A pair of naturally occurring antibodies may dampen complement-dependent phagocytosis of red cells with a positive antiglobulin test in healthy blood donors V. Alaia, B. M. Frey, A. Siderow, P. Stammler, M. Kradolfer & H. U. Lutz Institute of Biochemistry, Department of Biology, ETH Zurich, Zurich, and the Regional Blood Transfusion Service SRK, Schlieren, Switzerland Received: 4 April 2009, revised 4 June 2009, accepted 5 June 2009, published online 30 June 2009 Background and Objective It is known that red blood cells (RBC) from healthy blood donors with a positive direct antiglobulin test (DAT) for IgG continue to circulate despite carrying elevated numbers of IgG molecules. To unravel the properties of these RBC-bound IgG, we studied them not only on whole RBC populations, but also on density-fractionated RBCs. Materials and Methods The properties of acid-eluted RBC-bound IgG and plasma IgG were studied by ELISA for binding to RBC proteins and opsonins, and by blotting. In vitro phagocytosis was studied on density-separated RBCs. Results IgG-DAT-positive blood donors carried most IgG molecules on dense RBCs and had more RBCs of high density than DAT-negative controls. Their densest RBCs were older than the oldest RBCs of DAT-negative controls, based on the band 4Æ1a/b ratio. In vitro phagocytosis of senescent RBCs from IgG-DAT-positive donors was 1Æ5 to 2 fold higher than that of senescent control cells, but the same or less in the presence of physiological IgG concentrations, implying that RBC-bound IgGs impaired complement-dependent uptake. The IgG molecules on these DAT-positive RBCs comprised anti-band 3 naturally occurring antibodies (NAbs) and were two- to fivefold enriched in anti-C3 and framework-specific anti-idiotypic NAbs as compared to controls. Correspondingly, anti-C3 and framework-specific anti-idiotypic NAbs were proportionally elevated in the plasma of two-thirds of DAT+ donors. Conclusions Extra-binding of anti-C3 together with anti-idiotypic NAbs to senescent RBC-associated C3 fragments may suppress complement-dependent RBC phagocytosis and may prolong the in vivo life span of RBCs. Key words: complement, direct antiglobulin test, naturally occurring autoantibodies, phagocytosis, red blood cells. Introduction One in 9000 to 14 000 healthy blood donors (HBD) [1] has red blood cells (RBC) that agglutinate in the presence of anti- immunoglobulins and thus show a positive direct antiglobulin test (IgG-DAT+). A varying number of IgG-DAT+ donors also has a positive DAT for complement C3 fragments [1,2]. IgG-DAT+ RBCs from HBD have been studied primarily by characterizing eluted IgG with regard to blood group specificity and IgG subclasses [2–4]. RBCs from these donors have two to 10 times as many IgG molecules (200 to 1000 IgG/ RBC) on their surface as DAT– senescent RBCs [5]. IgG mole- cules eluted from RBCs of 18 of 20 IgG-DAT+ HBD inhibited binding of anti-Rhesus antibodies (anti-D, anti-E, anti-c) to homozygous antigen-positive RBCs largely irrespective of Correspondence: Dr Hans U. Lutz, Institute of Biochemistry, ETH Zurich, Schafmattstr. 18, CH 8093 Zurich, Switzerland E-mail: hlutz@bc.biol.ethz.ch This work was supported by grants to HUL from the ETH (TH 22/01-1) and the SNF (31000-105831). Vox Sanguinis (2009) 97, 338–347 ORIGINAL PAPER ª 2009 The Author(s) Journal compilation ª 2009 International Society of Blood Transfusion DOI: 10.1111/j.1423-0410.2009.001214.x 338