Evaluating anti-sickling therapies for sickle cell disease: a microfluidic assay for red blood cell-mediated microvascular occlusion under hypoxia

Sickle cell disease (SCD) is characterized by the polymerization of hemoglobin S (HbS) upon deoxygenation, leading to the formation of sickled red blood cells (RBCs) with reduced deformability. Under hypoxic conditions, the impaired RBC behavior significantly contributes to vaso-occlusive events, hemolysis, and end-organ damage. Consequently, RBC deformability serves as a pivotal hemorheological biomarker for evaluating disease severity and therapeutic response. The OcclusionChip, a microfluidic assay, measures RBCs deformability through microcapillary occlusion. However, its current hypoxic assay relies on a complex nitrogen gas setup, rendering it bulky, expensive, and unsuitable for point-of-care diagnostic use. Here, we optimized a chemically induced hypoxia assay using sodium metabisulfite (SMB) within the OcclusionChip platform and validated the hypoxia occlusion index (HOI) as a robust measure of RBC deformability in SCD. Optimal hypoxia conditions were established, replicating nitrogen-induced hypoxia without affecting RBC membrane integrity, reactive oxygen species (ROS) levels, or phosphatidylserine (PS) exposure. Under these conditions, RBCs from individuals with heterozygous (HbAS), HbSC, and HbSS genotypes showed significantly higher HOI compared to healthy controls (HbAA), correlating strongly with clinical biomarkers in SCD. Additionally, the HOI assay effectively assessed the efficacy of therapeutic agents, including hemoglobin-oxygen affinity modifiers (GBT021601, GBT440) and protein kinase R (PKR) activators (PKR-3, FT4202), which significantly reduced OI in SCD RBCs. Notably, combination therapies showed enhanced effectiveness, highlighting the assay's potential for optimizing treatment regimens. This study establishes the chemically induced hypoxia OcclusionChip assay as a reliable and clinically useful tool for evaluating RBC deformability in SCD, with significant potential to improve personalized treatment strategies and thus patient outcomes.