Quantifying hemolytic response in sickle cell disease blood to mechanical and hypoxic stressors

Abstract Introduction Sickle cell disease (SCD) leads to ongoing destruction of red blood cells (RBCs) within blood vessels, driven by the fragility and abnormal flow properties of sickle RBCs. Pinpointing how hypoxia and mechanical forces (blood flow shear) contribute to this hemolysis is crucial for advancing diagnostics and treatments. Here, we introduce SickleFlow, a standardized in vitro platform utilizing a 30-gauge needle to simulate physiological and pathological shear rates (5,000 to 10,000 s⁻¹) encountered by RBCs in the microcirculation. The primary goal of this study is quantifying the relative contributions of mechanical shear, hypoxia, and osmotic stress to hemolysis in SCD and healthy blood samples. Methods We collected whole blood from SCD patients and healthy donors and standardized it to a 20% hematocrit. Mechanical shear stress was applied by flowing the blood through the SickleFlow system at controlled rates. Hypoxic stress was induced by deoxygenating the blood with a 23 mM sodium metabisulfite solution to simulate physiologic hypoxia. To evaluate osmotic stress, we dehydrated RBCs using phosphate-buffered saline (PBS) at elevated osmolarities (700 and 1,000 mOsm/kg). Hemolysis was measured by spectrophotometric detection of free hemoglobin in plasma, and we calculated a hemolysis index (HI) as the ratio of free hemoglobin to total hemoglobin, adjusted for hematocrit (multiplied by 1 - hematocrit) for consistent comparisons. Results and Discussion SCD blood samples exhibited markedly higher hemolysis under hypoxic and osmotic stresses compared to healthy controls (n=6 for each group). Hypoxia emerged as the key trigger for hemolysis, with SCD samples showing greater vulnerability than healthy ones. Under normal oxygen levels (normoxia), SCD samples had an average HI of 0.07, more than double the 0.033 in healthy samples (p0.05), highlighting inherent RBC fragility in SCD. Deoxygenation amplified hemolysis in both groups, but the rise was steeper in SCD (p0.01). Under hypoxia, average HI values were similar with or without shear flow: 0.262 (flow) vs. 0.285 (stationary) for SCD (p=0.33), and 0.240 (flow) vs. 0.230 (stationary) for healthy samples (p=0.63). This result suggests that shear alone does not significantly worsen hemolysis. When hypoxic shear conditions were linked to clinical markers like lactate dehydrogenase (LDH), a strong positive correlation emerged (R²=0.78, p0.05). For instance, a patient with high LDH (600 U/L) displayed intensified hemolysis in SickleFlow, severe pain (score 6/10), and acute anemia (hemoglobin 6 g/dL). Patients with moderate LDH (400 U/L) showed intermediate hemolysis, while those with lower LDH (250 U/L) had minimal hemolysis in SickleFlow. These findings indicate that our hypoxic shear model can mirror real-world hemolytic risk, correlating with LDH levels to potentially predict disease severity and guide monitoring. Dehydration further intensified hemolysis in deoxygenated SCD samples at both osmolarities (HI=0.079 at 700 mOsm/kg, p=0.009; HI=0.097 at 1,000 mOsm/kg, p=0.003), but had little effect under oxygenated conditions. This result underscores how everyday factors like dehydration can exacerbate crises in low-oxygen environments. Conclusion This study highlights hypoxia as the dominant driver of hemolysis in SCD, with dehydration amplifying hemolysis risk. The integration of shear and hypoxic stresses in our model correlates with clinical indicators, including LDH, pain, and anemia, offering a tool to potentially assess individual hemolytic susceptibility. The SickleFlow platform provides a reliable way to simulate mechanohypoxic stresses, yielding insights that could personalize therapies and inspire new interventions to reduce hemolytic complications and improve patient outcomes.