Complement activation by human red blood cell antibodies: hemolytic potential of antibodies and efficacy of complement inhibitors assessed by a sensitive flow cytometric assay

BACKGROUND: Therapeutic intervention strategies in complement-mediated hemolytic diseases are still inappropriate, and lethal events cannot be reliably prevented. As an in vitro model of intravascular hemolysis, a sensitive flow cytometric assay was designed using red blood cells (RBCs) of patients with paroxysmal nocturnal hemoglobinuria (PNH) as target cells. Complement activation by human allo- and autoantibodies directed against RBC antigens and the effect of different complement inhibitors were studied. STUDY DESIGN AND METHODS: RBCs of patients with a PNH III RBC clone of more than 20% were coated with different human allo- or autoantibodies. Hemolysis was initiated with pooled normal human AB serum with or without the addition of complement inhibitors. Loss of PNH III RBCs was estimated by flow cytometry. RESULTS: RBC antibodies of 174 different patients representing 37 different specificities were tested for their potency to activate complement. In correlation with blood group specificities roughly three different patterns were observed: 1) strong and regular, 2) sporadic, and 3) weak or absent complement activation. Remarkably strong complement activators were among antibodies directed against high-prevalence blood group antigens. The C5 inhibitor eculizumab abrogated mild but not strong complement activation, even in presence of excess inhibitor. However, this residual complement activity could be further depressed by combining eculizumab with other inhibitors. CONCLUSION: The PNH hemolysis assay offers a sensitive tool for in vitro analyses of classical pathway mediated complement activation. The recognition of additive effects of complement inhibitors may guide novel intervention strategies against unwanted complement damage.