Exercise Induced Myoglobinuria is Determined by Haptoglobin Polymorphism.

BACKGROUND: In professional soccer players (n = 27), confounders of quantitative myoglobinuria following physical training were assessed in order to improve interpretation of post-exercise myoglobinuria. METHODS: Urine samples were collected in the morning before training sessions, 48 to 72 hours following a game. Urine myoglobin was assayed using immunoturbidimetry. Blood was drawn 48 hours following training session. Creatinine was assayed using a Jaffe method. Creatine kinase (CK) activity was assayed according to the IFCC reference method. Serum myoglobin was assayed using the same assay as the one used for urine. Hp polymorphism was assessed on hemoglobin supplemented serum. Serum Hp concentration was assayed nephelometrically. Training intensity was assessed using a wearable GPS tracking system. Physical load monitoring included the covered total distance, the distance at different speed zones, and the number of sprints/accelerations/decelerations/jumps. Multiple regression analysis was used to detect the determinants of post-exercise myoglobinuria. RESULTS: Myoglobinuria negatively correlated with serum haptoglobin (Hp) concentration. Athletes presented with Hp values, which were lower than the Hp phenotype reference ranges, which can be explained by depletion of circulating Hp stores. Myoglobinuria was most pronounced in players carrying a Hp 2-2 phenotype, which is associated with the lowest Hp reference range. Myoglobin clearance was inversely correlated with Hp 2-2 concentration. Correlation between myoglobinuria and biomarkers of muscle damage was weak. Neither age nor glomerular filtration rate were found to be confounders of myoglobinuria. When comparing myoglobinuria with training intensity, the number of sprints, average acceleration speed, and maximal speed were determining factors for predicting exercise-induced myoglobinuria. CONCLUSIONS: In athletes, plasma myoglobin binding capacity is depleted. Moderate myoglobinuria not only should be regarded as a muscle damage marker, but also should be interpreted as an indicator for Hp depletion. Apart from its significance as a biomarker for muscle damage and rhabdomyolysis, myoglobinuria in athletes should be a warning that the heme binding capacity of plasma Hp is depleted, indicating an exhausted defense against Fenton chemistry induced free radicals. Fenton chemistry is associated with free radical formation, which is to be avoided because of the causative relationship with inflammatory processes and tissue damage.

Minimal Coexpression of CD34+/CD56+ in Acute Promyelocytic Leukemia Is Associated With Relapse.

OBJECTIVES: Surface CD56 expression on leukemic cells in acute promyelocytic leukemia (APML) is considered an indicator of poorer outcome even in patients receiving conventional treatment. METHODS: In the present case, at initial diagnosis, the hallmark phenotype of APML was found (strong CD33 and cytoplasmic MPO expression, absence of HLA-DR expression). RESULTS: Both CD34 and CD56 antigen expression was considered negative. The patient relapsed 3 years after reaching complete remission, and the hallmark surface antigen combination for APML was again found. In contrast, the leukemic cells now clearly coexpressed CD34 and CD56. Retrospective analysis revealed the presence of small CD34+ and CD56+ populations at initial diagnosis (<20%). CONCLUSIONS: This case report suggests that the presence of a clone with minimal coexpression of CD34/CD56 in APML at initial diagnosis should not be neglected since it may be associated with earlier relapse.

An abnormally glycosylated isoform of erythropoietin in hemangioblastoma is associated with polycythemia.

BACKGROUND: Hemangioblastomas express erythropoietin and the patients often present with polycythemia. METHODS: Serum erythropoietin was measured using a commercial immunoassay, a functional erythropoietin assay and iso-electric focusing. RESULTS: Despite the polycythemia, serum erythropoietin remained low, while a functional erythropoietin-assay showed a 4-5 higher activity in serum compared to the immunoassay. Iso-electric focusing of serum erythropoietin indicated overrepresentation of highly sialylated erythropoietin isoforms produced by the tumor. As a result, altered affinity of the monoclonal antibody used in the immunoassay for the hypersialylated isoforms was suggested. CONCLUSION: Analysis of erythropoietin isoforms may be helpful in distinguishing the ectopic erythropoietin isoforms from normally glycosylated erythropoietin.

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

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.

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.

Microheterogeneity of serum ß-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 ß-hexosaminidase (ß-HEX) in chronic alcohol abusers in the framework of a driver's license regranting program. Studies have shown that increased serum activity of ß-HEX B (isoforms P, I, and B) may be a sensitive marker for chronic alcohol abuse. Here, we describe methodology, limitations, and correlation of ß-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 ß-HEX activity was assayed fluorimetrically following preparative IEF in 81 subjects. ß-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 ß-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 ß-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.

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 driver's 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 driver's 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 driver's 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 driver's 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.