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Emicizumab: In-house method validation

1Laboratorio de Hematología. Hospital Dr. César Milstein, Hospital de Pediatría Juan P. Garrahan, Buenos Aires, Ciudad Autonoma de Buenos Aires, Argentina, 2Laboratorio de Hemostasia y Trombosis. Servicio de Hematología y Oncología. Hospital de Pediatría Prof Dr Juan P Garrahan, Buenos Aires, Ciudad Autonoma de Buenos Aires, Argentina, 3Hospital de Pediatría Prof Dr Juan P Garrahan, Buenos Aires, Ciudad Autonoma de Buenos Aires, Argentina, 4 Hospital de Pediatría Prof Dr JP Garrahan, Buenos Aires, Ciudad Autonoma de Buenos Aires, Argentina, 5Servicio de Hematología y Oncología. Hospital de Pediatría Prof Dr Juan P Garrahan, Buenos Aires, Ciudad Autonoma de Buenos Aires, Argentina

Abstract Number: PB0229

Meeting: ISTH 2022 Congress

Theme: Hemophilia and Rare Bleeding Disorders » Novel Biotherapeutics in Hemophilia

Background: Emicizumab (Hemlibra®, ROCHE), is effective in the prevention of bleeding in severe Hemophilia A patients with (sHAinh) or without (sHA) inhibitors. To date, emicizumab plasma quantification (Epq) is not routinely recommended but, could be required to verify treatment adherence, suspected anti-Emicizumab antibodies. Epq method, calibrators and quality controls are not yet available.

Aims: To validate in-house Epq method using a modified one-stage assay (modOSA).

Methods: modOSA: 1:80 pre-diluted sample with Owren Koller Buffer(OKB). Calibration Curve: 1:1(1:80 pre-diluted home-made calibrator (hmCal) with OKB), 1:2,1:4,1:8,1:16,1:32. hmCal (150µg/mL of Emicizumab): FVIII deficient plasma (FVIIIdp) was spiked with Emicizumab. Home-made Controls: level 1=25µg/mL (hmL1), level 2=75µg/mL (hmL2). Reagents: Immunodef VIII, C.K. Prest, CaCl2 0.025M. STA Compact Max2, Diagnostica Stago.

Repeatability(CVr)= hmL1 and hmL2: 20 times in the same run. Within-laboratory Imprecision(CVwl)= hmL1 and hmL2 in triplicate for 5 days. Calibration curve: 6 levels in duplicate (range: 5–150µg/mL). Linearity: 11 levels in triplicate, using hmCal and FVIIIdp (0µg/mL). In addition, aPTT, One Stage FVIII Assay (OSA), FVIII Chromogenic Assay (CSA, Bovine reagent, Chromogenix) and OSA/Epc were measured in the 9 levels. Limit of quantification (LoQ): FVIIIdp, sHAinh and sHA samples were evaluated. Bias estimation: from an external quality assessment program (UK NEQAS). Interference: mixtures of Emicizumab with FVIII were evaluated (Unicalibrator, Diagnostica Stago as a source of FVIII). EP evaluator was used for statistical analysis.

Results: hmL1: CVr 4.8%(27µg/mL); CVwl 5.7%(26µg/mL), hmL2: CVr 3.1%(73µg/mL); CVwl 4.1%(74µg/mL). Calibration curve: r:-0.999, r2:0.998. Linearity: Table1, r2:0.997 (linear range 5-150µg/mL), Slope:1.011, Intercept:0.52. LoQ:5µg/mL. Bias:-4.0%. Interference: Table2.

Conclusion(s): modOSA showed a good performance in the Epq. aPTT was below the reference range and CSA did not interfere in the evaluated range of 5-100 ug/ml of Epq. FVIII surrogate activity is appoximately 7 times the level of Epq. In vitro FVIII acts as an interference in the method.

Table

Table 1

Table

Table 2

To cite this abstract in AMA style:

Sueldo R, Hepner M, Frontroth J, Annetta E, Cervio C, Bianco M, Sciuccati G. Emicizumab: In-house method validation [abstract]. https://abstracts.isth.org/abstract/emicizumab-in-house-method-validation/. Accessed October 1, 2023.

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