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

Abstract

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.

Flow cytometric analysis of human erythrocytes: II. Possible identification of senescent RBC with fluorescently labelled wheat germ agglutinin

“…Dockter et al (1986) andJennings et al (1985) previously established MCF/FLS (mean channel of fluorescence vs. forward lightscatter plot) as a useful means of following changes in RBC surface markers with change in cell size. We confirmed its usefulness in our previous study using FITC-labelled WGA, GAH-IgG, and GAH-IgM and the Coulter Epics 541 flow cytometer (Gutowski et al, 1991). In this study, using the BD FACScan, we again confirm the utility of this analytical method despite greater instrument sensitivity to changes in cell shape.…”

“…In our previous report (Gutowski et al, 1991) we, like Dockter et al (1986) and Jennings et al (1985), found that RBC could be analyzed in isotonic condition in the Coulter Epics. Furthermore, we demonstrated that smallest and largest cells did show the properties anticipated for oldest and youngest RBC in their reactivity with FITC-WGA and FITC-labelled goat-anti-humanIgG, and-IgM.…”

“…THE ~RST PAPER of a series we explored the application of the Coulter Epics 541 flow cytometer to follow changes in red blood cells (RBC) at the cellular level as they age in (Gutowski et al, 1991). The data obtained supported our hypothesis, that glycophorin or a glycophorin-like molecule on the surface of RBC undergoes time-dependent desialylation in vivo (Aminoff, 1988).…”

“…In the Epics 541, RBC were examined under isotonic conditions (Gutowski et al, 1991). In contrast, the FACScan clearly viewed the flow of ellipsoid, biconcave RBC as essentially two populations of cells under isotonic conditions (276 mOsmol) (Fig.…”

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Abstract --In the first paper of a series (Gutowski, et al, 1991) we discussed the use of flow cytometry to follow at the cellular level the aging of red blood cells (RBC) in circulation, using fluorescently labelled lectins and goat anti-human-IgG and -IgM. The Coulter Epics 541 was used for those studies.

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