BACKGROUND Red blood cell (RBC) deformability plays a critical role in RBC elongation and viscosity, and when reduced, perfusion of peripheral tissues and oxygen delivery are impaired. Intracellular ATP maintains water and ion homeostasis in RBCs; reduced ATP leads to water and ion loss, dehydration and loss of RBC elasticity. RBC dehydration increases mean corpuscular hemoglobin concentration (MCHC) and also affects distribution of RBC width (RDW). Patients with sickle cell disease (SCD) have reduced RBC ATP, and markedly reduced RBC deformability when compared to healthy individuals. Here, we employed Laser-Optical Rotational Red Cell Analyzer (LORRCA, RR Mechatronics) to evaluate deformability in RBCs from patients with SCD (HbSS) compared to RBCs from sickle cell carriers (HbAS) and ethnic-matched healthy controls (HbAA). We further analyzed how these LORRCA-derived parameters correlate with ATP, MCHC, and RDW. METHODS We enrolled adult subjects (age ≥ 18 years) of African-descent, and not recently transfused (within 8 weeks) under protocol NCT03685721 approved by the NHLBI Institutional Review Board. Fresh whole blood in EDTA was processed on the same day of collection. Cells were counted and diluted in polyvinylpyrrolidone (PVP) solution before undergoing LORRCA assays including deformability, osmoscan, and oxygenscan (HbSS subjects). The deformability assay uses the elongation Index (EI) to measure the cells' ability to undergo deformation with increasing shear stress at 0.95, 3, and 30 Pascals (Pa). The osmoscan assay measures the deformability under continuous osmotic changes (0-600mOsm/kg) and has several parameters: O min (RBC fragility); El max (maximum deformability and membrane flexibility); O max (osmolality where EI max is achieved) and O hyper (intracellular viscosity and maximum deformability in the hypertonic region). The oxygenscan assay measures the oxygen pressure at the time when RBCs start to become rigid (point of sickling). Whole blood levels of ATP were measured using LC-MS/MS with LLOQ at 50.0 μg/mL and converted to intracellular concentrations by dividing by the hematocrit (as a fraction). Descriptive statistics and correlation (Spearman) of ATP, RDW, and MCHC with each LORRCA parameter were performed using R (v4.2.3) and Prism(v9). RESULTS We studied 156 subjects which comprised of 63 HbSS (33 males), 61 HbAS (23 males) and 32 HbAA (8 males) genotypes. The average age in years (range) in HbSS was 32 (18-58); in HbAS, 43 (18-70), and in HbAA, 38 (19-72). As shear stress and osmotic pressure increased, the mean EI and osmolality were significantly (p<0.001, unpaired two-sample t-test) lower in the HbSS cohort compared to HbAS and HbAA (Figure 1). There was no significant difference in the mean EI and osmolality between HbAA and HbAS with increasing shear stresses and osmotic pressures. As shear stress increased, the correlation of deformability with reduced ATP got significantly stronger in the HbSS cohort (r= -0.351 at 0.95 Pa to r=-0.401 at 30 Pa) but the trend was reversed for the HbAS and HbAA cohort (Table 1). Under increasing osmotic pressure, a significant correlation was found between reduced ATP and maximal deformability (EI Max) in HbSS (p=0.008) compared to HbAS (p=0.896) and HbAA (p=0.114). MCHC was significantly correlated with RBC hydration state (O Hyper) in all 3 genotypic groups (HbSS, HbAS and HbAA). Similarly, RDW correlated with maximal deformability (EI Max) under increasing osmotic pressure in all 3 cohorts. In the HbSS subjects, there was a significant positive correlation of ATP and RDW with point of sickling (oxygenscan); MCHC was negatively correlated but was not significant. CONCLUSION RBC deformability was reduced in HbSS patients compared to HbAS and HbAA under increasing shear stress and osmotic pressure. ATP levels showed significant correlation with RBC deformability as measured by LORRCA, particularly in patients with SCD, suggesting increasing ATP levels as an important therapeutic strategy in patients with SCD. In keeping with published data, our analyses showed that RBC volume and size as well as intracellular hemoglobin concentration can affect deformability.
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