A FXIa-modified clot waveform analysis for estimation of factor VIII level and emicizumab concentration in hemophilia A Free

Background

Hemophilia A treatment options include factor VIII (FVIII) substitution and non-factor therapies, like emicizumab. The activated partial thromboplastin time (aPTT) is sensitive to the presence of emicizumab, but imprecise at quantifying emicizumab plasma concentrations, especially at clinically meaningful concentrations in treatment steady-state (>40 μg/ml). In clinical scenarios like peri-interventional management, where additional FVIII, is being used on top of emicizumab, limited options are available for estimating the treatment response.

We evaluated the treatment response of hemophilia A treatment options using a modified clot waveform analysis assay (CWA) by using FXIa + tissue factor (TF) and diluted aPTT + TF as a coagulation triggers in comparison to undiluted aPTT.

Methods

We used plasma samples (N=42) from patients with severe hemophilia A (endogenous FVIII level <1IU/dL, without inhibitor) on treatment with emicizumab and spiked FVIII (standard half-life, octocog alfa) to achieve target levels (50-100 IU/dL). Emicizumab concentrations were measured with a modified one-stage assay (r² Diagnostics). Additionally, FVIII-deficient plasma (Technoclone, Austria) was spiked to a wide range of precise FVIII and emicizumab levels for correlation analysis.

Clot waveform analysis was performed on a Sysmex CS-5100 automated blood coagulation analyzer. Clotting was initiated with three different reagent mixtures and CaCl₂: (1) FXIa 40 U/l (CoaChrom, Austria) + TF 0.25 pmol/L (Innovin, Siemens) + phospholipids (FXIa+TF-CWA), (2) diluted aPTT 1:9 (Actin FSL, Siemens) + TF 0.25 pmol/L (diluted aPTT+TF-CWA), and (3) undiluted aPTT Actin FSL (undiluted aPTT-CWA). CWA parameters were derived from light transmittance measured over time. Maximum velocity (first derivative of clotting time) and maximum acceleration (second derivative) were adjusted to intrinsic differences in transmittance between samples using the manufacturer-provided software.

To evaluate the sensitivity of different reagents at high emicizumab levels, CWA parameters were compared across reagents using one-way ANOVA. To assess the effects of additional FVIII or rFVIIa, linear regression models were used with spiking condition as a categorical predictor, stratified by reagent. Post-hoc pairwise comparisons of marginal means were conducted using the delta method. Statistical significance was defined as p < 0.05. All analyses were conducted using Stata (version 18.0).

Results

Increasing concentrations of FVIII and emicizumab in FVIII-deficient plasma showed a shortening of clotting time following an exponential decay function using all three reagents and an increase of CWA-velocity and acceleration following a logarithmic function.

However, at on-treatment levels of emicizumab (≥40 µg/mL) in patient plasma, FXIa + TF-CWA was significantly more responsive to concentration changes in terms of adjusted maximum velocity than both diluted aPTT + TF-CWA (Δslope = +0.059, p = 0.009) and undiluted aPTT-CWA (Δslope = +0.065, p = 0.005).

After spiking of FVIII to 50 IU/dL and 100 IU/dL levels into steady-state emicizumab samples (40 - 75 µg/mL), the triggers FXIa+TF and diluted aPTT+TF demonstrated statistically significant increases in adjusted maximum velocity (FXIa+TF: p=0.006 and p=0.002; diluted aPTT+TF: p=0.004 and p=0.001 for 50 IU/dL and 100 IU/dL FVIII levels, respectively). Similar trends were observed in adjusted maximum acceleration. Clotting time significantly decreased only with diluted aPTT+TF-CWA (p=0.007 and p=0.002 for 50 IU/dL and 100 IU/dL FVIII levels, respectively). In contrast, undiluted aPTT-CWA showed no statistically significant changes in clotting time, velocity, or acceleration across categories of additional FVIII (p>0.1).

Conclusion

We found the modified-CWA by FXIa + TF activation to be more responsive to emicizumab at typical steady-state treatment ranges than aPTT-CWA. FXIa+TF-CWA and diluted aPTT+TF-CWA were better at discriminating additional FVIII in emicizumab samples than undiluted aPTT-CWA. These findings indicate that FXIa+TF-CWA and diluted aPTT+TF-CWA better discriminate clotting dynamics of emicizumab and additional FVIII substitution, which may be of use in clinical scenarios like peri-interventional monitoring.