A new mechanism for immune destruction of red blood cells?

I n 1997, we reported three cases of autoimmune hemolytic anemia (AIHA) associated with immunoglobulin (Ig)M warm autoantibodies directed at determinants on or associated with glycophorins. The antibodies were anti-En, -Wr, -Pr. All three patients had severe hemolytic anemia with evidence of hemoglobinemia and hemoglobinuria. Two of the three patients (associated with the anti-En and -Wr) died as a result of the hemolysis. In this article we emphasized how the serology did not correlate well with the degree of in vivo hemolysis. The direct antiglobulin tests (DATs) were 3+, 1+, /2+ respectively, due to red blood cell (RBC)-bound C3; IgM was detected (1+) on the RBCs of one patient; and no RBC-bound IgG was detected in any of the cases. The sera of all three patients agglutinated RBCs at 37°C, but the anti-En and anti-Wr were dependent, or enhanced by, a low pH (6.5) or the presence of 30% albumin. The antibodies showed another unusual characteristic in that they reacted better at 30°C than at 37°C. In 2009, we published serologic findings in 49 cases of AIHA associated with IgM warm autoantibodies; 4 of 17 (24%), studied for specificity were antibodies directed against epitopes on or associated with glycophorins (anti-N, -Pr, -En, -Wr). Because of the above poor correlations with the serology and clinical status of the patients we studied, we were very interested to read two publications by Brain and colleagues in 2002 and 2004. Like us, Brain and colleagues were also puzzled by the unimpressive serologic findings in a patient with severe AIHA associated with an IgM anti-Pr autoantibody. The DAT was weakly positive due to RBC-bound C3; the serum contained only an anti-Pr cold agglutinin of low titer and high thermal amplitude (titer of 32 at 4°C, titer of 1 at 30°C, and nonreactive at 37°C). The patient had a life-threatening hemolytic anemia (HA) with a 3-day t1/2 of Cr-labeled RBCs. Dr Brain is an expert in the nonimmune hemolytic anemias and so approached the problem in a different way than a serologist. As Pr is present on glycophorins (mainly glycophorin A [GPA], but also present on glycophorin B [GPB] and possibly glycophorins C and D), he wondered if the anti-Pr was affecting GPA because, as earlier work had shown, attachment of bifunctional ligands (lectins and monoclonal antibodies) to GPA make RBCs more resistant to shear forces and reduce the mobility of Band 3 in the RBC membrane. Brain and coworkers went on to show that GPA-reactive lectins (Maclura pomifera and wheat germ), antibodies to GPA (human anti-Pr and mouse monoclonal anti-GPA), would cause hemolysis of human RBCs in vitro, without involving complement. The lysis appeared to be due to sodium, and probably calcium, entering the RBCs. They suggested that antibodies directed at epitopes on GPA might aggregate the GPA causing destabilization of the phospholipid bilayer of the RBC membrane, leading to an increase in cation permeability. In a later publication Brain and coworkers showed that calcium entered approximately 28% of the RBCs when the RBCs were under stress; they suggested that mechanosensitive channels could be activated by the changes in lateral pressure within the phospholipid bilayer of the RBC membrane, such as those caused by GPA–anti-GPA interactions. In this issue of TRANSFUSION, Brain and colleagues continue the story. They had another patient, a 29-yearold man, with a HA associated with cold agglutinins. The DAT was weakly positive with anti-C3 and negative with anti-IgG. The cold agglutinin titer (CAT) was abnormal, but not very high (titer of 256 at 4°C); the cold agglutinin reacted up to 30°C, thus fulfilling the criteria for cold agglutinin syndrome (CAS). The antibody had several unusual characteristics; it was a polyclonal IgG cold agglutinin with anti-Pr specificity. The case was very similar to one described by Curtis and coworkers, which stimulated Brain and coworkers to treat the patient with cyclophosphamide, as used by Curtis and coworkers, when they had little success with transfusions, plasma exchange, steroids, IVIG, and rituximab; Curtis and coworkers also used splenectomy. Both patients responded well and had complete remissions. Brain and colleagues confirmed that calcium entered RBCs when the RBCs were treated with the patient’s IgG anti-Pr and a rabbit IgG anti-GPA; high-titer IgM anti-I from a patient with CAS did not cause the same phenomenon. The authors added a new finding that after exposure to antibodies to epitopes on GPA, phosphatidylethanolamine (PE) was exposed on the RBC membrane. These changes appeared to occur only in a subpopulation of the RBCs. Nevertheless, Brain and colleagues feel that this antibody-induced mechanism could cause HA independent of complement-mediated hemolysis. There are many publications concerning anti-Pr in the literature; many of these were found in patients with CAS, but unfortunately, many of them concentrate on serologic and biochemical characteristics of the antibodies. Nevertheless, on reviewing the literature TRANSFUSION 2010;50:274-277.