Sickling times of individual erythrocytes at zero PO2

Abstract

A rapid reaction parallel plate flow channel was used to study the kinetics of erythrocyte sickling upon sudden deoxygenation with sodium dithionite. The erythrocytes were recorded on 16 mm film or video tape and visually tracked in time. Sickling was identified by morphologic criteria. At the flow rate used in these studies, the rate of sickling was a reaction limited process. There was no loss of cellular deformability or membrane flicker before the onset of sickling. Typical sickling times for sickle (SS) cells and trait (AS) cells at room temperature in isotonic buffer were 2.0 and 70 s, respectively. Increasing the buffer osmolality resulted in shorter sickler times and under hypotonic conditions the time required for sickling was prolonged. Between pH 6.4 and 7.0 there was little change in the time required for 50% of the originally discoidal cells to sickle (t[50]); whereas a marked increased in t(50) occurred between pH 7.4 and 7.6. Whole populations of AS and SS erythrocytes were separated into 3 fractions after centrifugation. The t(50) of the fractions progressively decreased from top to bottom, which paralleled an increase in mean corpuscular hemoglobin concentration (MCHC). The t(50) decreased as the temperature was increased from 13° to 34°C. This temperature effect was more pronounced for cells that had osmotically induced reductions in MCHC. A 2 step process for erythrocyte sickling is proposed: an initial lag phase, during which there is little or no change in internal viscosity, followed by a rapid phase of cellular deformation. The lag phase is altered by changes in MCHC, pH, and temperature.