Sickling in vitro of reticulocytes from patients with sickle cell disease at venous oxygen tension.

Changes in the degree of sickling in vitro of reticulocytes and nonreticulocytes from patients with sickle cell disease were studied under complete deoxygenation (PO2 = 0 mm Hg) and partial deoxygenation (PO2 = 30 mm Hg, the average PO2 in the venous circulation) conditions at pH 7.4. Degree of sickling was quantitated by image analysis after identification of reticulocytes by acridine orange staining. Sickling in vitro of reticulocytes and nonreticulocytes under complete deoxygenation was similar and relatively unchanged during a 2-hour incubation. In contrast, under partially deoxygenated conditions, at least two populations of reticulocytes were apparent, one more susceptible to sickling than the other; nonreticulocytes were generally less susceptible to sickling. Many of the severely deformed reticulocytes showed formation of long spicules during incubation. These data suggest that a subset of reticulocytes are more susceptible to sickling than nonreticulocytes, and that the degree of reticulocyte sickling in vitro increases dramatically with time even at constant partial oxygen pressures observed in the venous circulation. Since dehydration in sickled reticulocytes seemed to be proceeding, mechanisms of inhibition were also examined. We found that quinine, an inhibitor of the Ca(++)-activated K+ efflux, inhibited part of the reticulocyte sickling while okadaic acid, a K(+)-Cl- co-transport inhibitor, did not inhibit sickling under our experimental conditions. These phenomena observed at pH and oxygen tension similar to physiological venous conditions may be important in understanding the clinical course and pathophysiology of sickle cell disease.