FLOW CYTOMETRIC ANALYSIS OF HUMAN ERYTHROCYTES .2. POSSIBLE IDENTIFICATION OF SENESCENT RBC WITH FLUORESCENTLY LABELED WHEAT-GERM-AGGLUTININ

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. In this report we describe more extensive experiments using the Becton-Dickinson FACScan flow cytometer, and compare the results with those obtained with the Coulter Epics 541. By changing sample conditions from isotonic to hypotonic, compensation for differences of the two instruments was accomplished. We confirmed our previous observations that RBC react very strongly with fluorescein isothiocyanate labelled wheat germ agglutinin (FITC-WGA) and that there is little change in the intensity of fluorescence given by RBC of all sizes with the exception of the smallest. Reactivity with FITC-WGA is markedly decreased in the presence of competitive inhibitors of sialic acid or upon enzymatic removal of sialic acid from RBC. Removal of sialic acid is accompanied by increased reaction with peanut agglutinin (FITC-PNA). Flow cytometry was also used to monitor the enrichment of a population of smallest RBC (less than 0.05%), isolated from both counterflow centrifugation and the interface obtained from Histopaque separation. These smallest RBC showed low reactivity with FITC-WGA and higher binding of FITC-goat-anti-human-IgG, and -IgM, and therefore represent the most senescent RBC, just prior to their clearance from circulation by the reticuloendothelial system. These observations are in compliance with the hypothesis that physiological desialylation of glycophorin is responsible for clearance of senescent RBC from circulation (Aminoff, 1988).