The quantification of reticulocyte maturation and neocytolysis in normal and erythropoietin stimulated rats.

ABSTRACT

A technique has recently been proposed for obtaining the reticulocyte (RET) age distribution from the flow cytometric reticulocyte count. It allows for a quantitative characterization of reticulocyte dynamics. In this work this technique was applied to characterize the blood, bone marrow and spleen reticulocytes in homeostatic and erythropoietically stimulated rats in order to determine the reticulocyte maturation times in the bone marrow and blood; and to confirm the presence of ineffective erythropoiesis (neocytolysis). The latter was done by comparing the reticulocyte removal rate from blood with bilirubin formation after erythropoiesis stimulation. A single subcutaneous dose (4050 IU/kg) of recombinant human erythropoietin (rHuEPO) was administered to rats, then their reticulocytes were stained with thiazole orange and the distribution of the fluorescent signal measured using flow cytometry. The obtained signal distribution of the reticulocytes was transformed to the age distribution and a set of basic parameters reflecting reticulocyte dynamics was determined. Bilirubin concentrations were measured to directly assess the presence of reticulocyte irreversible removal. The bilirubin formation was found to be considerably modulated by rHuEPO and corresponded well to the determined reticulocyte removal rate. The initial increase and subsequent decrease of the reticulocyte maturation time in blood was quantitated and directly linked with RET mobilization from the bone marrow. A substantial number (60%) of reticulocytes is sequestrated during homeostasis in rats. This number increases and then decreases after rHuEPO administration, as also reflected by bilirubin formation. Flow cytometry seems to be an excellent method for studying RET dynamics and the presence of young RBC neocytolysis.