Laboratory Assays for Monitoring Rivaroxaban: In House in-Vitro Analysis and Review of Literature
Authors: Pathak Praveen Kumar , McRae Simon , Duncan Elizabeth , Lewis Ian D , To Luen Bik 
Published: October 2019

Abstract
Abstract 4320

Introduction:
Rivaroxaban an oral direct Xa inhibitor has recently been approved for VTE prophylaxis. It doesn’t require monitoring but in special circumstances like bleeds a monitoring tool will be useful. Also, it is prudent to know its effect on various routine coagulation tests. Currently, there is no commercially available method for monitoring of Rivaroxaban. Aim: We conducted an In house- in-vitro analysis to (a)determine the effects of rivaroxaban locally on a range of commercially available coagulation assays, lupus anticoaugulant, factor VIII level, thrombin generation time and (b) measure it by two anti-Xa assays. Materials: Rivaroxaban (Xeralto) tablet, strength-10 mg. Plasma samples: Citrated normal human platelet poor plasma procured and freezed at &minus;70 degree centigrade, Baxter sterile water.

Method:
The tablet was dissolved over 72 hours in a litre of sterile water to make 10 ug/ml of solution. Despite stirring a fine particulate compound remained which settled on standing. Samples were vortexed prior to each assay. This stock solution was used to spike citrated normal human platelet-poor plasma (PPP) at increasing concentrations of rivaroxaban from 0.1,0.2, 0.3, 0.4, upto 0.5 ug/ml to see the effect on the following tests and to determine rivaroxaban concentration by two 1.anti-Xa methods. All method were run using standard protocols taken from manufacturers’ instructions.Anti-Xa assay:a) BIOPHEN DiXaI kit for 2-stage chromogenic assay of Direct Xa inhibitors; Ref 221030 b) Rotachrome using 1:2 dilution and further using 1:10 and 1:20 dilutions; catalogue Number-00612.2.PT & INR (STAGO neoplastin CI Plus MNPT-13.2 s, ISI-1.30 s).3.aPTT (Triniclot aPTT reagent)4.LAC-dRVVT screening and confirm (STAGO).5.Factor VIII assay (Siemens- Factor VIII deficient plasma)6. TT (STAGO-Thrombin)7. Factor VIII assay- Helena SARP.8.TGT: Thrombinoscope 5 picomolar TF and standard CAT method.Values described for Xarelto/ Rivaroxaban on patients with thromboprophylaxis in orthopedic surgery are usually of about 0.1–0.2 ug /ml in plasma.Result & Discussion: Concentration dependent prolongation of PT was observed. We used a single reagent (STAGO neoplastin CI Plus) which was sensitive at 0.1ug/ml concentration. Samama et al found that the results of PT were variable with reagent and not corrected by conversion to INR.We found that aPTT at lower concentrations of Rivaroxaban was not significantly different. Hillarp et al used five different reagents and found a reagent dependent dose response of APTT to rivaroxaban.The effect on Thrombin Time was non concentration dependent with no prolongation of TT at lower levels.There was fall in FVIII level in concentration dependent manner with 35% fall at 0.5ug/ml. We did not come across any literature till date about FVIII levels in Rivaroxaban therapy and our conclusion emphasises the importance of drug history.The LAC by DRVVT test showed positive result at the lowest concentration with an ILR of 1.23 and a dose dependent prolongation of clotting time. Thus making thrombophilia screening difficult.Thrombin generation time showed dose dependent effect on all parameters with almost complete suppression of thrombin generation. The initiation and propagation phase of TGT was affected at the lowest concentration (Green et al).The two anti-Xa tests used for measuring the concentration of Rivaroxaban in spiked plasma showed that Biophen DiXAI method has a broad range of sensitivity (0.1–0.5ug/ml) with calibration curve almost identical to that of manufacturer’s with an excellent R value through out.The Rotachrome method at the standard 1:2 dilution was unable to measure concentration beyond 0.2ug/ml and at 1:20 dilution could only measure upto 0.4ug/ml. Thus, this method needs modification to determine a broad range of concentration.There was a variable result with 0.5ug/ml concentration of Rivaroxaban despite repeated vortex with unclear cause. This could be due to instability or settling of non-dissolved particles of the sample.The calibration curves were linear throughout with an acceptable R-value but there was a progressive drop in assay over a period of time.

Conclusion:
Anti-Xa assay with Biophen DiXAI method is a promising candidate for measurement of concentration of Rivaroxaban in special circumstances. Our findings show a trend towards prolongation of global clotting assays. The tablet is not stable as a calibrator.

Disclosures:
Pathak: BIOPHEN-DIXAI: Research Funding. McRae:BIOPHEN-DIXAI: Research Funding.