Rapid and reproducible characterization of sickling during automated deoxygenation in sickle cell disease patients

Abstract

In sickle cell disease (SCD), sickle hemoglobin (HbS) polymerizes upon deoxygenation, resulting in sickling of red blood cells (RBCs). These sickled RBCs have strongly reduced deformability, leading to vaso‐occlusive crises and chronic hemolytic anemia. To date, there are no reliable laboratory parameters or assays capable of predicting disease severity or monitoring treatment effects. We here report on the oxygenscan, a newly developed method to measure RBC deformability (expressed as Elongation Index ‐ EI) as a function of pO2. Upon a standardized, 22 minute, automated cycle of deoxygenation (pO2 median 16 mmHg ± 0.17) and reoxygenation, a number of clinically relevant parameters are produced in a highly reproducible manner (coefficients of variation <5%). In particular, physiological modulators of oxygen affinity, such as, pH and 2,3‐diphosphoglycerate showed a significant correlation (respectively R = ‑0.993 and R = 0.980) with Point of Sickling (PoS5%), which is defined as the pO2 where a 5% decrease in EI is observed during deoxygenation. Furthermore, in vitro treatment with antisickling agents, including GBT440, which alter the oxygen affinity of hemoglobin, caused a reproducible left‐shift of the PoS, indicating improved deformability at lower oxygen tensions. When RBCs from 21 SCD patients were analyzed, we observed a significantly higher PoS in untreated homozygous SCD patients compared to treated patients and other genotypes. We conclude that the oxygenscan is a state‐of‐the‐art technique that allows for rapid analysis of sickling behavior in SCD patients. The method is promising for personalized treatment, development of new treatment strategies and could have potential in prediction of complications.

Characterization of Sickling During Controlled Automated Deoxygenation with Oxygen Gradient Ektacytometry

“…Both EI values are measured at a pO 2 of 100-120 mmHg. This parameter reflects the capacity of RBCs that sickle during deoxygenation to reverse sickling during reoxygenation 22 . Parameters from duplicate measurements generally had a coefficient of variation (CV) <5% (median 1.83%).…”

“…This ensures better discrimination between the deoxygenation and reoxygenation curves and, hence, a better interpretation of the curve. This figure has been modified from Rab et al 22 Please click here to view a larger version of this figure. represents the baseline position and shows an overall deformability of the total RBC population.…”

“…To aid in the interpretation, all data points were connected in every individual experiment by a line to graphically present the results. This figure has been modified from Rab et al 22 Please click here to view a larger version of this figure.…”

“…Several well-known factors influence oxygen gradient ektacytometry parameters, namely pH, temperature, and osmolarity. Ektacytometry, especially PoS, is influenced by 2,3-diphosphoglycerate (2,3-DPG) 22 . Also, there is a clear correlation between %HbF and the EI min , and to a lesser extent PoS (Figure 5A-D).…”

Phytocomponents from Anacardium occidentale, Psidium guajava, and Terminalia catappa altered membrane osmotic stability of sickle erythrocytes

“…Accordingly, osmotic fragility index defines the capacity of the erythrocytes to withstand hemolysis when subjected to osmotic stress [27]. Osmotic fragility test, in which the population of hemolyzed erythrocytes is measured as a function of variability in osmolality of erythrocyte suspension, is primarily diagnostic of hereditary disorders of erythrocyte membrane cytoskeletal structures such as hereditary spherocytosis and other vast groups of the hemolytic anemias [27][28][29][30][31][32]. The extrinsic and intrinsic factors that influence erythrocyte osmotic fragility index are summarized elsewhere [27,30].…”