Effect of cell shape on extravascular hemolysis

Although it has been inferred that alteration of erythrocyte shape causes cell clearance, no direct measure of the effect of varying shape on clearance has been made. In this study, the protein oxidant, diamide, was employed to fix rat erythrocytes in altered shapes produced by the addition of different amphipaths or by changes in membrane structure. Clearance of cells in vivo as measured by ⁵¹Cr uptake in the spleen and liver correlated with the extent of shape alteration, either crenation or cupping. Whereas only 5%-10% of discoid cells were cleared by the spleen after 1 hr, there was 50%-80% clearance of severe echinocytes (crenated cells) and severe stomatocytes (cupped cells) primarily by the liver. This was not an effect of the crenating or cupping agents, since they were washed away prior to injection, and treatment of cells with the agents alone did not cause clearance. Further, if cells were crenated by ATP depletion through incubation without glucose for 12-16 hr, then conversion of their shape to a discoid form reduced clearance. To determine if serum components might be involved in the clearance, shape-altered cells were incubated in vitro in autologous plasma. With severe stomatocytes but not severe echinocytes, the binding of IgG was increased by 3-8 fold over controls, whereas no differences in C3 binding were observed. It is concluded that in diamide-fixed cells, the alteration of shape from a biconcave disc will increase in vivo clearance and, in the case of stomatocytes, the binding of IgG. Because diamide treatment alone altered the deformability properties of the discs, we suggest that the clearance of shape-altered cells results from their altered membrane surface properties rather than mechanical factors.