Analytical Performance of Modified Anti-FXa Assays for Andexanet Alfa-Containing Plasma Samples on the ACL TOP/TOP 50 Coagulation Analyzers

Background:

Assay parameters for anti-FXa activity assays for apixaban and rivaroxaban on the ACL TOP/TOP 50 analyzers have been modified to minimize the effect of sample dilution suitable for analyzing andexanet-containing samples with drug-specific calibrators [1].The modified assays showed a dose-dependent reversal of rivaroxaban and apixaban anti-FXa activity in andexanet-containing samples similar to the 96-well format anti-FXa activity assay used in the andexanet clinical studies. In the current study, further assay optimization and verification of analytical performance was performed for the modified apixaban and rivaroxaban assays.

Methods:

Calibration curves for the modified anti-FXa assays (apixaban, rivaroxaban) were further optimized to improve precision at 100 ng/mL. The calibration curves consisted of 5 dilution levels. The number of replicates at each level was increased from 3 to 4, and outlier removal was enabled to improve the % coefficient of variation (%CV). The limit of blank (LoB), detection (LoD) and quantitation (LoQ) for modified assays on the ACL TOP/TOP 50 were determined in accordance with the minimum requirements of two lots of calibrators and reagent, one instrument system, and three days in accordance with CLSI [2]. The linear ranges were tested for 3 lots of calibrators on 4 analyzers. The samples for the linearity tests consisted of a minimum of 9 to 11 levels extending below and above the anticipated linear range. A 5-day precision was performed with diluted controls encompassing the modified assay calibration range and andexanet-containing samples on a minimum of two analyzers with one lot of drug-specific calibrator to verify analytical performance.

Results:

Analytical performance with optimized calibration settings was evaluated with a minimum of 9 calibration curves generated with 3 calibrator lots and demonstrated improvement in the r² and %CV at each level. The analytical parameters for the modified apixaban assay generated with 3 lots of calibrator and reagent on 4 analyzers are as follows: LoB of ~2.1 ng/mL, LoD of 3.5 ng/mL, and LoQ of 5.9 ng/mL. The analytical parameters for the modified rivaroxaban assay were generated with 2 lots of calibrators and reagents with a LoB of 2.3 ng/mL, and LoD of 3.6 ng/mL. Three lots of calibrators and reagents were used to generate an LoQ of 5.4 ng/mL. The linear range was determined to be 10-100 ng/mL and met the acceptance criteria for r²≥ 0.95 and slope of 0.90 - 1.10 for both assays.

Conclusions:

Results of this study demonstrate that modified apixaban and rivaroxaban LoQ and linearity meet the assay requirements. Modified test parameters will be user-defined on the ACL TOP/TOP 50 analyzers and can be used to measure apixaban or rivaroxaban levels when andexanet is present in the samples.

References:

1. Khan, A. et al. Blood(2019) 134 (Supplement1): 1153. American Society of Hematology- Modified Anti-FXa assays for measuring the residual activity of apixaban and rivaroxaban in andexanet alfa-containing samples on the ACL TOP Family® coagulation analyzers.

2. CLSI EP17-A2.Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline - Second Edition.Wayne, PA: Clinical and Laboratory Standards Institute; 2012.