Quantitation of protein 3 content of circulating erythrocytes at the single-cell level

Abstract

The density and size of human erythrocytes has been roughly correlated with cell age, with the denser and smaller cells being older. Observations of this type have led to a hypothesis that the membranes of circulating erythrocytes are dynamic with respect to composition and that material is lost from the membrane during cell maturation and circulation. In this study, flow cytofluorimetry was used to investigate the distribution of the human erythrocyte anion transport protein (protein 3) in heterogeneous samples of circulating red cells. We verified that protein 3 can be specifically and quantitatively labeled in intact human erythrocytes with eosin-5-maleimide, a luminescent probe. Individual cells were accordingly analyzed for size by forward light scattering and for protein 3 content by quantitation of eosin fluorescence. Initial results indicated that the smallest erythrocytes had a protein 3 content equal to that of the largest circulating erythrocytes. This result was independently verified by light scatter-activated cell sorting; direct measurement of cell diameters by microscopy verified that the cell sizes of erythrocytes showing the 10% greatest and 10% smallest light-scattering signal were indeed distinct. Independent analysis of the size-sorted erythrocytes for protein 3 content was accomplished by gel electrophoresis of stroma from 150,000 large and small erythrocytes. Quantitative scanning densitometry of silver-stained gels of prepared stroma showed that protein 3 content of each set of fractionated cells was equal and did not vary as a function of cell size. Taken in combination with the reported correlation between increasing red blood cell age and decreasing cell size, these results indicate that any loss of membranous material during the cell aging process is not random.

Rapid flow cytometric test for the diagnosis of membrane cytoskeleton‐associated haemolytic anaemia

“…Eosin‐5‐maleimide binds stochiometrically to band 3 on intact red cells (Jennings et al , 1985). Thus, a reduction of fluorescence intensity is assumed to indicate a quantitative reduction of erythrocyte band 3 (Jarolim et al , 1994).…”

Flow cytometric analysis of human erythrocytes: I. Probed with lectins and immunoglobulins

“…FCP could be applied to detect differences between young and senescent RBC taking advantage of differences in their cell size. Some pioneers did attempt to correlate changes in cell surface parameters with change in cell size (Jennings et al, 1985;Dockter et al, 1986). Fluorescently labelled lectins also had been used to detect specific sugars on lymphocytes (Stein et al, 1973;Wiig et al, 1973;Kraemer et al, 1973;Steinkamp et al, 1974).…”

Evaluation of eosin‐5‐maleimide flow cytometric test in diagnosis of hereditary spherocytosis

“…Estimates ranging from 75 to 95% of EMA fluorescence were found to be associated with band 3 by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) (Nigg & Cherry, 1979;Jennings, Brown & Dockter, 1985) and the rest contributed by Kell GP, CD47, RhAG and the Rh blood group proteins of which the relative content of CD47, RhAG and the Rh blood group proteins was lower in HS than normal red cells (King, Smythe & Mushens, 2004). Thus, binding of EMA to the integral proteins (band 3, RhAG, CD47 and Rh proteins) implicated in close association with the red cell cytoskeleton underpins the high specificity of the flow cytometric test for HS (King, Smythe & Mushens, 2004).…”