Microfluidic capillary transit velocity as a functional measure for sickle cell disease and in vitro-derived red blood cells

Abstract

Emerging therapies in sickle cell disease (SCD) aim to restore healthy red blood cell (RBC) function, but they often yield heterogeneous cellular responses. There are no proven techniques to evaluate restored rheological functionality and heterogeneity in these RBCs. We present a biomimetic microcapillary network, high-speed imaging, and computational algorithms to analyze RBC capillary velocity profiles of the entire sample population at single-cell resolution. Using peripheral RBCs from SCD patients and healthy donors, we showed that RBC capillary transit velocity correlated with cell shape, hydrodynamic adaptability, and elongation index. Healthy RBCs exhibited a velocity distribution skewed toward higher values, whereas RBCs from individuals with SCD showed a shift toward lower velocities. SCD samples had a greater fraction of slow RBCs than healthy controls (42.1% ± 12.0% vs. 19.0% ± 4.9%, p < 0.0001). We tested mixtures of healthy and SCD RBCs to simulate heterogeneous therapeutic effects and demonstrated that the assay was sensitive to small fractions of abnormal RBCs. The slow RBC fraction emerged as a potential biomarker associated with SCD disease severity. This fraction significantly increased under hypoxia showing sensitivity to hypoxia-induced sickling. Finally, we assessed in vitro-derived RBCs and observed distinct velocity profiles for nucleated and enucleated cells. Processing methods to enrich enucleated RBCs improved the velocity profile, producing a distribution that was more comparable to that of peripheral RBCs. This platform's ability to assess individual RBCs and generate a velocity profile from a small number of cells makes it well suited for evaluating the rheological properties of in vitro-derived RBCs.