Optimization of Heparin Monitoring with Anti-FXa Assays and the Impact of Dextran Sulfate for Measuring All Drug Activity.

Heparins, unfractionated or low molecular weight, are permanently in the spotlight of both clinical indications and laboratory monitoring. An accurate drug dosage is necessary for an efficient and safe therapy. The one-stage kinetic anti-FXa assays are the most widely and universally used with full automation for large series, without needing exogenous antithrombin. The WHO International Standards are available for UFH and LMWH, but external quality assessment surveys still report a high inter-assay variability. This heterogeneity results from the following: assay formulation, designed without or with dextran sulfate to measure all heparin in blood circulation; calibrators for testing UFH or LMWH with the same curve; and automation parameters. In this study, various factors which impact heparin measurements are reviewed, and we share our experience to optimize assays for testing all heparin anticoagulant activities in plasma. Evidence is provided on the usefulness of low molecular weight dextran sulfate to completely mobilize all of the drug present in blood circulation. Other key factors concern the adjustment of assay conditions to obtain fully superimposable calibration curves for UFH and LMWH, calibrators' formulations, and automation parameters. In this study, we illustrate the performances of different anti-FXa assays used for testing heparin on UFH or LMWH treated patients' plasmas and obtained using citrate or CTAD anticoagulants. Comparable results are obtained only when the CTAD anticoagulant is used. Using citrate as an anticoagulant, UFH is underestimated in the absence of dextran sulfate. Heparin calibrators, adjustment of automation parameters, and data treatment contribute to other smaller differences.

The various assays for measuring activity states of factor VIIa in plasma and therapeutic products: Diagnostic value and analytical usefulness in various pathophysiological states.

The key coagulation factor FVII, and its activated form FVIIa, present a major interest for their role at the initiation phase of blood coagulation, and because they can activate all blood coagulation cascade, through the extrinsic, but also the intrinsic pathway. Blood activation initiated through FVII is first presented, as it is understood nowadays. Measurement of FVII and FVIIa were of main interest for epidemiological studies, but FVIIa contribution to assay results was only deduced. The introduction of specific FVIIa assays, functional or immunoassays, allowed measuring directly FVIIa without any interference of non-activated FVII, or other coagulation factors or their activated forms. The various methods available, and their characteristics are presented, with a special focus on two assays developed by our group for FVIIa (a clotting one and a chromogenic one). The FVIIa clotting assay shows evident superiority for measuring its activity in plasma, in pathophysiological conditions. The normal range is <2.5ng/ml, which represents less than 0.5% of the FVII protein. FVIIa is elevated in some pathological states. The chromogenic assay is of interest for assigning the potency of FVIIa concentrates, as it has a higher dynamic range. Both assays are fully automatable on laboratory instruments, and standardized in a satisfactory manner thanks to the use of the FVIIa concentrate WHO International Standard (NIBSC). The various applications and usefulness of FVIIa laboratory assays are discussed, for the measurement of therapeutic products, or for following recoveries in treated patients, including hemophiliacs with inhibitors, patients with severe bleeding risk (liver diseases, surgery, trauma, …), and lastly for measurement of its activity in therapeutic products.

Anti-Xa bioassays for the laboratory measurement of direct Factor Xa inhibitors in plasma, in selected patients.

In the past decade Direct Oral Anti-Coagulants (DOACs), targeting Thrombin or Factor Xa, have enormously facilitated the daily treatment of all relevant patients, including those requiring lifelong therapy. These DOACs have considerable advantages over the use of oral Vitamin K Antagonist (VKA) treatments, in view of having little interferences with food and other medications and also not requiring adjustment for age, gender or weight, with some well-defined exceptions. In this current What's Happening Section we focus on measurements of DiXaIs in plasma using anti-Xa assays, with the objective of providing a tribute to Professor Michel Meyer Samama, who was not only a real leader in this field but, in the past, both authors benefited from his wisdom, as a teacher who dedicated his scientific and professional life (among many other interests in hemostasis, thrombosis and fibrinolysis) to develop and promote methods and strategies for laboratory monitoring of anticoagulants. This review presents the performance characteristics of the Anti-Factor Xa assays (measuring Factor Xa inhibition by drugs), which are available for measuring Direct Factor Xa Inhibitors in plasma, and show good compliance of the results with the reference LC:MS method (which measures the mass of Direct Factor Xa Inhibitors). We also present the preparation and validation of drug specific plasma calibrators and controls which are requested for drug measurements. These assays are convenient and practical laboratory tools which can be used in any laboratory setting, and meet the requirements of regulatory bodies for making smart, quantitative, sensitive, accurate and ease of use assays for measuring DOACs when needed. The manuscript focuses mainly on the following areas of current interest: interference in coagulation assays; anti-Xa laboratory methods; development of calibrators and controls for DiXaIs; method validation and comparison with reference techniques (LC:MS); regulatory requirements and method registrations; newer clinical applications and experience on DiXaIs with Anti-Xa assays, and future perspectives.

An update on laboratory measurements of Dabigatran: Smart specific and calibrated dedicated assays for measuring anti-IIa activity in plasma.

Use of Direct Oral Anticoagulants (DOACs) is continuously increasing for clinical application. The first product released was Dabigatran, which was proposed for many preventive and curative applications, especially for prevention of stroke in patients with non-valvular atrial fibrillation. Although measurement of Dabigatran Anti-IIa activity in plasma is not requested on a routine basis, in some situations its measurement is clinically useful. Especially, before an emergency surgery in treated patients, where its presence at high concentrations, which will expose the patient at an increased bleeding risk, has to be excluded. Hence, smart, specific, rapid and accurate quantitative assays are warranted as an essential required. Hemoclot™ Thrombin Inhibitors and Biophen® DTI were specifically designed for these applications, and can be used on all automated instruments with a standard range protocol for measuring concentrations at peak, or with a low range protocol for testing residual concentrations. Both functional assays have a good correlation with the reference LC-MS/MS method, and concentrations measured are similar. Performances of these assays and interferences of various substances or drugs are discussed. Some differences in variations of clotting times are observed between mechanical or optical clot detection instruments, which could be explained by the fibrin clot structure, altered by direct Factor Xa inhibitors, and more especially Rivaroxaban. Both clotting and chromogenic assays offer a safe and accurate quantitative measurement of Dabigatran in plasma in all situations where this determination is requested. In short this manuscript provides an in depth update on current opinions on laboratory aspects of measuring Dabigatran concentrations in plasma, when required.