Bert Verbruggen

Hyperhomocysteinemia and other thrombotic risk factors in women with placental vasculopathy

Objective To investigate coagulation inhibitors and abnormalities of the homocysteine metabolism, which are related to an increased thrombotic risk, as risk factors for placental vasculopathy.

Meloxicam, 15 mg/day, spares platelet function in healthy volunteers.

To study the influence of meloxicam, a cyclooxygenase‐2 (COX‐2) preferential nonsteroidal anti‐inflammatory drug, on serum thromboxane and platelet function in healthy volunteers with use of the maximum recommended daily dosage of 15 mg/day.

Improvements in Factor VIII Inhibitor Detection: From Bethesda to Nijmegen

All methods for the detection of factor VIII (FVIII) inhibitors are based on the measurement of inactivation of FVIII in a mixture of the test plasma containing the putative inhibitor and an exogenous source of FVIII. Various types of assays have been developed since the first inhibitor was described in 1941. Nowadays, two methods are preferably used, the Bethesda assay and the Nijmegen assay. Although the Nijmegen assay shows a better specificity and intra- and interlaboratory variation, it is still hampered by several limitations related to assay characteristics (pH, temperature, and time of incubation), type of control sample, and the von Willebrand factor content of the assay mixture. Epitope specificity plays an important role in the reliability of functional assays because inhibitors against the C2 domain are more difficult to quantify compared with inhibitors against the A2 domain. Finally, lupus anticoagulants can interfere with inhibitor assays, resulting in aberrant results. This report describes in detail the various problems encountered with the assays used in the quantification of functional FVIII inhibitors.

The between-laboratory variation of factor VIII inhibitor testing: the experience of the external quality assessment program of the ECAT foundation.

The detection and quantification of factor VIII (FVIII) inhibitors is clinically important both for the identification of hemophilia A patients with inhibitors and for the management of immune tolerance treatment. Only limited data are available on the between-laboratory variation of FVIII inhibitor testing. This report describes the evaluation of the results of the large-scale external quality assessment program of the European Concerted Action on Thrombosis Foundation. This study includes the results of six different surveys for the period 2006 to 2008 with 100 to 170 participating laboratories. The overall between-laboratory variation ranged from 28% to 52% with a slightly lower variation for the Nijmegen assay (approximately 39%) on average than for the Bethesda assay (approximately 45%). The use of buffered normal pooled plasma as FVIII source showed better performance compared with the use of nonbuffered pooled plasma; likewise the use of FVIII-deficient plasma compared with the use of imidazole buffer. However, the combination of both was essential for lowest between-laboratory variation. The Nijmegen assay also showed better performance with respect to specificity and sensitivity than the Bethesda assay, although the results for neither were entirely satisfactory. In general, it can be concluded that the measurement of FVIII inhibitory antibodies with the Nijmegen assay should be favored over the use of the Bethesda assay. However, further improvement of the laboratory test for FVIII inhibitors is urgently needed.

Thrombocytopenia in early malaria is associated with GP1b shedding in absence of systemic platelet activation and consumptive coagulopathy.

Thrombocytopenia develops early in malaria, but the underlying mechanisms remain incompletely understood. We studied the aetiology of malaria‐associated thrombocytopenia in volunteers experimentally infected with Plasmodium falciparum malaria, in Indonesian malaria patients and in ex vivo studies. In experimental human malaria, the decrease in platelet counts was associated with a concurrent rise in young platelets (immature platelet fraction) and thrombopoietin. D‐dimer concentrations were moderately elevated without a prolongation in the activated partial thromboplastin time or decrease in fibrinogen. There was no increase in expression of the platelet surface markers CD62P, PAC‐1 and CD63 and in plasma concentrations of the platelet factors P‐selectin, CXCR4, CXCL7, RANTES and CD40L. In contrast, concentrations of soluble glycoprotein‐1b (sGP1b), the external domain of the platelet receptor for von Willebrand factor (VWF), increased early. Indonesian malaria patients also had elevated concentrations of sGP1b, which correlated with VWF concentrations. Finally, incubation of platelets with parasitized erythrocytes in vitro failed to induce platelet aggregation or activation. We concluded that neither compromised platelet production nor platelet activation or consumptive coagulopathy were responsible for the early thrombocytopenia in malaria. We hypothesize that the increase in sGP1b concentrations results from VWF‐mediated GP1b shedding; a process that may prevent excessive adhesion of platelets and parasitized erythrocytes.

Diagnosis and quantification of factor VIII inhibitors.

Summary.  The laboratory detection of factor VIII inhibitors is invariably performed by methods that measure the inactivation of factor VIII in mixtures of test plasma and exogenous factor VIII, e.g. normal pooled plasma. Unfortunately the intra‐ and inter‐laboratory variation of the inhibitor assays is rather high often resulting in unreliable results. The pH of the mixtures of test plasma and pooled plasma, incubation time and temperature, type of control sample, von Willebrand content of factor VIII deficient plasma that is used in the assay and the presence of lupus anticoagulant all influence and/or interfere with the results of inhibitor testing. In this review these assay characteristics, pitfalls and limitations of the assays are discussed.

A model for the harmonisation of test results of different quantitative D-dimer methods.

The numerical test results of different D-dimer assays vary widely. Because of the complexity of the analyte of target as well as the variability in specificity of different D-dimer assays, only harmonisation of the test results seems to be feasible. The use of a single conversion factor does not take into account for several methods the lack of commutability between test results and consensus values at different D-dimer levels. This is probably related to the mutually different response of methods to high and low levels. We therefore designed a harmonisation model based on the transformation of a method-specific regression line to a reference regression line. We used the data for the measurement of a set of plasma samples with different D-dimer levels by 353 different laboratories using 7 of the most frequently used quantitative D-dimer methods. For each method we calculated the method-specific consensus value for each sample. The overall median value was also estimated. Per method linear regression was applied throughout the method-specific consensus values using the amount of patient pooled plasma added to the different plasma samples as the independent variable. The line through the overall median values of all 7 methods was used as the reference line. Harmonisation between the methods was obtained by transformation of the method-specific regression line to the reference line. This harmonisation resulted in a reduction of the variability between the method-specific consensus values from about 75% to about 5.5%. Clinical validation of this concept had shown significant improvement of the test result comparability. We conclude that this model is a feasible approach in the harmonisation of D-dimer methods. If the harmonisation procedure is included in the calibration procedure by the manufacturers, customers will automatically obtain harmonised test results.

Diagnosis of factor VIII deficiency

Summary.  The correct diagnosis of factor VIII deficiency and the assessment of severity of the disease are essential for a patient‐tailored treatment strategy. An optimal diagnostic procedure comprises sensitive and specific screening methods and factor VIII activity assays. Different screening reagents show variable characteristics and receiver operator characteristic curves are presented showing the relation between sensitivity and specificity of eleven activated partial thromboplastin time reagents. The details of the three methods for factor VIII activity assay, one‐stage and two‐stage assay and chromogenic assays, are discussed. The chromogenic assay seems to be more sensitive than the one‐stage assay with regard to the detection of severe haemophilia. Discrepant results obtained with one‐stage and two‐stage assays are reviewed and discussed.

A novel hemostasis assay for the simultaneous measurement of coagulation and fibrinolysis

Abstract Thrombin and plasmin are the key enzymes involved in coagulation and fibrinolysis. A novel hemostasis assay (NHA) was developed to measure thrombin and plasmin generation in a single well by a fluorimeter. The NHA uses two fluorescent substrates with non-interfering fluorescent excitation and emission spectra. The assay was tested in vitro using modulators like heparin, hirudin, epsilon-aminocaproic acid, gly-pro-arg-pro peptide and reptilase and validated by measurement of prothrombin fragment 1+2 and plasmin-alpha2-antiplasmin levels. Intra- and inter-assay coefficients of variation were <9% and 6–25%, respectively. Interplay between coagulation and fibrinolysis was demonstrated by the effect of tissue-type plasminogen activator on thrombin generation and by the different responses of activated protein C and thrombomodulin on fibrinolysis. The last responses showed the linkage between coagulation and fibrinolysis by thrombin activatable fibrinolysis inhibitor. In conclusion, this strategy allows detection of coagulation, fibrinolysis and their interplay in a single assay.

Laboratory testing for factor inhibitors

Inhibitor assays are performed when patients present with unexplained prolonged routine coagulation test times and unexpected and/or unusual bleeding (potential for acquired haemophilia) as well as being a part of normal congenital haemophilia management and monitoring, particularly when bleeding occurs on therapy, or when increments in factor levels post‐factor replacement remain lower than expected. In this article, we will describe the assays used, as well as their development, pitfalls in testing such as inter‐laboratory variability and false negative/positive results, as well as some strategies for overcoming these pitfalls and potential alternative test approaches. The inter‐laboratory coefficient of variation often approaches (and sometimes exceeds) 50%, as evidenced by various external quality assessment groups, and this variability has not improved over recent years. Additional important considerations include appropriate interpretation of test results, repeat testing for confirmation, and assessment of recovery as part of the diagnostic process.