Use of ektacytometry to determine red cell susceptibility to oxidative stress

Abstract

To define a more sensitive and reliable method to determine changes in the overall cellular characteristics of erythrocytes after oxidative damage, we used a viscodiffractometric method (ektacytometry) to measure the effect of oxidative stress. Erythrocytes were incubated in the presence of hydrogen peroxide, t-butyl hydroperoxide, or cumene hydroperoxide in phosphate buffer. This treatment resulted in decreased cellular deformability of the intact erythrocytes. In addition, deformability and fragility measurements of the erythrocyte ghost membranes indicated an increased membrane dynamic rigidity and altered-mechanical stability as a consequence of oxidant stress. These changes were observed before the onset of hemolysis. The observed decrease in deformability was accompanied by oxidation of hemoglobin, alterations of membrane proteins, and lipid peroxidation. To continuously measure the time course of the decrease in deformability in intact erythrocytes under oxidative stress, a new ektacytometric method was developed. Erythrocytes were oxidatively challenged within the viscometer at a constant osmolality and shear stress. The change in deformability was monitored and a typical range was defined for erythrocytes from normal individuals. Comparison of erythrocytes from patients with sickle cell disease with those from normal individuals demonstrated a higher susceptibility of sickle red cells toward oxidative stress.