Assessment of primary hemostasis with an acoustic biosensor using shear dependent kinetics behavior: principle and limitations

Objective Primary hemostasis involves in-flow interactions between platelets and sub-endothelial matrix at the wall of the damaged vessel. Assessing primary hemostasis defects would benefit from evaluation of the whole sequence of processes involved in platelet plug formation. We propose a novel label-free approach based on characterization of shear-dependent kinetics to evaluate the early stages of primary hemostasis. We developed a quartz crystal microbalance (QCM) biosensor to measure the amount of platelet deposited over time. With experiments and numerical simulations, we investigated the relevance of this approach and its limitations.

Material and Methods We designed and built an acoustic biosensor based on a QCM whose gold surface was functionalized with Horm® collagen and used as the floor of a microfluidic chamber. We recorded with an impedance analyzer the variations of the QCM sensor resonance frequency during a 5-minutes perfusion through the chamber with anticoagulated whole blood from two healthy donors. The real-time QCM measurements performed at 500 - 1500/s range shear rate were supplemented with atomic force microcopy (AFM) observation at the end of the perfusion to evaluate the final morphology of the deposit and the surface coverage. Numerical simulations were used to understand the influence of deposit topology on the acoustic response.

Results For analyzing the complex kinetics profile of the frequency shift, we defined three metrics: total frequency shift, lag time, and growth rate. These metrics enabled the characterization of the kinetics of platelet deposition with good repeatability. We showed that these parameters measured at different shear rates, gave precise indications on the processes involved in the early stage of primary hemostasis, opening the way to analyze abnormal behavior.

However we observed that the frequency shift was not always a direct measure of the platelet amount and depends on the surface topology of the deposit, which varies with the shear rate. The numerical simulation confirmed that if a platelet deposits is modeled as a structured viscoelastic load, the surface coverage affects the frequency shift of the sensor.

Conclusion Shear-dependent kinetics assays seems to be a promising method for studying primary hemostasis and its defects. We showed that QCM sensor measurements have to be combined with a precise evaluation of deposit topology to be fully usable.

Fig. 1 Photograph of the whole blood perfusion chamber with an installed QCM biosensor (a) and the experimental setup (b)

Fig. 2 QCM sensor admittance frequency shift (Hz) recorded during the blood perfusion during five minutes for two donors (donor1 (a) and donor2 (b)) at a shear rate of 500s-1 (blue), 770s-1 (orange), 1000s-1 (green) and 1500s-1 (yellow).

Fig. 3 Bar charts of the three defined metrics as a function of shear rates for two healthy donors: TFS (a); lag time (b); growth rate (c). Error bars show the absolute deviation from the mean value at the same shear rate the tests were made for the same donor on three different days.

Publication History

Article published online:
18 June 2021