Evaluation of emicizumab calibrator and controls with a modified one-stage FVIII assay on an automated coagulation analyzer
ISTH Academy. Shinohara S. Jul 9, 2019; 264496; PB1305
Dr. Sho Shinohara
Dr. Sho Shinohara
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Evaluation of Emicizumab Calibrator and Controls with a Modified One-stage FVIII Assay on an Automated Coagulation Analyzer

S. Shinohara1, T. Saito1, M. Noguchi-Sasaki2, T. Ishiwata2, M. Morris3
1Sysmex Corporation, Kobe, Japan, 2Chugai Pharmaceutical Co., Ltd., Tokyo, Japan, 3r2 Diagnostics, Indiana, United States

Main Topic: Diagnostics and OMICs
Category: Laboratory Diagnostics – Bleeding

Background: Emicizumab is a bispecific antibody that bridges activated factor IX and factor X to restore the function of missing activated factor VIII (FVIII) that is needed for effective hemostasis in persons with hemophilia A. Emicizumab has been approved in several countries for the use on Hemophilia A patients with and without inhibitors. Recently, emicizumab calibrator and controls became commercially available for emicizumab concentration measurements, but its instrument applications and evaluation data are limited.
Aims: To develop an application of emicizumab concentration for an automated coagulation analyzer and evaluate its analytical performance.
Methods: Assay parameters were developed for the measurement of emicizumab concentration on the Sysmex CS-5100 analyzer. The assay was tested with 2 different activated partial thromboplastin time (APTT) reagents: Actin FSL (Siemens, Germany) and APTT-SLA (Sysmex, Japan). Immuno-depleted FVIII deficient plasma was used with both reagents. Following evaluations were performed: Calibration curve, linearity, limit of quantification (LoQ), interference, repeatability and within-laboratory imprecision. Emicizumab calibrator and controls (r2 Diagnostics, USA) were prepared according to manufacturer's instruction. Contrived samples were prepared by spiking emicizumab into congenital FVIII deficient plasma. Recombinant full-length factor VIII (rFVIII), recombinant activated factor VII (rFVIIa) and activated prothrombin complex concentrate (aPCC) were used as interfering substances.
Results: Emicizumab concentration assay was developed by modifying one-stage FVIII activity assay of the analyzer. Following modifications were made: Sample dilution ratio was determined as 1/80, and the assay was calibrated against emicizumab calibrator with dilution ratios of 1/1, 1/2, 1/4, 1/10 and 1/64. Summary of evaluation results are shown in Table 1.
Conclusions: Emicizumab concentration assay on Sysmex CS-5100 analyzer showed good analytical performances. The assay further needs to be validated with samples from emicizumab dosed patients.

Calibration curve r2 = 0.9991 r2 = 0.9996
(up to 300 µg mL-1)
r2 = 0.9980
y= 1.01 x - 3.76
r2 = 0.9769
y= 0.96 x - 2.26
LoQ 10 µg mL-1 10 µg mL-1
(±20 µg mL-1 absolute)
rFVIII: Affected from 100 IU dL-1
rFVIIa: Not affected
aPCC: Not affected
rFVIII: Affected from 100 IU dL-1
rFVIIa: Not affected
aPCC: Not affected
Repeatability (CV%) Level 1 (26.2 µg mL-1): 4.7
Level 2 (75.0 µg mL-1): 2.6
Level 1 (26.2 µg mL-1): 2.3
Level 2 (75.0 µg mL-1): 2.2
Within-Laboratory imprecision (CV%) Level 1 (26.2 µg mL-1): 4.7
Level 2 (75.0 µg mL-1): 3.3
Level 1 (26.2 µg mL-1): 2.9
Level 2 (75.0 µg mL-1): 2.9
[Summary of evaluation results]

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