Accelerated removal of antibody-coated red blood cells from the circulation is accurately tracked by a biotin label

Red blood cells (RBCs) can be removed from the bloodstream at an accelerated rate by antibody-mediated mechanisms in a number of immunologic conditions including the following: 1) autoimmune hemolytic anemias, 2) transfusion of incompatible allogeneic RBCs into a recipient whose blood contains a corresponding alloantibody, 3) transfusion of passive antibodies into a recipient whose RBCs express the cognate antigen, 4) transplacental transfer of maternal allogeneic RBC antibodies into a fetus with RBCs expressing the cognate antigen resulting in alloimmune hemolytic disease of the fetus and newborn, and 5) posttransplantation transfer of viable donor lymphocytes capable of producing antibodies directed against recipient RBCs (e.g., group O donor into group A recipient). When caring for patients with these disorders, having an accurate and safe method for determining in vivo RBC kinetics would be of considerable value for establishing the diagnosis, defining the pathophysiology, and assessing the response to therapy. The availability of RBC survival (RCS) methods that address safety issues (e.g., avoidance of radioactivity) is particularly important for vulnerable patient populations including fetuses, neonates, children, and pregnant women. Direct assessment of RCS requires the ability to distinguish cohort-labeled RBCs from the remaining RBCs in the circulation and to express their number as the relative proportion of labeled RBCs to total RBCs in that blood sample. The internationally recognized reference method for measuring RCS uses autologous or allogeneic RBCs labeled with chromium 51 (51Cr) and is based on concentration of 51Cr per unit volume of blood.1 Because transfusion of 51Cr-labeled RBCs exposes the recipient to radiation, this approach is viewed as unacceptable for research studies in the human fetus, neonate, and pregnant woman. Our laboratory and others2-4 have developed methods for measuring RCS in humans based on labeling of RBCs with biotin at one5-10 or more11 densities. Here the term density refers to biotin labels per RBC and is determined by our selection of the concentration of biotinylating reagent per milliliter of RBCs in the biotinylation reaction mixture. Biotin-labeled RBCs (BioRBCs) are quickly and easily enumerated by flow cytometry.11 The biotin RBC method has the following safety and technical advantages over the standard 51Cr method: 1) study subjects including vulnerable populations are not exposed to radiation; 2) small blood volumes (e.g., 20 μL) are required for monitoring; and 3) multiple, independent RCS measurements can be made simultaneously in the same individual by labeling RBCs at more than one biotin density. The aim of this study was to determine whether RBCs labeled with biotin at a single density can be used to accurately measure shortened RCS of RBCs experimentally coated (opsonized) with antibody simulating the situation present in immune-mediated hemolytic anemia. We hypothesized that short-term and long-term RCS measured using BioRBCs would agree with RCS determined using RBCs labeled with 51Cr.