1). Various methods have been introduced for assaying CDT in serum, including isoelectric focusing, ion-exchange chromatography, HPLC, capillary zone electrophoresis (CZE), and latex enhanced immunonephelometry (1, 2). Measuring the CDT percentage (%CDT) in a forensic context demands CZE or HPLC methodology, because it provides high-resolution separation of serum transferrin (Tf) isoforms and allows the detection of genetic variants and glycosylation disorders. In some cases, the interpretation of CDT results is hampered by the presence of mutant Tf (3). In addition to wild-type Tf (C), D variants (cathodal to C) and B variants (anodal to C) have been described (1). The allele frequencies of the Tf subtypes vary among populations of different ethnicities (4). Exact measurement of D variants of CDT is difficult, however, because di- and trisialylated Tf may coelute with the tetrasialylated D peak …

Microheterogeneity of serum β-hexosaminidase in chronic alcohol abusers in a driver's license regranting program.

BACKGROUND Carbohydrate-deficient transferrin (CDT) is one of the best indicators for chronic alcohol abuse and detection of relapse. In this study, we explore the microheterogeneity of β-hexosaminidase (β-HEX) in chronic alcohol abusers in the framework of a drivers license regranting program. Studies have shown that increased serum activity of β-HEX B (isoforms P, I, and B) may be a sensitive marker for chronic alcohol abuse. Here, we describe methodology, limitations, and correlation of β-HEX isoforms with CDT. METHODS CDT was assayed at the central laboratory of the Ghent University Hospital by capillary zone electrophoresis, measured on the Capillarys 2™ system and was expressed as a percentage of total serum transferrin (%CDT). Serum of chronic alcohol abusers was compared to nonheavy drinkers using agarose gel isoelectric focusing (IEF). Total β-HEX activity was assayed fluorimetrically following preparative IEF in 81 subjects. β-HEX isoforms were investigated and compared between nonheavy drinkers and heavy drinkers. RESULTS Agarose gel IEF shows additional cathodal bands in serum of chronic alcohol abusers. Mean total β-HEX activity between pH 6.8 and 7.7, designated as HEX-7, showed the highest correlation with %CDT (r = 0.70, p < 0.0001, n = 68). In a selected subgroup, where CDT could not be quantified (n = 13) because of an atypical electropherogram, HEX-7 was in concordance with either estimated %CDT value or liver enzyme activities. CONCLUSIONS In this proof-of-concept study, we introduce a novel approach to quantify β-HEX isoforms using preparative IEF and fluorimetry. A highly significant correlation of HEX-7 and %CDT has been found. Because of exclusion of the P isoform, HEX-7 could be a useful supplementary marker for detecting chronic alcohol abuse.

Impact of the routine implementation of automated indirect immunofluorescence antinuclear antibody analysis: 1 year of experience.

*Corresponding author: Carolien Bonroy, Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospital (2P8), De Pintelaan, 185, 9000 Ghent, Belgium, Phone: +32 9 332 36 31, Fax: +32 9 332 49 85, E-mail: carolien.bonroy@uzgent.be Sylvie M.N. Mulliez and Thomas M. Maenhout: Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospital, Ghent, Belgium Letter to the Editor

Detecting doping use: more than an analytical problem

Abstract The recent Armstrong case, where more than 250 negative doping tests are confronted with the athlete’s confession of erythropoietin use, blood doping, steroid, and growth hormone abuse, illustrates the limitations of current laboratory tests in detecting doping in sport. Despite numerous doping controls and simultaneous indications of common doping abuse among professional athletes in the last two decades, the number of positive urine tests for recombinant human erythropoietin (rHuEPO) remains remarkably low. Athletes are using various masking strategies, among them protease inhibitors, intravenous injections of rHuEPO and alternative erythropoiesis stimulating agents. As one of the countermeasures, the Athlete’s Biological Passport has been introduced. The sensitivity of the Athlete’s Biological Passport is limited if the effect of a low-dose doping remains within the intra-individual reference range. A possible solution could be the use of a novel Epo test (MAIIA Diagnostics). Another performance-enhancing strategy is the return to ‘old’ doping techniques, such as autologous blood transfusions. Several indirect methods to detect autologous blood transfusions have been proposed with the majority relying on changes in erythropoiesis-sensitive blood markers. Currently, an algorithm based on the haemoglobin (Hb) level concentration and the percentage of reticulocytes (OFF-hr model; Hb(g/l)–60·√%ret) is approved by the World Anti-Doping Agency. Genetic factors have been identified which may interfere with test interpretation. A large inter- and intra-ethnic variation in testosterone glucuronidation and excretion has been described. Consideration of genetic variation should improve performance of the testosterone doping test. Taking into account the pre-analytical care and better tailoring of the threshold values could increase test sensitivity. Anti-doping laboratories should routinely adjust for multiple testing as failure of doping control to detect cheaters could lead to more frequent controls. Finally, despite the huge technological progress, there is a need for increased collaboration between physiologists, analytical chemists, biostatisticians, and ethicists to reduce doping in sport.