Novel Single-cell Measurements Suggest Irreversibly Sickled Cells Are Neither Dense Nor Dehydrated.

Abstract

In sickle cell anemia, deoxygenation causes erythrocytes to distort, while reoxgenation permits them to recover a normal biconcave disk shape. Irreversibly sickled cells (ISCs) cells remain distorted when reoxygenated and have been thought to have among the highest intracellular hemoglobin concentration of the sickle red cell population and therefore the greatest vulnerability to vaso-occlusion. Using a new optical method, which we describe, we have made precise measurements of the intracellular hemoglobin concentration, and intracellular O2 saturation, of ISCs, as well as oxygenated sickle cells with a normal biconcave disc shape, and cells with shapes distorted by the sickle fibers they contain. This method also provides good estimates of cell volumes, and hemoglobin per red cell. The concentration distribution of the ISCs is found to be similar to normal, discoid cells. Average ISC volumes exceed their discoid counterparts, with a much broader distribution, arguing against dehydration as their origin. The concentration distribution of the polymer-laden sickled cells is significantly higher in mean value, and their volume distributions indicate some dehydration. Previous assumptions about ISCs may have thus been colored by the presence of sickle cells that did contain polymer, and that true ISC's may be much more benign than once thought, which underscores the importance of accurate measurement on individual cells. This method could be used to follow changes in individual cell properties under various specific perturbations, and where characterization by flow cytometry is infeasible.