APTT-Based Clot Waveform Analysis to Assess FVIII and FVIII Mimetic Therapies in Hemophilia A

Introduction Laboratory assessment of hemostasis in hemophilia remains challenging, particularly in the era of non–factor and factor-mimetic therapies. Conventional one-stage clotting assays based on the activated partial thromboplastin time (APTT) provide limited information on overall clot dynamics. Clot waveform analysis (CWA), derived from optical clotting measurements, allows evaluation of the kinetics of clot formation beyond clotting time and may better capture differences in coagulation potential among patients receiving factor and factor-mimetic therapies. Method A total of 175 plasma samples from persons with hemophilia A were analyzed, including 50 samples with endogenous FVIII activity, 45 from patients receiving emicizumab, and 80 obtained during FVIII substitution therapy. CWA was performed on a Sysmex CS-5100 automated coagulation analyzer. Clotting was initiated with two reagent combinations and CaCl₂: (1) FXIa+TF (Innovin, Siemens)+phospholipids (FXIa+TF-CWA) and (2) diluted aPTT 1:9 (Actin FSL, Siemens)+TF (diluted aPTT+TF-CWA). Clot formation kinetics were recorded as changes in light transmittance over time. The maxima of the first and second derivatives of the clotting curve (maximum velocity and maximum acceleration) were derived using manufacturer-provided algorithms and adjusted for individual baseline plasma turbidity. Chromogenic FVIII activity and emicizumab plasma concentrations (r²- Diagnostics) were determined as comparators using validated assays. Correlation between CWA parameters and log-transformed FVIII activity or emicizumab concentration were evaluated using Pearson’s correlation coefficients ( r ) and simple linear regression ( R² ). Results CWA parameters showed strong correlations with log-transformed chromogenic FVIII activity in samples containing endogenous or substituted FVIII ([ Fig. 1 ]). Among endogenous FVIII samples, the highest associations were observed for diluted aPTT+TF-CWA (maximum velocity: r =0.86, R² =0.74; maximum acceleration: r =0.83, R² =0.69). In FVIII substitution samples, correlations remained high, particularly for diluted aPTT+TF-CWA (maximum velocity: r =0.89, R² =0.79). In emicizumab-treated samples, CWA parameters correlated moderately with log-transformed emicizumab plasma concentrations measured by the R² Diagnostics assay, with the strongest relationships seen for FXIa+TF-CWA (clotting time: r =–0.72, R² =0.52; maximum velocity: r =0.69, R² =0.48). Conclusion APTT-based clot waveform analysis provides an assessment of coagulation kinetics that reflects FVIII activity with high analytical precision and moderate responsiveness to emicizumab. Overall, the diluted aPTT+TF and FXIa+TF reagent combinations provided responsive and consistent profiles across treatment groups. While these findings support the potential of reagent-optimized CWA as a complementary tool for assessing hemostatic function in hemophilia, its clinical applicability for routine monitoring remains to be determined. Fig. 1 Correlation between CWA parameters and endogenous FVIII level, FVIII substitution level, and emicizumab concentration using different CWA trigger reagents Publication History Article published online: 17 February 2026 © 2026. Thieme. All rights reserved. Georg Thieme Verlag KG Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany