Density ultracentrifugation of sickle cells during and after pain crisis: increased dense echinocytes in crisis.Blood 1984; 64(2):507-15Blood
An increase in the number of irreversibly sickled cells (ISCs) in pain crisis has been found by some investigators but not others. We have used the technique of discontinuous arabinogalactan density-gradient ultracentrifugation of whole blood to study ISCs from patients with sickle cell anemia (SCA) during pain crisis and again when pain free (5-331 days after crisis). Nine patients have been studied through ten episodes of pain crisis. Five layers with densities from 1.128 g/mL to 1.158 g/mL have been used. Careful classification of the cells using Nomarsky optics demonstrated highly significant changes occurring in the layers of the gradient. The changes involve the appearance of an increased percentage of echinocytic ISCs and echinocytic cells that were not ISCs, especially in the denser gradient layers, during crisis, and their replacement by normal-appearing discocytes in the pain-free state. There was no change in ISCs that were not echinocytic. Data collected previously demonstrated that reduced glutathione activity correlated with increased echinocytosis in our gradient layers. This indicates that the echinocytic change may occur as a result of oxidant stress. Hemoglobin F levels and the percentage of hemoglobin F from reticulocytes showed no consistent change to coincide with the rise in normal-appearing discocytes in the lightest layers after crisis. Our data indicate that pain crisis occurs in association with an echinocytic change, which may be induced by oxidant injury. The rise in normal-appearing cells after crisis may reflect increased hemoglobin F production in some patients but mainly relates to the disappearance of these echinocytic erythrocytes.