Anti-Xa assays for unfractionated heparin monitoring: should we use a reagent with or without dextran sulfate? Insights from two recent studies

Introduction: In recent years, anti-Xa assays have become the preferred option for monitoring unfractionated heparin (UFH) because they are less affected by non-anticoagulation-related variables than the activated partial thromboplastin time (aPTT) and are associated with a faster time to reach the therapeutic range with fewer adjustments. However, limited agreement between commercially available anti-Xa assays has been consistently reported, with potentially important clinical implications including changes in treatment decisions. A major difference between anti-Xa assays is the addition of dextran sulphate (DS) in some reagents. DS is used to displace, at least in part, UFH from Platelet Factor 4 (PF4) released in vitro from platelets into plasma after blood collection, to recover that part of UFH.

Method: Based on the results of two studies we recently published [1] [2], we aim to revisit the issue of DS in anti-Xa assays.

Results: In the first study, we analyzed 4,546 anti-Xa values of 165 patients divided in four groups [cardiopulmonary bypass (CBP) after heparin neutralization, n=39; cardiothoracic intensive care unit (ICU) after CPB, n=35; medical ICU, n=53; other medical inpatients, n=38] using seven reagent/analyzer combinations, including two without DS. Median anti-Xa levels were consistently higher (+30.2% to+296%) when measured with reagents containing DS, whatever the patient group ([Fig. 1]). The greatest effect was observed in CBP patients after UFH neutralization with protamine (0.32 vs. 0.05 IU/mL, with and without DS). In the second study, we spiked normal pool plasma with increasing concentrations of UFH (up to 1 IU/mL) and of DS (Sigma Aldrich, ~8000Da). We found that anti-Xa levels increased with increasing concentrations of DS. It reached a plateau at approximately 160 μg/mL DS (well above the amounts in the commercial reagents) at which the apparent anti-Xa level had almost doubled. In the presence of added protamine sulphate, the addition of DS increased anti-Xa levels, consistent with the dissociation of UFH-antagonist complexes in vitro ([Fig. 2]).

Fig. 1 Anti-Xa values. Anti-Xa values measured in plasma samples with reagents containing or not dextran, and proportion of values below the lower limit of quantification, according to patient groups.

Fig. 2 Anti-Xa values according to UFH concentrations. Anti-Xa levels according to UFH concentrations, using a kit that contains dextran sulfate (Biophen Heparin LRT (Hyphen Biomed); left panel) and a kit that does not (Liquid anti-Xa (Stago); right panel)). Factor Xa inhibition was measured in normal pool plasma (n=5) after addition of increasing UFH concentrations (up to 1.0 IU/mL), and heparin antagonists hexadimethrine bromide (25 μg/mL) or protamine sulfate (in a 1U/1U ratio to UFH). Dashed lines: lower limit of quantification of the assay (0.05 IU/mL for Biophen Heparin LRT and 0.1 IU/mL for Liquid anti-Xa).

Conclusion: Our results suggest that in addition to displacing UFH from PF4 released in vitro from platelets into plasma after blood collection, DS likely also displaces UFH from complexes formed in vivo with proteins or with protamine, after UFH neutralization in the setting of cardiac surgery under CBP. This may lead to an overestimation of the actual anticoagulant effect of UFH in such settings and may alter treatment decisions, as this bound UFH is not available for interaction with antithrombin in vivo. We therefore call for action to improve the availability and use of appropriate and concordant anti-Xa assays, and to resolve the issue of whether dextran should be added or not to anti-Xa reagents, and if so, at the appropriate concentration.

Publikationsverlauf

Artikel online veröffentlicht:
13. Februar 2025

• References

• 1 Lastname FN, Lastname FN, YLasne D, Toussaint-Hacquard M, Delassasseigne C. et al. Factors Influencing Anti-Xa Assays: A Multicenter Prospective Study in Critically Ill and Noncritically Ill Patients Receiving Unfractionated Heparin (DEXHEP). Thromb Haemost 2023; 123 (12) 1105-1115
• 2 Hardy M, Cabo J, Deliège A. et al. Reassessment of dextran sulfate in anti-Xa assay for unfractionated heparin laboratory monitoring. Res Pract Thromb Haemost. 2023 7. 08 102257).YYY, ‘Article’, Journal, Edition, Page, Place of publication: publishers