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

Abstract

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.

Factors Influencing Anti-Xa Assays: A Multicenter Prospective Study in Critically Ill and Noncritically Ill Patients Receiving Unfractionated Heparin

“…The results are in line with those of previous studies. 8,10,19,20 However, in those previous studies, data came from external quality assessment programs using lyophilized plasma samples, plasmas spiked with UFH, or left-over plasma samples from patients receiving different doses of UFH or LMWH, making their clinical relevance difficult. The magnitude of the effect of dextran may differ among clinical settings with variable inflammatory status and platelet Fig.…”

“…7 Recent external quality assessment programs such as External quality Control of diagnostic Assays and Tests (ECAT) using lyophilized samples have shown a substantial inter-laboratory variability of anti-Xa levels according to reagents, especially for the low range of values. [8][9][10] Different parameters might potentially contribute to the heterogeneity of anti-Xa levels, among which are the type of blood collection tube, the presence or absence of dextran sulfate in reagents, the addition of exogenous antithrombin, the calibrator, and the calibration curve mathematical processing. Some manufacturers chose to add dextran in reagents in order to displace UFH from proteins released ex vivo after blood sampling, notably platelet factor 4 (PF4) following platelet activation; thus, UFH released from those neutralizing proteins recovers its anti-Xa level.…”

Pharmacology of Heparin and Related Drugs: An Update

“…Like heparin it can be quantified by its interaction with protamine (Gordon et al, 2021), and it is cleared from the circulation by the HARE scavenger of endothelial cells (Weigel, 2020). DexS can be used to improve the performance of anti-Xa assays of heparin in plasma, improving recovery and avoiding the impact of heparin binding to neutralizing plasma proteins (Amiral et al, 2021). As an aside, DexS-induced colitis in mice is a frequently used model of inflammatory bowel disorder (Xie et al, 2021).…”

Editorial for the Special Issue of Monitoring Anticoagulants

“…Clotting or chromogenic assays are available for evaluating the anticoagulant activity of these polysaccharide anticoagulants [ 5 , 6 ]. The standard approach for drug monitoring tends to rely on anti-factor Xa kinetics assays, and must accurately measure all types of anticoagulants, with a like to like calibrator [ 1 ]. Side effects concern mainly HIT, produced by the development of heparin dependent platelet factor 4 (PF4) antibodies.…”

“…This issue contains eight articles reporting today’s assay methods available for measuring anticoagulant drug concentrations in plasma and managing their side effects when present. The published reports firstly concern heparin [ 1 ] and its complications [ 2 , 3 ], such as heparin-induced thrombocytopenia (HIT), or direct oral anticoagulants (DOACs) [ 4 ], which specifically and directly inhibit thrombin or factor Xa (FXa). Other articles review the various approaches for testing ancient, current, and new anticoagulants [ 5 , 6 ]; alternatively, they discuss the specific issues related to the management of critically ill patients who are anticoagulated for prophylaxis or for curative treatment of thromboembolic diseases [ 7 ].…”