session

84 - Oral Communication
Diagnostic laboratory: Today & tomorrow
Feb. 26, 2021, 1:45 p.m. - 3:15 p.m., Sydney

Abstract

2
Heparinase treatment to remove the inhibitory heparin effect on the thrombin generation assay
A. Weber, M. LLusa, N. B. Binder, H. Gritsch, Presenter: A. Weber (Vienna)

Background and Objective
Clinically, reversal of anticoagulation by vitamin K antagonists (VKAs) is obtained by administration of four-factor prothrombin complex concentrates (4F-PCCs). Continuous monitoring of thrombin activity, also known as thrombin generation assay (TGA) is a suitable method for evaluating the hemostatic potency of coagulation factor concentrates in plasma milieu. Commercially available 4F-PCCs contain heparin at different levels, thus TGA results will be biased by the inhibitory effect of heparin and may not fully reflect the clinical efficacy of 4F-PCCs observed. Removal of heparin by anion exchange adsorption has been described but could potentially alter the complex composition of 4F-PCCs, while heparin neutralization with protamine sulphate requires exact titration in order not to alter the hemostatic potency. Here, we describe the use of heparinase I from Flavobacterium heparinum which results in a fast, enzymatic heparin fragmentation associated with the loss of anticoagulant activity.
Methods
The heparin-containing 4F-PCC sample (Prothromplex Total, Baxter) was mixed with Heparinase I (Sigma) and incubated for 5 min at room temperature. Then, the hemostatic potential was measured by Technothrombin TGA assay (Technoclone) using the CeveronĀ® reagents. Normal and VKA anticoagulated plasma was used at a final heparinase I concentration of 1.25 U/mL in the TGA at two trigger levels provided by the TGA reagents RCLow and RCHigh. The optimal heparinase I concentration was determined by investigating a concentration series of heparinase I, ranging from 0.25 to 2.5 U/mL.
Results
Heparinase I at a concentration of 1.25 U/mL in the final TGA reaction mix completely removed the inhibitory influence of heparin. At this level, there was only moderate influence on the characteristic TGA readouts lag time, peak thrombin, area under the curve (endogenous thrombin potential) and velocity index for all conditions tested. In contrast, the heparin-containing 4F-PCC concentrate demonstrated dose-dependent TGA response after heparinase I treatment, which was not the case when this treatment was omitted.
Conclusion
Enzymatic breakdown of heparin by heparinase I efficiently removed the inhibitory effect of heparin observed during the TGA of 4F-PCCs. Heparinase I treatment can easily introduced in existing TGA test procedures and will increase the significance of data generated with TGA.
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