PB1187 - A Rapid, Fully Automated and Highly Reproducible Factor VIII-Inhibitor Assay, Insensitive for Emicizumab, a Lowest Level of Quantification of 0,2BU and Lacking Need for FVIII Calibration

Background: Currently, Factor VIII inhibitors are measured using Nijmegen or Bethesda assays which are labour and resource expensive, lack sensitivity for low-titre inhibitors and show high variations in quality surveys mainly because of manual assay procedures.

Aims: Development of a Rapid Inhibitor Assay (RA) by using rFVIII as substrate in VWF-free assay-matrix, which enables automatization.

Methods: Heat-inactivated (30 min/56°C) patient and reference plasma (FVIII/VWF-deficient) are analysed by a predefined application in a coagulation analyser capable of three subsequent sample dilution steps and three reagent additions (Ceveron s100, Technoclone). In summary: mixtures of equal volumes of sample and rFVIII (1.0U/mL) are incubated 10 minutes/37°C, whereafter remaining FVIII-activity in the mixture is analysed with a kinetic chromogenic assay allowing inhibitor-activity calculation without preceding FVIII-activity calibration. “Residual FVIII-Activity” is expressed as quotient of extinction-change/minute of patient and reference samples, and Bethesda values determined by the equation: {2-log 100x(Residual FVIII-Activity}/log2.
Analyses were performed in 16 inhibitor-positive samples with the RA and Nijmegen Assay using chromogenic test for residual FVIII activity (NA-CS), and in 36 samples from non-haemophilic persons with normal FVIII, with RA.
Reproducibility was tested by analysing seven samples in three laboratories on five days, twice daily.
Emicizumab dependency was tested in spiked inhibitor-positive-samples.

Results: The mean titre in non-haemophilic persons was 0.0BU (SD 0.1) yielding a Limit-of-Blank of 0.1BU and a Lower-Limit-of-Quantification (LLOQ) of 0.2BU.
Combined data of patient-samples (Table 1) and non-haemophilic-samples showed very high positive and negative predicting values compared to NA-CS.
Emicizumab did not influence outcome of RA in spiking experiments.
CV of all samples of the reproducibility tests were < 15%.

Conclusion(s): A rapid and fully automated FVIII-inhibitor assay was developed with a LLOQ of 0,2BU, low interlaboratory CV, insensitivity to Emicizumab, and good correlation with NA-CS. The Rapid Assay may be a gamechanger as it may be used as STAT assay.