Meulenbroek Elisabeth M, Wouters Diana, Zeerleder Sacha
Department of Immunopathology, Sanquin Research and Landsteiner Laboratory Academic Medical Center, University of Amsterdam.
Department of Hematology, Academic Medical Center, University of Amsterdam;
J Vis Exp. 2014 Jan 29(83):e51161. doi: 10.3791/51161.
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.
抗红细胞（RBC）抗体可导致补体激活，进而通过肝脏中的补体受体加速清除（血管外溶血），或导致红细胞血管内溶解。导致补体激活的同种抗体（如ABO）或针对红细胞抗原的自身抗体（如自身免疫性溶血性贫血，AIHA中所见）具有潜在危害，尤其当导致血管内溶解时可能致命(1)。目前，通过使用反映补体在红细胞上沉积的库姆斯试验或反映红细胞溶解的非定量溶血试验，在体外评估红细胞上（自身）抗体引起的补体激活(1-4)。然而，为了评估补体抑制剂的疗效，必须采用定量技术。在此，我们描述两种这样的技术。首先，介绍一种检测患者血清中抗体诱导的红细胞上C3和C4沉积的试验。为此，使用流式细胞术分析，采用荧光标记的抗C3或抗C4抗体。接下来，描述一种定量溶血试验。在该试验中，利用分光光度法检测释放的血红蛋白，测量患者血清诱导的补体介导的溶血。这两种试验都具有高度可重复性和定量性，有助于研究抗体诱导的补体激活。
