Biophysical characterization of red blood cells in sickle cell trait reveals heterogeneous and intermediate pathophysiological profiles

Abstract Introduction: Sickle hemoglobin (HbS) results from a single nucleotide substitution in the β-globin gene. Individuals with homozygous HbS (HbSS) develop sickle cell anemia, while heterozygous carriers (HbAS) are said to have sickle cell trait (SCT). Although SCT has historically been considered benign, emerging evidence suggests that red blood cells (RBCs) from individuals with SCT may exhibit functional abnormalities in environments of high physiological stress such as hypoxia, dehydration, or inflammation. Despite this, systematic biophysical profiling of HbAS RBCs remains limited. Here, we utilize microfluidic platforms, including the OcclusionChip and sickle cell disease (SCD) Biochip, to investigate RBC deformability and adhesion under both normoxic and hypoxic conditions. Additionally, we used RHODA (Rapid Hemoglobin Oxygen Dissociation Assay), a novel optical hemoglobin-oxygen (Hb-O2) dissociation assay, to measure Hb-O2affinity. HbS content and clinical markers were also analyzed to contextualize biophysical findings. Methods: We analyzed RBC biophysical properties from N=100 individuals with SCT (HbAS) enrolled in the National Alliance of Sickle Cell Center AUNT (Achieving Understanding of the Natural History of Sickle Cell Trait) study and compared them to RBC from healthy controls (HbAA) (n=20) and HbSS (n=30). RBC deformability and microvascular obstruction were assessed using the OcclusionChip, a microfluidic device mimicking capillary networks. Washed RBCs (20% hematocrit in PBS) were perfused under constant pressure, and post-wash imaging was used to quantify the Occlusion Index (OI), a standardized metric of network blockage. RBC adhesion under normoxic and hypoxic conditions was evaluated using the Adhesion Biochip, a laminin-coated microfluidic assay that captures adherent cells under flow. Hb-O2affinity was assessed using RHODA, an optical assay for rapid quantification of O2 unloading by induction of physiological acidic pH (6.9). Hb composition (HbS, HbA, HbF) was measured using high-performance liquid chromatography (HPLC), and additional clinical markers, including reticulocyte count, hematocrit, and pyruvate kinase (PK) and G6PD enzyme activity, were also measured to support the phenotypic analysis. Results: Under hypoxia (oxygen partial pressure (pO)₂ of 44 mmHg (~83% oxygen saturation (SO2)), HbAS samples exhibited a markedly elevated microvascular occlusion index compared to HbAA controls (3.3 ± 2.5 vs. 0.8 ± 0.5; p 0.001) and were substantially lower than HbSS samples (35.8 ± 15.6). RBC adhesion to laminin was found to be significantly higher in HbAS versus HbAA under both normoxic (0.8 ± 0.5 vs. 0.3 ± 0.1; p 0.001) and (~83% SO2) hypoxic (1.99 ± 2.4 vs. 0.6 ± 1.6; p 0.001) conditions, although below adhesion levels observed in HbSS. Considerable heterogeneity was noted among HbAS individuals, especially under hypoxia. The HbS content of HbAS samples averaged 36.62 ± 3.65 %. In RHODA assays, HbAS cells demonstrated reduced Hb-O2 affinity relative to HbAA, as reflected by an increased optical shift. A subset of HbAS samples showed lower Hb-O2 affinity than HbSS patients on transfusion therapy or hydroxyurea. HbS% did not correlate significantly with adhesion or occlusion indices but did correlate with Hb-O2 affinity (Pearson r = 0.70). No significant associations were found between G6PD activity and any biophysical metric. However, PK activity (below 12.4 Ug Hb) showed moderate positive correlation with RBC adhesion under both normoxia and hypoxia (Pearson r = 0.58). Conclusions: These findings demonstrate that RBCs from individuals with SCT display an intermediate biophysical phenotype profile compared to RBCs from HbAA or HbSS, including elevated occlusion, increased adhesion, and altered oxygen affinity, especially under low oxygen conditions. Notably, these parameters vary significantly across HbAS subjects, suggesting that some carriers may be more susceptible to microvascular complications than others. This variability is not specific to the HbS% but may reflect additional biochemical and genetic modifiers such as co-inheritance of α-thalassemia or other globin-gene variants known to influence RBC behavior in SCD. Our results, therefore, emphasize the need for a more nuanced clinical understanding of SCT, including the current prospective study accounting for both HbS fraction and other modifying factors, rather than treating trait carriers as a uniformly homogenous population.