title: Methods for quantitative detection of antibody-induced complement activation on red blood cells.
abstract: Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal(1). Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis(1-4). However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.
authors: Department of Immunopathology, Sanquin Research and Landsteiner Laboratory Academic Medical Center, University of Amsterdam.
authors: Elisabeth M
authors: EM
authors: Meulenbroek
authors: Department of Immunopathology, Sanquin Research and Landsteiner Laboratory Academic Medical Center, University of Amsterdam.
authors: Diana
authors: D
authors: Wouters
authors: Department of Immunopathology, Sanquin Research and Landsteiner Laboratory Academic Medical Center, University of Amsterdam.|Department of Hematology, Academic Medical Center, University of Amsterdam; s.s.zeerleder@amc.uva.nl.
authors: Sacha
authors: S
authors: Zeerleder
mesh_terms: D000744:Anemia, Hemolytic, Autoimmune / Q000097:blood / Q000276:immunology*; D001963:Bromelains / Q000494:pharmacology; D003167:Complement Activation / Q000276:immunology*; D050718:Complement C1 Inhibitor Protein / Q000494:pharmacology; D003176:Complement C3 / Q000276:immunology*; D003181:Complement C4 / Q000276:immunology*; D004912:Erythrocytes / Q000737:chemistry / Q000187:drug effects / Q000276:immunology*; D006461:Hemolysis / Q000276:immunology; D006801:Humans
chemical_list: D050718:Complement C1 Inhibitor Protein; D003176:Complement C3; D003181:Complement C4; D001963:Bromelains