Reproducible in vitro preparation of intermediate C3d coated red blood cells

As a standardizing reagent and/or as a positive control for the anti-C3d reactivity of antiglobulin reagents, test red blood cells (RBC) reproducibly coated with "C3d only" (i.e. lacking other complement components and immunoglobulins) are essential. We have prepared RBC coated by intermediate amounts of C3d. Two approaches to varying the amount of C3d bound to RBC were studied: a) variation in Mg++ concentration and b) dilution of donor plasma. The amount of C3d bound to RBC was assessed both by agglutination reactions with serial dilutions of a standard anti-C3d serum and by quantitation of bound anti-C3d with 125I-labeled anti-antiglobulin serum. Marked individual donor differences were encountered in response to varying Mg++ concentration and to dilution of donor plasma; no single set of conditions could be employed to produce a desired intermediate C3d-coated RBC from all donors. Examples of variations to be expected with both manipulations are illustrated, along with studies of conditions under which the standard deviation for bound C3d on intermediate C3d-coated RBC made from 5-donor pools was less than 10%.