Biologic Assessment of RBC Biology and Neutrophil Activation: Correlation with Sickle Cell Disease Activity

Sickle cell disease (SCD) has pleiomorphic effects on affected individuals, most typically including both hemolysis and vaso-occlusive events (VOE). However, previous reports have suggested that these two paradigmatic presentations of SCD represent two poles of a spectrum of disease activity, so that patients with the highest frequency of VOEs are at one end, and patients with high hemolytic rates and organ damage, such as nephropathy and pulmonary hypertension, are at the other end. We performed an observational study of erythroid (RBC) factors, neutrophil activation, and clinical parameters, including vaso-occlusion event (VOE) frequency, in order to determine which factors were related to each other and to either hemolytic rate or VOE frequency. 115 patients in steady state, at least 3 weeks after last VOE, with either HbSS, HbSß +/0 thalassemia, or HbS/HPFH, were enrolled from 10/20/2020 - 10/25/2022. Erythrocytes and plasma were isolated from whole blood and analyzed for RBC deformability, intracellular RBC [ATP], RBC phosphatidylserine exposure, and plasma matrix metallopeptidase 9 (MMP9), a product of neutrophil activation and degranulation. A subset of samples were examined for RBC reactive oxygen species (ROS) content and adhesivity to laminin with and without stimulation by epinephrine. Clinical data collected included complete blood count parameters, LDH, total bilirubin, reticulocyte (retic) counts, and health care utilization for VOE in the preceding year. Deformability was measured as maximal elongation index (EImax) at Pa=30 by laser optical rotational red cell analyzer (Lorrca). RBC ROS was quantitated using 2′,7′-dichlorofluorescein and expressed as nM/mg Hb. MMP9 was measured by commercial ELISA assay. We found (N=102 with complete data) that EImax was directly proportional to total [Hb] (P