Walter-Michael Halbmayer

Interference of the new oral anticoagulant dabigatran with frequently used coagulation tests

Abstract Background: Dabigatran etexilate is a new oral anticoagulant for the therapy and prophylaxis of venous thromboembolism and stroke prevention in patients with atrial fibrillation. To investigate the extent of interactions of this new anticoagulant with frequently used coagulation assays, we completed a multicenter in vitro trial with Conformité Européenne(CE)-labeled dabigatran-spiked plasma samples. Methods: Lyophilized plasma samples with dabigatran concentrations ranging from 0.00 to 0.48 μg/mL were sent to the coagulation laboratories of six major Austrian hospitals for evaluation. Coagulation assays were performed under routine conditions using standard reagents and analyzer. Results: Dabigatran led to a dose-dependent prolongation of the clotting times in coagulometric tests and influenced the majority of the parameters measured. Statistically significant interference could be observed with the prothrombin time (PT), activated partial thromboplastin time (aPTT) and PT/aPTT-based assays (extrinsic/intrinsic factors, APC-resistance test) as well as lupus anticoagulant testing. Even non-clotting tests, such as the colorimetric factor XIII activity assay and to a minor extent the amidolytic antithrombin activity assay (via factor IIa) were affected. Conclusions: This multicenter trial confirms and also adds to existing data, demonstrating that laboratories should expect to observe strong interferences of coagulation tests with increasing concentrations of dabigatran. This finding might become particularly important in the elderly and in patients with renal impairment as well as patients whose blood is drawn at peak levels of dabigatran.

Platelet size, fibrinogen and lipoprotein(a) in coronary heart disease

BackgroundAn increase in mean platelet volume has been reported to be associated with arterial thrombosis and myocardial infarction. A larger mean platelet volume has been regarded as an independent risk factor for recurrent myocardial infarction. We therefore investigated whether it is also increased in patients with coronary heart disease examined a few days before cardiac surgery. MethodsFour hundred and twenty-six patients with coronary heart disease who were waiting for cardiac surgery and 125 healthy individuals were included in the study. Mean platelet volume and other platelet parameters were obtained from a routine blood count procedure using a flow cytometric haematology analyser. ResultsMean platelet volume did not differ significantly between patients and controls; however, as expected from the literature, patients had significantly elevated levels of fibrinogen, cholesterol, triglyceride, apolipoprotein B and apolipoprotein(a). Furthermore, we observed no significant difference in mean platelet volume between patients without myocardial infarction and those who had survived at least one myocardial infarction. ConclusionOur findings suggest that, using a routine laboratory procedure, mean platelet volume cannot be used as a predictive marker for coronary heart disease or myocardial infarction.

HCV genotypes and age distribution in patients of Vienna and surrounding areas

BACKGROUND Chronic hepatitis C (CHC) can result in liver cirrhosis and hepatocellular carcinoma. Determination of the hepatitis C virus (HCV) genotype/subtype may be of prognostic value to estimate the risk of development of liver cirrhosis. OBJECTIVE The HCV genotype/subtype was determined in patients with CHC and possible associations with age, source of HCV transmission, duration of HCV infection, and development of liver cirrhosis were investigated. STUDY DESIGN A total of 250 consecutive patients with CHC were studied. HCV genotypes/subtypes were determined with a commercially available assay based on the reverse-hybridization principle. Source of HCV transmission and duration of HCV infection were taken from the patient documentation and liver cirrhosis was diagnosed by clinical, biochemical, and sonographic data. RESULTS HCV genotypes 1, 2, 3, 4, and 5 were found in 74.8, 2.8, 16, 5.2, and 0.4% of the patients. Most frequent subtypes were 1b (54%), 1a (15.6%), and 3a (15.6%). Patients with genotype 1 (mean, 52.8 years) or 2 (mean, 51.0 years) were significantly older than patients with genotype 3 (mean, 37.2 years) or genotype 4 (mean, 37.2 years). Patients with subtype 1b (mean, 58.1 years) were significantly older than patients with subtype 1a (mean, 40.8 years) or 3a (mean, 37.5 years). The main sources of HCV infection were intravenous drug abuse in 30.0% of all patients (genotype 1 in 53.3%; genotype 3 in 40%) or transfusion of blood and blood products in 21.6% of all patients (genotype 1 in 83.4%). The source of transmission, however, remained unknown in 44.8% of all patients. The prevalence of genotype 1 was significantly higher in patients with long duration (more than 20 years) of CHC. In none of the patients with genotype 2 or 3, duration of CHC for more than 20 years was observed. The prevalence of genotype 4 was significantly higher in patients with short duration (less than 10 years) of CHC. Liver cirrhosis was diagnosed in 13.6% of all patients (97.1% of patients with genotype 1). Patients with liver cirrhosis were significantly older compared to asymptomatic patients (mean, 63.8 vs. 51.3 years). CONCLUSION HCV subtype 1b was found to be the main subtype in the investigated population and is currently the major contributor to liver cirrhosis. Patients infected with subtype 1a, however, are at comparable risk for development of liver cirrhosis. In future, subtype 3a and genotype 4 may also become an increasing problem.

Relation of homocysteine, vitamin B12, and folate to coronary in-stent restenosis

Coronary in-stent restenosis represents a clinical problem. Because homocysteine is being discussed as a new risk factor for atherosclerosis and thrombosis, this study investigated the relations of homocysteine, folate, and vitamin B(12) to the rate of in-stent restenosis. Patients undergoing successful percutaneous transluminal coronary angioplasty of native coronary lesions with stent implantation were investigated for fasting total serum homocysteine, folic acid, and vitamin B(12). The rate of in-stent restenosis was determined angiographically after 6 months, or earlier if clinically indicated. Of 292 enrolled patients, 262 (90%) (189 men and 73 women) underwent control angiography on an average of 6.3 +/- 1.0 (SD) months after intervention. The rate of in-stent restenosis was 36%. Univariate and multivariate analyses revealed no significant differences between patients with or without restenosis with regard to total homocysteine (median [interquartile range]: 12.9 [11.2 to 14.8] and 12.4 [10.3 to 15.4] micromol/L, respectively), folate (16.1 [12.4 to 20.5] and 15.4 [12.5 to 19.5] nmol/L, respectively), or vitamin B(12) (239.0 [182.5 to 322.1] and 258.4 [205.8 to 330.5] pmol/L, respectively). These results suggest that homocysteine, folate, and vitamin B(12) are not related to the angiographically determined rate of coronary in-stent restenosis after 6 months.

Prevalence of factor XII (Hageman factor) deficiency among 426 patients with coronary heart disease awaiting cardiac surgery.

BackgroundSeveral case reports of myocardial infarction in patients with factor XII deficiency have been published. In the present study we investigated the prevalence of this condition. MethodsFactor XII activity (one-stage clotting assay), fibrinogen (derived method), and lipoprotein (a) (enzyme-linked immunosorbent assay) were measured in the plasma of 426 consecutive patients with coronary heart disease awaiting cardiac surgery. ResultsAmong the 426 patients, 44 (10.3 %) were found to be moderately deficient in factor XII (factor XII activity 17–50%, antigen 15–57%). The prevalence of factor XII deficiency was significantly higher (P < 0.0001) among patients with coronary heart disease than among 300 healthy blood donors (2.3%). Among coronary heart disease patients with this deficiency, elevated levels of fibrinogen, lipoprotein (a), and blood pressure were no more prevalent than in those without the deficiency; nor were cigarette smoking or a positive family history of thromboembolism more prevalent. ConclusionsCoronary heart disease patients showed a 10% prevalence of factor XII deficiency. However, the pattern of atherosclerotic risk factors did not differ between patients with or without the deficiency.

Homocysteine, Folate and Vitamin B12 in Patients with Coronary Heart Disease

Background/Aims: Homocysteine and possibly also folate and vitamin B12 are involved in the pathogenesis of cardiovascular disease. We investigated the prevalence of hyperhomocysteinemia in patients with coronary heart disease (CHD), as well as folate and vitamin B12, the main nutritional factors determining the level of homocysteine. Methods: Patients with angiographically documented CHD were prospectively investigated (n = 315, 70% male, mean age 61 [range 36–81] years). Fasting total serum homocysteine was determined by high-performance liquid chromatography and fluorescence detection. Folic acid and vitamin B12 were measured with AxSYM® Systems. Results: Median homocysteine concentrations for homocysteine, folate and vitamin B12 were 12.8 µmol/l, 6.8 ng/ml and 345 pg/ml, respectively. Homocysteine levels >10 µmol/l were found in 82% of men and 73% of women. In 19% of the patients serum folate was <3 ng/ml and 22% of the patients had serum vitamin B12 values <250 pg/ml. In a multivariate linear regression model, folate and vitamin B12 showed significant negative correlations to homocysteine, explaining 5 and 3% of its variability. Age and creatinine were the most important determinants for serum homocysteine, contributing 12 and 7%, respectively. Discussion: The main determinants of total homocysteine in patients with CHD are higher age and increased creatinine. The association of lower levels of folate and vitamin B12 with higher levels of homocysteine may indicate poor dietary habits in these patients.

Comparison of a New Automated Kinetically Determined Fibrinogen Assay with the 3 Most Used Fibrinogen Assays (Functional, Derived and Nephelometric) in Austrian Laboratories in Several Clinical Populations and Healthy Controls

A new automated kinetically determined fibrinogen assay was measured in plasmas of healthy subjects and three clinical cohorts (acute-phase reaction, liver cirrhosis and fibrinolytic therapy) that were expected to show normal, high and low levels of fibrinogen. The results were compared with the results of fibrinogen measurement using the derived method, the method according to Clauss and an immunological-nephelometric method. Altogether, the best correlation was achieved between the kinetic and the derived method. However, results from the derived method were generally higher than values obtained through the kinetic method. This was particularly true for high concentration levels above 400 mg/dl (patients with acute phase reaction) as well as for plasmas containing fibrin(ogen) degradation products and low concentrations of fibrinogen (below 150 mg/dl). Fibrinogen determinations in several commercial plasma pools with declared fibrinogen levels show remarkable heterogeneity when different methods were applied. To improve the discernment of fibrinogen determinations we suggest adjustment of standard preparations to international reference materials and the specification of the method used. Furthermore the attending physician is asked to cast a critical eye on fibrinogen values with regard to the used method of determination.

A hitherto unknown splice site defect in the protein S gene (PROS1): the mutation results in allelic exclusion and causes type I and type III protein S deficiency

A hitherto unknown splice site mutation, in the splice acceptor of intron B (tctag to tctgg), was identified in a symptomatic patient with type III protein S deficiency. The mutation co‐segregated with type I/III protein S deficiency in the patients family. RNA analysis showed allelic exclusion of the mutant transcript in affected individuals. The apparent type III deficiency in the propositus was not associated with the protein S Heerlen variant.

Reference materials (RMs) for analysis of the human factor II (prothrombin) gene G20210A mutation

Abstract The Scientific Committee of Molecular Biology Techniques (C-MBT) in Clinical Chemistry of the IFCC has initiated a joint project in co-operation with the European Commission, Joint Research Centre, Institute of Reference Materials and Measurements to develop and produce plasmid-type reference materials (RMs) for the analysis of the human prothrombin gene G20210A mutation. Although DNA tests have a high impact on clinical decision-making and the number of tests performed in diagnostic laboratories is high, issues of quality and quality assurance exist, and currently only a few RMs for clinical genetic testing are available. A gene fragment chosen was produced that spans all primer annealing sites published to date. Both the wild-type and mutant alleles of this gene fragment were cloned into a pUC18 plasmid and two plasmid RMs were produced. In addition, a mixture of both plasmids was produced to mimic the heterozygous genotype. The present study describes the performance of these reference materials in a commutability study, in which they were tested by nine different methods in 13 expert laboratories. This series of plasmid RMs are, to the best of our knowledge, the first plasmid-type clinical genetic RMs introduced worldwide.

No evidence of hepatitis B virus activity in patients with anti-HBc antibody positivity with or without anti-hepatitis C virus antibody positivity.

BACKGROUND The serological pattern of anti-HBc antibody positivity without both, HBsAg and anti-HBs antibody positivity may be present in up to 4% of the population of Europe and the United States. OBJECTIVES The aim of the present study was to determine the hepatitis B virus (HBV) activity by detection of serum HBV DNA in patients with anti-HBc antibody positivity only and with confirmed anti-hepatitis C virus (anti-HCV) antibody positivity or without anti-HCV antibody positivity. STUDY DESIGN A total of 141 patients positive for anti-HBc antibodies only, were investigated on serum HBV DNA load. Patients were classified into two groups: patients with confirmed positive anti-HCV antibodies (group 1) and patients without anti-HCV antibodies (group 2). RESULTS Demographic data of patient groups were similar. In 66 of 70 patients with anti-HBc antibodies and anti-HCV antibodies (group 1), serum HCV RNA was detected; the remaining 4 patients were HCV RNA negative but the presence of anti-HCV antibodies was confirmed by the line probe assay. In none of the patients, with anti-HBc antibodies and without anti-HCV antibodies (group 2), serum HCV RNA was detected. In none of the patients, serum HBV DNA was detected. CONCLUSION In this study, serum HBV DNA could not be detected in patients with anti-HBc antibodies only. There seems to be no need for determination of serum HBV DNA in patients without clinical evidence of chronic liver disease. Nevertheless, it would be useful to test patients with progressive liver disease and those, which belong to high-risk groups such as hemophiliacs, intravenous drug abusers, patients on hemodialysis, and immunocompromised patients.