Longer in vivo survival of CD59- and decay-accelerating factor-almost normal positive and partly positive erythrocytes in paroxysmal nocturnal hemoglobinuria as compared with negative erythrocytes: a demonstration by differential centrifugation and flow cytometry

Three populations of erythrocytes have been shown by flow cytometric analysis on complement regulatory proteins: CD59 and decay-accelerating factor (DAF) on erythrocytes in paroxysmal nocturnal hemoglobinuria (PNH). CD59 and DAF in PNH may be completely deficient in CD59- and DAF- negative erythrocytes, they may be decreased varyingly in partly positive erythrocytes, and they may be approximately normal in almost normal positive erythrocytes. Control erythrocytes are always CD59- and DAF-normal positive. CD59- and DAF-negative erythrocytes have been shown to be most sensitive to complement lysis in vitro. However, it has not yet been elucidated whether CD59- and DAF-almost normal positive and partly positive erythrocytes in a patient have a longer in vivo survival than negative erythrocytes. Blood from controls and PNH patients was separated in five fractions by differential centrifugation. CD59 and DAF on the fractionated erythrocytes were determined by flow cytometry using specific antibodies. Ratios of CD59- and DAF-almost normal positive and partly positive cells to negative erythrocytes were increased progressively from the top fraction to the bottom. The erythrocytes in the top fraction are younger and reticulocyte-rich, while those in the bottom are older and reticulocyte- poor. Hence, the present results indicate that CD59- and DAF-partly positive erythrocytes as well as almost normal positive erythrocytes in patients may have a longer in vivo survival than negative erythrocytes.