Abstract Number: PB1186
Meeting: ISTH 2022 Congress
Background: Patients with hemophilia A may benefit from self-monitoring their coagulation status and adjusting their medication real-time based on their physiological status without a long laboratory turnaround time. Despite Factor VIII (FVIII) levels being above 1% trough levels, this does not guarantee a proper hemostatic response. The Thrombin Generation Assay (TGA) provides a global and overall understanding of the hemostatic balance of a patient, however, it requires specific lab equipment, as well as relatively large quantities of plasma. The use of a chemiluminescent-based TGA would allow for assay volume reduction as well as monitoring non-replacement and FVIII bypassing agents.
Aims: Developing a chemiluminescent-based assay for real-time TGA analyses applicable for near-patient platform technology.
Methods: A chemiluminescent-based TGA was constructed using an aminoluciferin-coupled thrombin substrate which releases aminoluciferin upon cleavage by formed thrombin. Almost instantaneously luciferase oxidizes aminoluciferin (in the presence of ATP and Magnesium) resulting in the generation of photons. The created TGA uses recalcified plasma and the addition of 1 pM Tissue Factor. Different plasma mixtures with FVIII activity (ranging 0-100%) were measured to determine the correlation between FVIII and TGA parameters, analysed with the Pearson correlation coefficient.
Results: The chemiluminescent-based TGA displays the typical TGA characteristics; lag-time, propagated thrombin formation, and inhibition of formed thrombin. The assay readout is real-time without the need for mathematical derivation as is required in assays with fluorogenic substrates. Concerning FVIII dependency, peak height ranges from 1.5*106-6.2*106 Relative Light Units and showed a strong correlation with FVIII levels (r=0.97). Strong correlations were also found for the velocity index (r=0.99), and time to peak (r=-0.92).
Conclusion(s): This chemiluminescent-based TGA allows for FVIII dependent thrombin generation and can therefore be used for personalized hemophilia A near-patient test application. The dynamic range and signal intensity offer the potential to miniaturize the assay to a volume off less than 1 µL.
To cite this abstract in AMA style:van Engelshoven L, van de Ridder C, Steeghs D, van Geffen M, van 't Veer C, van Heerde W. A novel chemiluminescent-based Thrombin Generation Assay for near-patient monitoring. [abstract]. https://abstracts.isth.org/abstract/a-novel-chemiluminescent-based-thrombin-generation-assay-for-near-patient-monitoring/. Accessed December 6, 2023.
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