PB0694 - A chemiluminescent-based multiparameter platform for simultaneous measurement of factor VIII activity and thrombin generation

Background: A near-patient platform containing a chemiluminescent-based quantitative Factor VIII (FVIIIact) assay and a Thrombin Generation Assay (TGA) could enable patients with hemophilia A (PwHA) to make informed treatment decisions. Chemiluminescent-based assays in combination with sensitive photon-sensors allow for miniaturization from micro- to nanoliter volumes.

Aims: To develop a nanoliter chemiluminescent-based FVIIIact (FVIIIlum) assay and TGA (TGAlum) on a microfluidic cartridge with dried-in reagents and a read-out with optical sensors.

Methods: FVIIIlum and TGAlum assays respectively operate via caged aminoluciferin substrates coupled to a factor Xa or thrombin-specific peptide sequence. Cleavage of the substrate releases aminoluciferin which generates photons by added luciferase. Assay-specific reagents were spotted (FIXa and FX, and tissue factor respectively) and dried on a multichannel microfluidic cartridge in picoliter volumes. The addition of prediluted citrated normal pooled plasma (NPP) to the microfluidic cartridge solubilized the reagents and started reactions due to recalcification. Additionally, different FVIII bypassing agents were also added to the plasma. Generated photons were detected with Single Photon Avalanche Diode detectors. Both assays were measured in quadrupole per microfluidic cartridge, in a total of eight detection chambers of 110 nanoliters.

Results: The addition of NPP and/or FVIII deficient plasma to the microfluidic cartridges with dried-in reagents showed luminescence in both assays in an FVIII-dependent manner over a range of < 1-120% FVIII. The FVIIIlum reported an intra- and inter-cartridge coefficient of variation of 14% (n=4) and 12% (n=6), respectively. The TGAlum reported an intra- and inter-cartridge coefficient of variation of 16% (n=4) and 38% (n=6), respectively. The TGAlum also shows FVIII bypassing agent dependency. Spotted cartridges were stable for at least two months if stored at room temperature.

Conclusion(s): Proof of concept demonstrated simultaneous measurement of FVIII activity and TGA using a multichannel microfluidic cartridge with dried-in reagents. This combination allows for treatment monitoring of PwHA including FVIII bypassing therapy.