Abstract Number: PB1185
Meeting: ISTH 2022 Congress
Background: Patients with severe hemophilia A require frequent injections with Factor VIII (FVIII). Due to the variability in the clearance of FVIII, these injections do not guarantee an adequate hemostatic response. A self-test would allow patients to monitor their FVIII levels. Luminescent-based assays can be incorporated in a handheld device without the need for a light source or filters, providing a linear signal over several orders of magnitude.
Aims: To develop a chemiluminescent FVIII activity assay to measure FVIII activity levels in small volumes.
Methods: A chemiluminescent-based FVIII activity assay (FVIIIlum) was constructed based on tenase complex formation. The activation of FX is made limited by FVIII by adding the other components of the Xase-complex in excess. FVIII activity was determined by measuring the amount of Factor Xa (FXa) formed using a FXa-specific substrate, consisting of a tetrapeptide coupled to a luciferin-moiety. Upon production of FXa, the specific substrate is cleaved, releasing aminoluciferin. Free aminoluciferin is converted by luciferase to oxyluciferin and a light quant. The Michaelis-Menten constant and Vmax were determined using 1.6 nM FXa. The assay was optimized to differentiate between 0.01-1.0 IU/ml FVIII activity in a total reaction mixture of 30 µl. FVIIIlum was compared to the FVIII:C BIOPHEN assay using samples from ECAT surveys.
Results: The FXa-specific substrate showed low cross-reactivity ( < 10%) for thrombin and FIXa at physiological concentrations. The Michaelis-Menten kinetics demonstrated a Km of 535 µM. FVIIIlum correlated well with the FVIII:C BIOPHEN with a slope of 0.819 and an R2 of 0.943, tested in ECAT samples (n=8) within the range of < 1% to 67%.
Conclusion(s): A chemiluminescent-based assay sensitive for FVIII activity was developed for use in small plasma volumes. Furthermore, a good correlation was demonstrated with the standard chromogenic FVIII:C assay. As the FVIIIlum demonstrated high RLU signals, this technique is suitable for further miniaturization.
To cite this abstract in AMA style:Steeghs D, van Geffen M, van Heerde W, van 't Veer C. Novel miniaturized Chemiluminescent-based Factor VIII activity assay [abstract]. https://abstracts.isth.org/abstract/novel-miniaturized-chemiluminescent-based-factor-viii-activity-assay/. Accessed February 28, 2024.
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