Development of a New Automated Assay for Measurement of the Oral Direct Factor Xa Inhibitor Edoxaban Plasma Concentration
Authors: Herve Tristan , Beaufils Sandra , Kochan Jarema , He Ling , Depasse Francois 
Published: November 2019

Edoxaban is a reversible orally active factor Xa inhibitor approved in Japan for Venous ThromboEmbolism (VTE) prophylaxis in major orthopedic surgery and submitted for approval in multiple markets for Stroke Prevention in non-valvular Atrial Fibrillation (SPAF) and VTE treatment and recurrence prevention. Although routine monitoring is not required, determination of anti-Xa activity with results expressed in edoxaban plasma concentration may be helpful in some special clinical settings such as urgent invasive procedures or in cases of bleeding.

We have developed a specific, automated, user-friendly assay for measuring plasma edoxaban-related anti-Xa activity using the STA® Liquid Anti-Xa with an edoxaban dedicated test set-up, along with specific edoxaban calibrator and control sets, namely STA® Edoxaban Calibrator and STA® Edoxaban Control, on the STA® line analyzers. These calibrator and control sets are freeze-dried in vitro edoxaban spiked citrated plasmas. Test results are expressed in ng/mL of edoxaban.

Assay performances including Limit of Blank (LOB), Lower Limit of Detection (LLOD) both according to CLSI EP-17-A guideline, Lower Limit of Quantification (LLOQ), Upper Limit of Quantification (ULOQ) according to CLSI EP6-A guideline, with and without automated re-dilution of plasma sample, and within and between-run reproducibility have been determined.

Anti-Xa assay results were compared to those obtained with Mass Spectrometry Liquid Chromatography (LC-MS) reference method to evaluate assay recovery.

All study assays were performed using freeze-dried of frozen in vitro edoxaban-spiked citrated plasma samples.

Main potential interferences, i.e., hemoglobin, non-conjugated bilirubin, and lipemia, have been assessed.

Assay performance results are summarized in Table I.

Table I: Main Edoxaban assay performances as determined during test development Parameter Results obtained with prototype reagent batch and test set-up LOB 10 ng/mL LLOD 15 ng/mL LLOQ (preliminary estimation) 20 ng/mL ULOQ Without sample re-dilution 150 ng/mL With sample re-dilution 450 ng/mL Reproducibility Within run (n = 21) Freeze-dried controls 40 ng/mL ≤ 4.5% 120 ng/mL ≤ 6.0% Frozen spiked samples 50 ng/mL ≤ 7.1% 100 ng/mL ≤ 4.9% 200 ng/mL ≤ 4.3% 350 ng/mL ≤ 4.0% Between run (n = 10) Freeze-dried controls 40 ng/mL ≤ 7.0% 120 ng/mL ≤ 3.6% Frozen spiked samples 50 ng/mL ≤ 5.0% 100 ng/mL ≤ 4.0% 350 ng/mL ≤ 4.6% Recovery(freeze-dried samples) 40 ng/mL 87.4% 120 ng/mL 101.9% Edoxaban calibrator and control stability Calibrators Onboard 4 hours Controls Onboard 24 hours +2 – 8°C 7 days Interferences Hemoglobin None up to 1 g/L Non-conjugated bilirubin None up to 200 µM Lipemia None up to 2.5 g/L (as Intralipid® concentration)

In conclusion, the proposed edoxaban assay developed using STA® Liquid Anti-Xa reagent with a dedicated test set-up and specific STA® Edoxaban Calibrator and STA® Edoxaban Control sets allows an accurate, reproducible, automated, and user-friendly, edoxaban plasma concentration determination. Further studies are required to confirm assay performance in ex vivo samples.

Disclosures

Herve: Diagnostica Stago: Employment. Beaufils:Diagnostica Stago: Employment. Kochan:Daiichi Sankyo: Employment. He:Daiichi Sankyo: Employment. Depasse:Diagnostica Stago: Employment.