PB1122 - Limits on the correction of inner filter effect and substrate consumption by the calibrated automated thrombinography algorithm: an edge of failure study

Background: Fluorogenic thrombin generation (TG) is a global hemostasis assay that provides a more physiological representation of hemostasis potential. However, the accurate detection of thrombin activity in plasma may be affected by artifacts inherent to the assay-specific fluorogenic substrate. The significance of the fluorogenic artifacts or their corrections has not been studied in hemophilia treatment applications.

Aims: To investigate TG in hemophilia plasma samples under typical and worst-case fluorogenic artifact conditions and assess the limitations of artifact correction algorithm known as calibrated automated thrombinography.

Methods: Severe hemophilic plasma with or without added Factor VIII were evaluated using commercially available and in-house TG reagents, instruments, and three software packages: one commercial TG software, an in-house Origin-based software, and a publicly available National Institute for Biological Standards and Control (NIBSC) Shiny App. The inner filter effect (IFE) was mimicked by spiking increasing amounts of fluorophore 7-amino-4-methylcoumarin (AMC) into plasma prior to the TG experiment. Substrate consumption was modeled by adding decreasing amounts of Z-Gly-Gly-Arg-AMC (ZGGR-AMC) to plasma and performing TG in antithrombin deficient plasma.

Results: The modeled IFE significantly affected the TG curves in all of our studied correction algorithms, failing to return, or overestimating TG results at high AMC concentrations. Similarly, correction algorithms failed to correct TG curves in experiments with extreme substrate consumption in antithrombin deficient plasma but were effective in correcting for reduced starting substrate concentration. Comparatively, normalization of TG values in hemophilia plasma to the values in a respective factor-supplemented sample resulted in a more accurate correction of substrate artifacts.

Conclusion(s): Correction algorithms may be effective in situations of moderate but not severe artifacts.