PB1324 - Evaluation of the analytical performance of a modified one-stage FVIII assay for the plasmatic quantification of Emicizumab

Background: Emicizumab is a bispecific antibody that bridges activated factor IX and factor X to restore the function of missing activated factor VIII (FVIII). Emicizumab has been approved for the prophylaxis of patients with Hemophilia A with and without inhibitors. In Argentina, the Emicizumab calibrator and controls are not commercially available for the measurement of Emicizumab plasma concentration and data on the evaluation of the analytical performance of the modified one-stage FVIII assay (mOSA) are limited.

Aims: To evaluate analytical performance of mOSA for the plasmatic quantification of Emicizumab on an automated coagulation analyzer.

Methods: mOSA: 1:80 pre-diluted sample with CA System Buffer, carried out automatically by the coagulometer. Calibration Curve (0.6 – 150 µg/mL of Emicizumab): 1:1 (1:80), 1:2, 1:8, 1:32, 1:64, 1:128, 1:256. Homemade Calibrator (calEMI: 150 µg/mL of Emicizumab): FVIII deficient plasma (FVIIIdp) was spiked with Emicizumab. Homemade Controls: level 1=25µg/mL (L1EMI), level 2=75µg/mL (L2EMI). Reagents: FVIIIdp, aPTT Pathromtin SL, CaCl2 0.025M; Analyzer: Sysmex CS-2500, Siemens Healthcare, Germany. Repeatability (CVr)= L1EMI, L2EMI were measured 20 times in the same run. Within-laboratory Imprecision (CVwl)= L1EMI, L2EMI were measured in triplicate for 5 days. Linearity: 6 levels in triplicate, using calEMI and FVIIIdp. Bias estimation: from an external quality assessment program (ECAT Foundation). EP evaluator was used for statistical analysis.

Results: CVr: L1EMI: 6.4% (28 µg/mL), L2EMI: CVr 3.2% (86 µg/mL). CVwl: see Table 1. Calibration curve: r2: 0.993. Linearity: see Table2, r2: 0.998 (linear range 1-150 µg/mL), Slope: 1.0098. Bias: 5.0%.

Conclusion(s): mOSA showed good analytical performances, with acceptable imprecision and bias. The method was shown to be linear over a range of 1 to 150 µg/ml emicizumab. mOSA on this platform could allow monitoring of patients with a simple laboratory test, but the assay must be validated with samples from patients who received doses of emicizumab.