Analysis of Red Blood Cells Oxidative Stress Susceptibility in Sickle Cell Disease Variants Using the Roxyscan

Background: Red blood cells (RBCs) are biconcave-shaped cells with viscoelastic membranes optimally adapted for oxygen delivery and gas exchange. RBCs have the antioxidant capacity to protect against reactive oxygen species (ROS). If this capacity is exceeded, damage can be inflicted, which disrupts cellular deformability, structure, and membrane properties. Sickle cell disease (SCD) is a monogenetic disorder characterized by RBCs that deform from biconcave to typically sickle-shaped upon deoxygenation, leading to reduced deformability. SCD-RBCs suffer from high levels of oxidative stress, which may exhaust their physiological antioxidant capacity. Therefore, there is a need for biomarkers to evaluate differences in withstanding oxidative stress among patients and to monitor treatment response.

Aim: To evaluate the key parameters of RoxyScan as novel biomarkers that reflect the ability of SCD RBCs to withstand oxidative stress.

Methods: Patients with HbSS, HbSβ0, HbSβ+, or HbSC were eligible to participate in the study. Patients who received blood transfusions <3 months before blood collection were excluded. RoxyScan was used to assess susceptibility to oxidative stress. This novel application of Lorrca (RR Mechatronics, Zwaag) measures the deformability of RBCs in response to exposure to cumene hydroperoxide (CHP, 90µM) during continuous shear (30 Pa). T-POD was defined as the time (seconds) required to reach a 10% decrease in deformability (expressed as EI, Elongation Index), calculated from a fitted curve. EIMin was characterized by the minimum elongation index reached during the assay. RoxyScan parameters were correlated to RBC sickling tendency (Point of Sickling, PoS, assessed by oxygen gradient ektacytometry), complete blood count (Cell Dyn Sapphire, Abbott), hemolysis markers and hemoglobin subfractions (HbF/HbS/HbSC) (HPLC, Tosoh G8). Additionally, ex vivo treatment with l-glutamine (2mM, 1h) was performed and evaluated using RoxyScan.

Results: Fifty adult SCD patients (HbSS/Sβ0, N=26; HbSC, N=20; HbSβ+ N=4) and 21 healthy controls (HC) were included. HbSS/Sβ0 and HbSC RBCs showed a significantly lower T-POD than HC, indicating a faster deformability loss in response to the same level of oxidant exposure (HC 1739s (SD 238s), HbSS/Sβ0 776s (SD 328s), HbSC: 1031s (SD 225s)). HbSC RBCs showed substantially more pronounced homogeneity in the response. When correlating T-POD to hemolysis parameters in HbSS/Sβ0, a trend of inverse correlation with reticulocyte count (p=0,09), lactate dehydrogenase (LDH, p=0,10), and bilirubin (p=0,068) was found. Furthermore, T-POD was significantly correlated with PoS in HbSS/Sβ0 patients, showing a direct link between RBC sickling tendency and ability to withstand oxidative stress. No significant T-POD correlations with sickling and hemolysis parameters were observed in patients with HbSC. Ex vivo treatment with l-glutamine showed a significant improvement in T-POD and EIMin despite substantial variation in individual patient responses.

Conclusion: In this study, we demonstrated the applicability of RoxyScan as a novel method for assessing the susceptibility of RBCs to oxidative stress in SCD. Patients with HbSS/Sβ0 and HbSC showed an earlier and more pronounced loss of deformability than HC did. Furthermore, correlations between T-POD and laboratory parameters in HbSS/Sβ0 patients compared to HbSC patients demonstrated different characteristics in terms of oxidative stress. The results of l-glutamine experiments showed the benefit of RoxyScan in evaluating treatment response and could help to identify patients benefiting from treatment with l-glutamine. Future studies are needed to explore the clinical applicability of RoxyScan.

van Wijk:RR Mechatronics: Consultancy; Pfizer: Research Funding; Agios Pharmaceuticals: Research Funding. Traets:Agios Pharmaceuticals: Research Funding. Van Beers:Agios Pharmaceuticals, Inc.: Consultancy, Research Funding. Rab:Agios Pharmaceuticals: Research Funding; RR Mechatronics: Research Funding. Rijneveld:Vertex: Other: Advisory board.