Abstract
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.
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) | |||
| 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.
Herve:Diagnostica Stago: Employment. Beaufils:Diagnostica Stago: Employment. Kochan:Daiichi Sankyo: Employment. He:Daiichi Sankyo: Employment. Depasse:Diagnostica Stago: Employment.
Author notes
Asterisk with author names denotes non-ASH members.
