Determination of an international sensitivity index of thromboplastin reagents using a WHO thromboplastin as calibrator for plasma spiked with rivaroxaban

Abstract

Rivaroxaban and other direct factor Xa inhibitors are used at fixed doses without drug monitoring and dose adjustment. Patients may require determination of the anticoagulant effect during treatment. The aim of this study was to develop a method to reduce the differences between thromboplastin reagents and coagulation analysers for determination of the anticoagulant effect of rivaroxaban in human plasma. Purity of rivaroxaban extracted from commercially available drug was confirmed by mass spectrometry, elemental analysis and 1H-NMR spectroscopy. Coagulation times of pooled human plasma spiked with 50-900 ng/ml rivaroxaban were analysed. Thromboplastin reagents, WHO RBT/90, Innovin, RecombiPlasTin 2G, STA Neoplastin Plus, Technoclot PT Plus and Thromborel S, the manual Kolle-Hook method and the KC10 analyser were used. An international sensitivity index (ISI) was determined for each reagent and coagulation method using the RBT/90 thromboplastin reagent as reference. The orthogonal, used for the determination of the ISI of coumarin plasmas, and ordinary regression analyses were compared. The results showed than increasing concentrations of rivaroxaban prolonged coagulation values of all thromboplastin assays linearly (r (2)= 0.96 and r(2) = 0.99, respectively). The coefficient of variation between the slopes of the dilution curves and the ratios of the thromboplastin reagents were reduced using the international normalized ratio (INR) and ISI calculated for rivaroxaban. The ISIs of the thromboplastin reagents ranged from 0.73 to 1.67 as compared with the WHO reagent using the manual technique. The coefficient of variations between the thromboplastin reagents comparing the orthogonal and the ordinary regression analysis were 6.8 versus 3.7% (Kolle-Hook method, P = 0.0011) and 8.5 versus 4.8% (KC10 method, P < 0.0001). Using ISI for vitamin-K antagonist and rivaroxaban, the INRs for the rivaroxaban-containing samples were significantly different for one of five commercial thromboplastin reagents. In conclusion, the determination of an ISI for rivaroxaban using a WHO thromboplastin reagent is required for commercial thromboplastin reagents. The manual Kolle-Hook method and an ordinary linear regression analysis should be adopted.

The laboratory and the direct oral anticoagulants

“…Moreover, standardization as assessed in a collaborative survey is poor: the average interlaboratory variation of results obtained while measuring the same rivaroxaban-spiked plasmas by means of local methods was high (ie, 25%). 18 Conversely, the PT is readily available even in an emergency and its standardization could be obtained as described 19,20 by expressing results as PT ratio (patientto-normal) corrected by means of a sensitivity index. Such an index can be determined for working thromboplastins relative to an international standard.…”

Laboratory Testing in the Era of Direct or Non–Vitamin K Antagonist Oral Anticoagulants: A Practical Guide to Measuring Their Activity and Avoiding Diagnostic Errors

“…In contrast to dabigatran, the PT is more generally sensitive to rivaroxaban than the aPTT, although differential reagent sensitivity is observed. 4,19,24,25,[54][55][56][57][58][59][60][61][62][63][64][65][66][67][68][69] All TT and ecarin-based assays (i.e., standard TT, dTT, HTI, ECT, and ECA) are insensitive to rivaroxaban. 24,35,55,61,63 The PICT assay was also reported as sensitive, although this assay may require some major degree of optimization.…”

Interpretation of coagulation test results under direct oral anticoagulants

“…As the PT assay is sensitive to the prothrombinase complex, the PT is influenced by rivaroxaban in a concentration-dependent manner with an increase in seconds [4][5][6]. Because the PT assay is widely available in routine clinical laboratories and known as monitoring system for vitamin K antagonists, there have been attempts to use this test method to monitor rivaroxaban and even to adapt the INR system for this new oral factor Xa inhibitor [7]. However, it has been shown that the classical INR fails to correct the thromboplastin variability in response to rivaroxaban.…”