Sickle cell rheology is determined by polymer fraction–not cell morphology

Abstract

Sickle cell disease pathophysiology is mediated by acute and chronic impairment of cell flexibility due to the formation of intracellular sickle hemoglobin (Hb S) polymer as cells are partially deoxygenated in the microcirculation. We have recently developed a method to measure the relationship between the formation of intracellular polymerized Hb S and cell filtration. In this study, we have used this method to examine whether sickle cell morphology, independent of Hb S polymer fraction, had an effect on cell rheology. We primarily use sickle trait (AS) and Hb S-beta(+)-thalassemia (S-beta(+)-thal) erythrocytes with low hemoglobin F levels, which have normal membranes and few or no dense cells, to remove these confounding effects. We find that the relationship between filtration and the percentages of each "type" of morphological deformation of AS erythrocytes was different from that of the S-beta(+)-thal erythrocytes. In addition, we find that while the filtration of AS erythrocytes as a function of oxygen saturation was similar, whether measured during deoxygenation or reoxygenation, the relationship between the percentages of each type of deformed erythrocyte and oxygen saturation demonstrated hysteresis during oxygenation-deoxygenation experiments. Transmission electron microscopy, for both elongated and irregularly shaped cells, showed that similarly distorted cells could have very different amounts and alignment of polymer. These results suggests that cell morphology per se is not strongly related to filtration, whereas calculated intracellular Hb S polymer fraction predicts loss of filtration of AS and S-beta(+)-thal erythrocytes well. Measured or calculated polymer fraction values would appear to be a better parameter for the study of sickle cell disease pathophysiology and response to treatment than cell morphology studies.

Kinetics of sickle cell biorheology and implications for painful vasoocclusive crisis

“…Shape change was found to be a reliable marker for cell sickling in hypoxia-induced sickled human RBCs at 37°C (36,37), although an earlier study argued otherwise based on experiments done at 24°C (47). Through imaging flow cytometry, the shape change was confirmed to be highly correlated with the existence of intracellular HbS polymers identified by transmission electron microscopy (TEM) in a recent study (37).…”

Effects of short supramaximal exercise on hemorheology in sickle cell trait carriers

“…This type of result has already been observed in previous works (Brandao et al 2003;Monchanin et al 2005;Connes et al 2006, in press). It has been reported that RBCs from SCT carriers and subjects with SCA present impaired filtration in an oxygenated environment even with normal cell morphology, suggesting that cell morphology is not directly related to the abnormal rheological behavior (Hiruma et al 1995). Embury (2004) explained that with the passage from the arterial to the venous circulation substantial numbers of RBCs containing HbS become grossly sickled.…”

Exposure of blood from patients with sickle cell disease to air changes the morphological, oxygen‐binding, and sickling properties of sickled erythrocytes

“…Mozzarelli et al [31] reported the presence of hysteresis between the sickling and unsickling curves of SS cells on the basis of the assumption that unsickling begins at a PO 2 of about 40 mmHg. We [1] and Hiruma et al [30] also showed that the PO 2 -dependent morphological changes of SS cells during oxygenation and deoxygenation significantly differ. We previously reported that the shape of fully deoxygenated SS cells depended on the rate of deoxygenation of oxygenated SS cells [16].…”

“…The existence of hysteresis between the sickling and unsickling curves of SS cells with respect to PO 2 has been reported [24,25,30]. Mozzarelli et al [31] reported the presence of hysteresis between the sickling and unsickling curves of SS cells on the basis of the assumption that unsickling begins at a PO 2 of about 40 mmHg.…”

“…Approximately 5 mg of sodium dithionite was added to this cuvette. An aliquot of the hemolysate was added into the spectrophotometric cuvette in such a way that the concentration of deoxyHb S would be 120% of its solubility (at 30 C), and this solution in the cuvette was quickly warmed to near 30 C. The cuvette was placed in the cuvette holder of a Hitachi UV-Vis spectrophotometer in which the cuvette holder was previously heated to 30 C. Polymerization of deoxy-Hb S was determined by the change in absorption at 700 nm. An aliquot of the hemolysate was transferred into another test tube, and was exposed to air on ice to depolymerize the deoxy-Hb S polymers.…”