Availability of the inner surface of the red cell membrane to antibodies in digitonin ghosts. Studies on D-negative cells with anti-D.

Studies were done to determine whether antibodies can detect antigens on the inner red cell stromal membrane. When albumin, anti-A, anti-D, or IgG were combined with varying volumes of intact O, Rh-negative red cells (RBCs), these proteins diluted, on the average, to 76 percent of the total volume, which was almost identical to the volume of the supernatant fluid (mean 77%). In contrast, when the protein markers were combined with packed stroma prepared from O, Rh-negative RBCs by digitonin, they diluted, on the average, to 94 percent of the total reaction volume rather than to the volume of the supernatant (mean 70%). Similar dilution was observed in the fluid volume harvested from stromal suspensions by ultracentrifugation, indicating that the protein markers occupied the total fluid volume of the reaction mixture (inside and outside the stroma). Nonspecific adsorption of the protein markers to stroma did not occur since their dilution was unaffected by doubling the stromal volume and since they could be totally recovered in the fluid harvested by ultracentrifugation. These data indicate that antibodies easily traverse the membrane of RBC digitonin stroma but not the membrane of intact RBCs. Therefore, antibodies may be used to detect antigenic determinants on the inner stromal membrane. When anti-D was incubated with Rh-negative stroma, we did not observe consumption of this antibody. Thus, our data did not indicate that the D antigen is present on the cytoplasmic membrane of Rh-negative RBCs.