FLOW CYTOMETRIC ANALYSIS OF HUMAN ERYTHROCYTES .1. PROBED WITH LECTINS AND IMMUNOGLOBULINS

A recent review (Aminoff, 1988) summarized the evidence for and against our hypothesis for the role of glycophorin in the senescence and clearance of mammalian red blood cells (RBC) from circulation. This hypothesis postulates the loss of sialic acid from RBC surface in two forms: (a) as vesicles containing the sialoglycoprotein glycophorin, and (b) as free sialic acid residues from glycophorin molecules remaining on cell surface. In this report we demonstrate the applicability of flow cytometric procedures to explore, at the cellular level, time-dependent changes on RBC surface with change in cell size, and with in vivo age. The RBC are probed with fluorescein isothiocyanate (FITC) labelled lectins and goat anti-human-IgG and -IgM. The relative intensity of fluorescence is correlated to the change in RBC size as measured by forward lightscatter. Reactivity of RBC with FITC-labelled wheat germ agglutinin can be inhibited with either 0.2M N-acetylglucosamine or by removal of sialic acid residues with neuraminidase. The properties of the smallest RBC correspond to those of the oldest RBC in their: (a) decreased reactivity with FITC-labelled lectins that recognize sialic acid residues, wheat germ and Limax flavus agglutinins, and (b) increased reactivity with FITC-labelled goat anti-human-IgG and -IgM. These results are compatible with our glycophorin hypothesis. Moreover, they suggest that the initial loss of sialic acid as glycophorin containing vesicles is gradual, while the subsequent step involving the loss of sialic acid residues is rapid and exposes multiple disaccharide galactose beta(1-3)N-acetylgalacosaminyl residues. These unmasked disaccharide sites are recognized by autoimmune IgG, IgM, and lectin-like receptors on macrophages resulting in the clearance of senescent RBC from circulation.