PB0169 - Proof of principle of a fast and fully automated FVIII functional inhibitor test

Background: Laboratory detection of Factor (F)VIII inhibitors is preferentially performed with the Nijmegen assay. It requires 2-hours incubation of test sample/FVIII-source mixture because of slow FVIII inactivation, due to its reversible binding to von Willebrand Factor (VWF) delaying inhibitor action.
Extended incubation time results in non-specific FVIII inactivation, that, together with complicated liquid handling, may contribute to the considerable variability (CV:30–40%) seen in inter-laboratory surveys (e.g. ECAT, UKNEQAS). We hypothesize that testing in a VWF-free assay matrix using recombinant (r)FVIII can dramatically lower incubation time that, together with full automation, will substantially improve standardisation.

Aims: Test proof of principle of a fast, fully automated FVIII inhibitor test using VWF-free rFVIII and a dedicated analyser.

Methods: As in the original Nijmegen assay, test samples are heated for 90 minutes at 58°C and centrifuged for 10 minutes to destroy residual FVIII.
The coagulation analyser employed must provide on board ability of three subsequent sample dilution steps and three reagent additions. An application was defined on a Ceveron s100 (Technoclone), as below.
After loading the heat inactivated samples, sequential automated analytical steps occur as follows:
1. Predilution with heat inactivated FVIII/VWF deficient plasma (if needed)
2. Mixing with rFVIII (1.0 IU/mL)
3. Incubation for 5-10 minutes at 37°C
4. Dilution of incubated samples 1:10 with Imidazole buffer, pH 7.3. and analysis for residual rFVIII activity

Results: Two samples whose inhibitor activities with the original Nijmegen assay were 0 and 14 NBU, were analysed with the automated method resulting in activities of 0 and 11 NBU respectively. Incubation for 5 and 10 minutes yielded similar results.

Conclusion(s): Rapid, fully automated FVIII-inhibitor testing can be performed with a dedicated coagulation analyser using rFVIII in a VWF-free matrix. Automation and reduced assay time improve viability and availability of a normally protracted assay, permitting a more rapid and informed clinical response.