Donald F Brophy1, Erika J Martin1, Marisa Ninivaggi2, Bassem M Mohammed1,3, John C Barrett4, Janice Kuhn4, Melinda E Nolte4, Emily K Waters2, Mirella Ezban2. 1. Coagulation Advancement Laboratory, Department of Pharmacotherapy & Outcomes Science, Virginia Commonwealth University (VCU), Richmond, Virginia. 2. Novo Nordisk A/S, Bagsvaerd, Denmark. 3. Department of Clinical Pharmacy, Faculty of Pharmacy, Cairo University, Cairo, Egypt. 4. Internal Medicine/Division of Haematology/Oncology, VCU, Richmond, Virginia.
Abstract
INTRODUCTION: The thrombin generation assay (TGA) can be used to monitor factor replacement therapy in patients with haemophilia. The TGA assay is typically performed using tissue factor as the reaction activator; however, activating with FIXa or FXIa can enhance assay sensitivity when FVIII < 1%. AIMS: To evaluate the sensitivity of the TGA when FIXa (5 nmol/L) and FXIa (0.22 nmol/L) are used to activate the assay in platelet-poor plasma and to compare these data to the one-stage and chromogenic assays. METHODS: Plasma from 10 severe FVIII-deficient subjects was supplemented with FVIII (0%, 0.1%, 0.4%, 1.2%, 4%, 11% and 33%), using either Novo Eight® , Advate® , Eloctate® , turoctocog alfa pegol or a control standard. The one-stage and chromogenic assays quantified the FVIII levels. The TGA assay was activated using either FIXa or FXIa. RESULTS: Both FIXa- and FXIa-activated TGA were sensitive across FVIII concentrations, with intra-assay coefficient of variation (CV) < 10%. The FXIa-activated assay had 25% CV at the lowest level of FVIII compared to 10% CV with FIXa activation. There were strong correlations between the FIXa- and FXIa-activated TGA tests (R2 = 0.9912) and between the one-stage and chromogenic assays (R2 = 0.9469). However, there were poor relationships between the TGA tests and one-stage and chromogenic assays. CONCLUSIONS: Both FIXa- and FXIa activation results in similar TGA profiles across a FVIII range of 0.1%-33%; however, FIXa activation was more robust at the lowest levels of FVIII compared with FXIa activation.
INTRODUCTION: The thrombin generation assay (TGA) can be used to monitor factor replacement therapy in patients with haemophilia. The TGA assay is typically performed using tissue factor as the reaction activator; however, activating with FIXa or FXIa can enhance assay sensitivity when FVIII < 1%. AIMS: To evaluate the sensitivity of the TGA when FIXa (5 nmol/L) and FXIa (0.22 nmol/L) are used to activate the assay in platelet-poor plasma and to compare these data to the one-stage and chromogenic assays. METHODS: Plasma from 10 severe FVIII-deficient subjects was supplemented with FVIII (0%, 0.1%, 0.4%, 1.2%, 4%, 11% and 33%), using either Novo Eight® , Advate® , Eloctate® , turoctocog alfa pegol or a control standard. The one-stage and chromogenic assays quantified the FVIII levels. The TGA assay was activated using either FIXa or FXIa. RESULTS: Both FIXa- and FXIa-activated TGA were sensitive across FVIII concentrations, with intra-assay coefficient of variation (CV) < 10%. The FXIa-activated assay had 25% CV at the lowest level of FVIII compared to 10% CV with FIXa activation. There were strong correlations between the FIXa- and FXIa-activated TGA tests (R2 = 0.9912) and between the one-stage and chromogenic assays (R2 = 0.9469). However, there were poor relationships between the TGA tests and one-stage and chromogenic assays. CONCLUSIONS: Both FIXa- and FXIa activation results in similar TGA profiles across a FVIII range of 0.1%-33%; however, FIXa activation was more robust at the lowest levels of FVIII compared with FXIa activation.