Sickling times of individual erythrocytes at zero Po2

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 sickling 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 (t50); whereas a marked increase in t50 occurred between pH 7.4 and 7.6. Whole populations of AS and SS erythrocytes were separated into three fractions after centrifugation. The t50 of the fractions progressively decreased from top to bottom, which paralleled an increase in mean corpuscular hemoglobin concentration (MCHC). The t50 decreased as the temperature was increased from 13 degrees to 34 degrees C. This temperature effect was more pronounced for cells that had osmotically induced reductions in MCHC. A two-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.