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Autoradiographic estimation of red cell D antigen content using 125I-anti-D and 125I membrane labeling.

The D antigen content of red cells in the venous circulation of normal adult donors was estimated autoradiographically with 125I–anti-D. The distribution of silver grains on individual cells deviated significantly from a Poisson distribution, indicating that there was nonuniform labeling. Neuraminidase and papain modification of red cells prior to antibody binding resulted in increased anti-D binding and a labeling pattern that more closely approximated the Poisson, demonstrating that part of the labeling heterogeneity was associated with membrane properties affecting antibody binding. Membrane iodination of intact red cells with lactoperoxidase, which served as a nonantibody membrane label, produced a labeling pattern indistinguishable from that found with immunologically bound anti-D. Membrane iodination with iodine monochloride produced a significantly different labeling pattern with a large proportion of unlabeled and heavily labeled cells. Neuraminidase and papain modification prior to membrane iodination did not produce an increase in the quantity of 125I–incorporated into the membrane nor did it significantly change the non-Poisson grain count distribution. Anti-D labeling heterogeneity was associated with immunologic and technical factors associated with autoradiography. The results, however, could not conclusively rule out differences in intrinsic D antigen content of circulating red cells.