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.

Identification of F-reticulocytes by two-stage fluorescence image cytometry.

Precise determination of reticulocytes (young red blood cells) containing fetal hemoglobin (Hb F), F-reticulocytes, is important for assessment of the efficacy of drugs such as hydroxyurea and butyrate in elevating the levels of Hb F in patients with sickle cell disease (SCD) and beta-thalassemia. We developed a reliable and easily applicable method for determining F-reticulocytes using fluorescence image cytometry. Reticulocytes were first identified by preparing a monolayer smear of blood stained by acridine orange. Images of reticulocytes and of all cells were obtained for a selected area on the smear. After removing the acridine orange, cells containing Hb F (F-cells) in the same area were then identified by immunofluorescence. Using images of F-cells, reticulocytes, and all cells for the same fields it was possible to identify F-reticulocytes. To assess the validity of our two-stage staining method, we compared our results with those obtained by traditional methods. There was significant correlation of our method with the conventional immunofluorescence staining method for F-cells (r2 = 0.99; slope = 0.99) and with the accepted brilliant cresyl blue method for reticulocytes (r2 = 0.97; slope = 0.96). Heretofore, the ability to determine F-reticulocyte levels has been limited to a small number of laboratories possessing special equipment and techniques. The method presented here should be of great interest to many basic science and clinical investigators involved in studies evaluating synthesis of Hb F.

Survival of F-reticulocytes in sickle cell disease.

Fetal hemoglobin, Hb F, is known to be an important factor for clinical course of sickle cell disease, as it suppresses polymerization of sickle hemoglobin. To investigate the effect of Hb F on the survival of sickle reticulocytes (young red cells) in circulation, Hb F levels in individual reticulocytes and mature erythrocytes were quantified via fluorescence image cytometry. We first examined unfractionated SS cells from 3 patients with different Hb F levels, and found that Hb F levels in reticulocyte populations were always lower than those in erythrocyte populations. This suggests that subsets of reticulocytes with lower Hb F levels are removed during maturation while those with higher Hb F levels tend to survive to become erythrocytes. The distribution of Hb F in reticulocytes was different among these patients and seems to strongly affect the survival of F-reticulocytes. We also analyzed density-separated fractions, and found that Hb F levels in reticulocytes found in the densest fraction were lower than those in lighter fractions. This suggests that reticulocytes with lower Hb F levels are susceptible to quick dehydration within their maturation period (1-2 days) in circulation.

Estimation of fetal hemoglobin levels in individual red cells via fluorescence image cytometry.

A method for estimating fetal hemoglobin (Hb F) levels in individual red blood cells was developed. Cell smears were prepared using a slide maker to ensure uniform thickness and were then stained with immunofluorescence. An antifading gel was applied to preserve a stable fluorescence. The total fluorescence intensities from the same number of red cells in different slide specimens correlated with their hemolysate Hb F levels, which were determined via column chromatography (R = 0.95). Hb F level in individual cells was estimated from fluorescence intensity and cell area, which were determined via image analysis techniques and the hemolysate Hb F level. Blood from a normal subject, a subject with hereditary persistence of fetal hemoglobin, and from sickle cell patients with varying Hb F levels was analyzed. Our analyses showed a wide distribution of Hb F among cells for the normal subject and a gaussian distribution with a peak at the hemolysate Hb F level for the subject with hereditary persistence of fetal hemoglobin. The Hb F distributions were unique to the patients with sickle cell disease. Because Hb F level in individual sickle cells is crucial to the inhibition of cell sickling, the unique hb F distribution may be important in determining the clinical course of this disease.