Acquired immune hemolytic anemia associated with IgA erythrocyte coating: investigation of hemolytic mechanisms

We have investigated the hemolytic mechanisms in a patient with acquired immune hemolytic anemia whose red cells appeared to be coated with IgA alone. The clinical course was similar to that of patients with hemolytic anemia mediated by warm-reacting IgG antibody. Splenic sequestration of red cells was demonstrated, and marked reduction of hemolysis occurred after corticosteroid therapy. Antibody was eluted from the patient's red cells and used to sensitize normal red cells in vitro. These sensitized red cells were not lysed by fresh autologous serum, nor did they fix detectable amounts of C3. However, red cells sensitized by eluted antibody were lysed by normal human peripheral blood monocytes in a system designed to demonstrate antibody-dependent cell-mediated cytotoxicity. Monocyte-mediated hemolysis of sensitized red cells was inhibited by the addition of low concentrations of normal serum IgA to the system, but not by IgG. The ability of the eluate to induce monocyte-mediated hemolysis was abolished by its adsorption on Sepharose-bound anti-IgA, but not by preincubation with Sepharose-bound anti-IgG. In addition, normal human monocytes were demonstrated to ingest eluate-sensitized red cells. These data demonstrate an in vitro interaction of IgA-sensitized red cells with leukocytes and suggest a possible mechanism for the patient's hemolysis.     

Extravascular hemolysis following the administration of cefamandole

Hemolytic anemia occurred in a 70-year-old female after a five-day course of intravenous cefamandole. The patient's serum contained an IgG antibody which was reactive with red blood cells which had been coated in vitro with cefamandole but not with uncoated cells. An in vitro assay of allogeneic mononuclear phagocytosis of cefamandole-coated red cells sensitized with the patient's anti-cefamandole indicated that the anti-cefamandole could induce significant phagocytosis. The anti-cefamandole was easily inhibited in vitro by cefamandole as well as by a variety of related cephalosporins indicating broad cross-reactivity, with the antigenic site primarily the 7-amino-cephalosporanic acid nucleus. Penicillins could inhibit the anti-cefamandole but only when using concentrations 3-10 X those of cephalosporins. Eleven examples of anti-penicillin tested failed to react with cefamandole-coated red cells. Screening of 344 random sera from hospitalized patients found only five (1.5%) reactive with cefamandole-coated red cells; three of these sera were also reactive with penicillin-coated red cells. The patient's hemolysis subsided following cessation of the drug. This is the first report of anti-cefamandole-induced hemolytic anemia.     

Multiple myeloma with hemolytic anemia and thrombocytopenia

We report a 59 year old female patient who was diagnosed as having IgG kappa myeloma with hemolytic anemia and thrombocytopenia simultaneously. Although M-protein was suspected to contribute to the hemolysis, the IgG purified from the patient's serum did not bind to red blood cells. Therefore, massive non-specific binding of M-protein to blood cells might contribute to high levels of red blood cell-associated IgG and platelet-associated IgG in the patient.     

Autoimmune Hemolytic Anemia Associated with Autoanti-Ge

The first reported example of autoimmune hemolytic anemia due to an autoanti-Gerbich is described. The patient's red blood cells exhibited a strongly positive direct antiglobulin test with both IgG and complement antiglobulin reagents. The serum contained a potent antibody which produced agglutination of red blood cells as well as a positive indirect antiglobulin test. Treatment of the serum with 2-mercaptoethanol demonstrated that the antibody contained both IgG and IgM components. The serum antibody and the antibody eluted from the patient's red blood cells had anti-Gerbich specificity. The patient's cells typed as Gerbich-positive with saline-agglutinating anti-Gerbich sera. Of great interest was the fact that the patient's mother also has acquired immune hemolytic anemia, but the IgG antibody in her serum and eluted from her red blood cells had anti-pdl specificity.     

Cephalosporin-induced Hemolytic Anemia in a Sicilian Child; Erythropoiesis

A 27-month-old child developed acute hemolysis on two occasions after the administration of cephalosporin. On the first occasion, hemolysis was intravascular and was due to the formation of complexes between antibodies and the drug, which bound to red blood cells and caused severe hemolysis. On the second occasion, hemolysis was extravascular and was probably due to antibody-dependent cell-mediated cytotoxicity. Marked increases in levels of CD(19) (+) and CD(57) (+) CD(8) (+) cells were detected among the subpopulations of the patient's lymphocytes but only in the level of CD(19) (+) cells from the patient's father, after incubation of a sample of whole blood with a solution of cephalosporins. These results might explain the differences between the immune response of the patient and those of other members of his family and of an unrelated control.     

Rheumatoid arthritis and pure red cell aplasia

Three patients with severe, deforming, and long-standing rheumatoid arthritis developed pure red cell aplasia that did not remit after withdrawal of medications, ran a chronic course, and in two patients remitted only after cytotoxic immunosuppressive treatment. An IgG inhibitor of autologous erythroid colony-forming and burst-forming unit growth in vitro was found in the serum of one patient. This specific erythropoietic inhibitor persisted in lower titer in the patient's serum even after an azathioprine-induced remission of pure red cell aplasia, indicating the possible need for maintenance immunosuppressive therapy. Chronic pure red cell aplasia may be another extra-articular manifestation of rheumatoid arthritis and should be considered when severe anemia develops in the absence of blood loss or hemolysis.     

Autoimmune Hemolytic Anemia Associated with an IgA Autoanti-Gerbich

A third patient with autoimmune hemolytic anemia due to autoantibodies against Gerbich antigens is described. The patient's serum contained strong hemagglutinating antibodies of the IgA plus IgG classes which reacted with all red blood cells (RBC) tested, but not with Gerbich-negative cells. Although the patient was typed as Gerbich positive, his serum failed to react with his own RBC, and the sensitization of his erythrocytes with autoantibodies was only demonstrable if eluates of his RBC were used. The failure of the autoantibodies to react with autologous RBC at the peak of hemolysis most likely reflects a weakening of Gerbich antigens during the course of autoimmune hemolytic anemia.     

Autoimmunity and the Kell Blood Groups: Auto-Anti-Kpb in a Kp(a+b-) Patient

Abstract. An example of auto-anti-Kp^b in a Kp (a + b-) patient is described. The antibody present in the patient's serum and in eluates from her red cells was IgG. It did not bind complement, and did not cause in vivo hemolysis. 9 months after recognition of the autoimmune state the direct antiglobulin test had become negative and anti-Kp^b was no longer detectable. It is postulated that autoimmunity involving the Kell blood group may be precipitated by antigens or enzymes of microbial origin.     

Methyldopa-induced autoantibodies against red blood cells

Methyldopa therapy results in the formation of red cell autoantibodies in 10-20% of patients taking the drug for longer than 4 months. These red cell antibodies are true autoantibodies, that is they are directed against an autoantigen on the red blood cell membrane and not against the drug or against a drug-altered antigen. The target membrane antigen is usually within the Rhesus system, although often the antibody specificity cannot be defined. Red cell antibody is usually detectable in the patient's sera as well as on the red cells. The autoantibody is usually a warm reacting IgG antibody. Most patients who develop these autoantibodies do not go on to develop hemolytic anemia in spite of high titres of antibodies on their red cells. In addition, these patients do not tend to develop hemolysis if methyldopa therapy is continued. Rarely patients develop hemolytic anemia which can be severe. Differences in antibody characteristics, including subclass restriction, complement-binding ability, or titre do not explain why some patients with autoantibody hemolyze while most do not. One group of investigators found that hemolyzing patients had IgM on their red cells while those who did not had IgG only. But while this observation could explain why some patients (IgM-sensitized red cells) hemolyze, it does not explain why most patients with IgG-sensitized red cells do not hemolyze. Why the autoantibody forms is not known but some investigators have proposed that the drug may directly affect B or T cells with resulting impairment of immune tolerance.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diphenylhydantoin-induced pure red cell aplasia

The pathogenesis of diphenylhydantoin-induced pure red cell aplasia was investigated in the case of a 32-year-old man who developed pure red cell aplasia while he was under treatment with diphenylhydantoin. The patient's serum IgG purified from serum drawn at the time of diagnosis suppressed normal allogeneic marrow colony-forming (CFU-E) and burst- forming (BFU-E) and autologous blood BFU-E growth in vitro only in the presence of diphenylhydantoin. This IgG-diphenylhydantoin complex had no effect on CFU-GM growth in vitro. Normal IgG or patient's IgG purified from serum drawn after the remission of red cell aplasia had no effect on erythroid colony formation in vitro in the presence of diphenylhydantoin. The IgG-diphenylhydantoin complex exerted no direct cytotoxic effect on normal marrow erythroblasts, CFU-E, and BFU-E, nor did it interfere with the action of erythropoietin on marrow erythroblasts. These studies suggest that diphenylhydantoin-induced red cell aplasia is immunologically mediated through an IgG inhibitor, which requires the presence of the drug to suppress erythroid colony formation in vitro. This inhibitor seems to exert its effect on erythroid progenitors at or beyond the stage of differentiation of CFU- E, but not on erythroblasts.     

Characterization of multiple quinine-dependent antibodies in a patient with episodic hemolytic uremic syndrome and immune agranulocytosis.

A 23-year-old woman experienced six distinct episodes of severe combined neutropenia and thrombocytopenia. At least one of the episodes was accompanied by hemodialysis-requiring acute renal failure and fragmentation hemolysis (hemolytic uremic syndrome [HUS]). In retrospect, all episodes were probably associated with the ingestion of quinine. Quinine-dependent antibodies to platelets, neutrophils, T lymphocytes, and red blood cells (RBCs) were detected in the patient's serum. By a monoclonal antibody antigen capture assay, the patient's serum contained IgG antibodies, which in the presence, but not absence, of quinine reacted with platelet glycoprotein (GP) complexes Ib/IX and IIb/IIIa, but not Ia/IIa. By immunoprecipitation assay, the serum, after addition of quinine, reacted strongly with an 85-Kd neutrophil membrane protein and weakly with 130- and 60-Kd moieties. Serum adsorbed with RBCs in the presence of quinine continued to react with platelets and neutrophils, and serum that was absorbed with platelets continued to react with neutrophils and RBCs, indicating that the antigenic targets were different on platelets, neutrophils, and RBCs. Since platelets and endothelial cells share some antigens, we tested patient serum for antibodies to human umbilical vein endothelial cells (HUVEC); no quinine-dependent antibodies to HUVEC were detected. However, her quinine-dependent antibodies not only bound to platelets and neutrophils, but also activated neutrophils. Thus, the patient's serum with quinine aggregated neutrophils, but neither agent alone caused activation. Moreover, the patient's serum with quinine (but not without) augmented the adherence of neutrophils to HUVEC. Treatment of the patient's serum with staphylococcal protein A removed the quinine neutrophil aggregation cofactor, suggesting it was due to IgG. In both neutrophil aggregation and adherence assays, decomplementation of the patient's serum markedly blunted its effect. Furthermore, the patient's serum failed to aggregate formalin-inactivated neutrophils, suggesting neutrophil activation, probably by activated complement, was necessary for aggregation and adhesivity to endothelium. We conclude that our patient's neutropenia, thrombocytopenia, lymphopenia, and anemia were due to quinine-dependent antibodies, and that these antibodies recognized epitopes that were different in the three target cell populations. We further suggest that HUS was likely secondary to the activation and adhesion of neutrophils to endothelium.     

IgG4 Autoantibodies against Erythrocytes, without Increased Haemolysis: a Case Report

A patient is described who, notwithstanding a strongly positive direct antiglobulin test with anti-IgG serum, apparently did not suffer from haemolytic anaemia. The survival of the patient's red cells, measured with 51Cr, was only slightly decreased. In vitro, the sensitized cells of the patient showed only a minimal tendency to adhere to monocytes. The patient's spleen functioned normally, since 51Cr-labelled donor erythrocytes, either sensitized with IgG-anti-D or damaged by heating, were eliminated rapidly from the circulation and sequestered in the spleen. These apparently contradictory findings could be explained by the fact that the patient's red cells were sensitized with autoantibodies, mainly belonging to the IgG4 subclass. Only weak IgG1 and IgG3 autoantibodies were detectable. Since previously the patient had suffered from a severe haemolytic anaemia, it is postulated that a switch has occurred from 'active' to 'inactive' IgG autoantibodies, perhaps induced by prednisone therapy.     

Rh blood group-specific antibodies in immune hemolytic anemia induced by nomifensine

Nomifensine (Merital, Alival; Hoechst, Frankfurt, FRG), an antidepressant drug, may cause immune hemolytic anemia (IHA) of the so- called immune complex type that is believed to occur by means of an innocent-bystander mechanism. In this report we describe findings that are not consistent with this mechanism in a patient with nomifensine- induced intravascular IHA associated with renal failure. In vitro studies showed a transitory positive direct antiglobulin test (DAT) due to IgG, IgM, and C3 fixation. The causative antibodies were found to be a drug-independent IgM antibody in the serum and eluate that reacted only with E-positive RBC, although the patient's RBC were E-negative; an IgG antibody in the serum and initial eluates that showed a stronger reaction with e-positive than with e-negative or Rhnull RBC, but only in the presence of ex vivo antigen (ie, urine containing the drug and all its metabolites); and an IgM antibody in the serum and initially also on the patient's RBC that, in the presence of ex vivo antigen as well as in the presence of known metabolites of the drug, agglutinated all RBC equally strongly, but was hemolytically more active against E- positive than E-negative cells. Within a few days of stopping the drug the hemolysis rapidly resolved without administration of prednisone, the DAT became negative with anti-IgG and anti-IgM, and the drug- independent anti-E disappeared, but both metabolite-dependent antibodies remained detectable in the patient's serum. We conclude that the production and specificity of the causative antibodies in this case were controlled by a larger antigenic site, presumably consisting of the drug and/or its metabolites plus RBC antigens, rather than by epitopes of the drug or metabolites alone.     

Acute immune hemolysis induced by a degradation product of amphotericin B

Summary We report here on an eight-year-old boy who first developed acute intravascular hemolysis following therapy with amphotericin B (AmB) and subsequently a delayed hemolytic transfusion reaction due to alloantibodies. Although there is as yet no evidence for metabolism of AmB in vivo, the hemolysis appeared to be the result of sensitization against a degradation product of the drug. The patient's serum contained a hemagglutinating IgM antibody that reacted with all red blood cells (RBC) tested in the presence of plasma obtained from patients receiving AmB (ex vivo antigen), but not in the presence of their urine, AmB itself, or with AmB-pretreated RBC. These findings indicate that the antibody was directed against a degradation product of AmB, presumably a trace metabolite, that has not yet been identified.     

Analysis of antibody to neutrophils associated with autoimmune neutropenia: possible recognition of Fc-receptor-related molecules

The serum of a 50-year-old male with neutropenia and a bout of bacterial infection was studied. Anti-neutrophil IgG antibody was detected by indirect immunofluorescence using a laser flow cytometry system. Purified IgG from our patient's serum did not inhibit chemotaxis of neutrophils, but inhibited rosette formation of neutrophils with ox red blood cells (ORBC) coated with anti-ORBC rabbit IgG dose-dependently. Surface iodination of neutrophils followed by their immunoprecipitation by purified IgG and sodium dodecylsulfate polyacrylamide gel electrophoresis showed a single band that corresponded to 45 kilodaltons. Possibly the IgG antibody in our patient's serum recognizes molecules related to Fc receptors.     

Autoimmune Hemolytic Anemia Associated with an IgG Cold Incomplete Antibody1

Abstract. Serologic studies are reported on a patient with severe autoimmune hemolytic anemia, whose red cells were strongly coated with IgG and with α2D component of C3. Direct and indirect Donath-Landsteiner reactions were negative. In addition to a typical IgM anti-I cold agglutinin of modest titer, the serum contained a lambda IgG incomplete antibody which bound more strongly to normal red cells at 4°C (1/64) than at 37°C (1/4). The IgG antibody did not require complement for binding but could bind complement and cause hemolysis with papainized red cells. Once bound, the IgG antibody did not dissociate appreciably at room temperature. The antibody eluted poorly from the patient's cells at 56°C but was readily dissociable from cell stroma at pH 3.5. The eluate exhibited no blood group specificity with a diagnostic red cell panel. In particular, equally strong reactions were obtained with O, A₂, B, A₁B, P + P₁-negative, Rh-null, adult and cord red cells. Reactivity was not impaired by prior papainization of the cells. Although the patient improved following splenectomy, neither IgG nor IgM antibodies were detectable in a concentrated splenic extract. The properties of the present cold IgG incomplete antibody are compared with those previously reported; possible clinical implications of these antibodies are briefly discussed.     

Reduced Survival of Isotope-Labelled Rh(D)-Negative Donor Red Cells in a Patient with Anti-LWab

The serum of an 85-year-old Caucasian male with no history of blood transfusion contained an IgG3 antibody with anti-LWab specificity. The antibody failed to react with dithiothreitol-treated red cells, and there was a marked reduction in titre of the antibody with pronase-treated cells, findings consistent with an antibody having this specificity. High association values were obtained in a mononuclear phagocyte assay when LW-positive red cells, sensitised in vitro with the patient's serum antibody, were incubated with peripheral blood monocytes from the patient. In vivo red cell survival studies demonstrated that 99mTc-labelled rhesus-negative (rr), LW-positive red cells had 53% survival at 1 h. The IgG subclass of the antibody, mononuclear phagocyte assay results and in vivo survival studies predicted a significant reduction in the posttransfusion survival of therapeutic volumes of rhesus-negative (rr), LW-positive red cells.     

An Autoantibody with Activity Dependent on Red Cell Age in the Serum of a Patient with Autoimmune Haemolytic Anaemia and a Negative Direct Antiglobulin Test

A red cell IgM autoantibody with anti-e specificity was identified in the serum of a rhesus-negative (rr) patient presenting with haemolytic anaemia and a negative direct antiglobulin test. The autoantibody strongly agglutinated normal allogeneic rhesus-negative (rr) red cells in saline at 37 degrees C but had only weak activity for autologous red cells. Incubation of the patient's serum with subpopulations of normal allogeneic red cells obtained by density fractionation, demonstrated that the strong agglutinating activity of the autoantibody was for red cells with density greater than 1.090 g/ml. Young red cell subpopulations of lower density gave weak reactions and low titration scores equivalent to those obtained with autologous red cells during the haemolytic episode. The patient's red cells during remission however, when the patient's haemoglobin level had returned to normal, were strongly agglutinated by serum samples taken during the haemolytic episode; as was the case with normal allogeneic red cells, the strong activity was for patient red cells with density greater than 1.090 g/ml, red cell populations of lower density giving low titration scores. The findings in this case indicate that the patient's red cells which had survived haemolysis during the haemolytic episode were young red cells (density less than 1.090 g/ml), the weak sensitization of these cells being insufficient to cause their destruction by macrophages. Furthermore, these findings, together with observations that IgG autoantibodies may also bind less strongly to young red cells [Gray et al., Br. J. Haemat., 55: 335-345, 1983; Branch et al., Blood 63: 177-180, 1984].(ABSTRACT TRUNCATED AT 250 WORDS)     

Autoimmune hemolytic anemia and the Kell blood groups

Approximately one in 250 people with autoimmunity involving their red cells have IgG autoantibodies with specificity in the Kell blood groups. Red cells of these individuals have an acquired temporary weakening of their Kell antigens. Some of the patients also have allo-anti-K in their serum. This report presents a case in which an IgG autoantibody may define a new high-incidence red cell antigen related to the Kell blood groups. The patient's Kell blood group antigens are depressed, and his serum contains allo-anti-K. It is postulated that reduced red cell Kell antigenicity is caused by enzymatic degradation, possibly of bacterial origin, and that the acquired loss of Kell antigens, the Kell-specific autoimmune state, and the serum all0-anti-K, are all related aspects of one phenomenon.     

Two cases of immune haemolytic anaemia,associated with anti-piperacillin,detected by the 'immune complex' method

BACKGROUND AND OBJECTIVES: Sera containing antibodies to penicillin and penicillin-related drugs are typically thought to react with drug-coated red blood cells (RBCs) (drug adsorption method), but not when the sera are added to drug and RBCs in the same tube ('immune complex' method). Two cases of immune haemolytic anaemia caused by anti-piperacillin have been previously described. Serological details were given in only one patient. In that subject, the antibody was immunoglobulin (Ig)M + IgG and reacted by both the drug adsorption and 'immune complex' methods. MATERIALS AND METHODS: Two patients with cystic fibrosis developed positive direct antiglobulin tests (DATs) and haemolytic anaemia after 11-12 days of piperacillin therapy. Serological studies were performed with piperacillin, Zosyn (piperacillin + tazobactam) and penicillin by using the drug adsorption and 'immune complex' methods. RESULTS: The first patient's serum contained an IgG, complement-activating anti-piperacillin that reacted by the 'immune complex' method only. The second patient's IgM + IgG, complement-activating anti-piperacillin reacted by the 'immune complex' method and agglutinated piperacillin-treated RBCs. An eluate from the patient's RBCs reacted weakly with all RBCs tested without the presence of drug. This patient had evidence of intravascular haemolysis and died. CONCLUSIONS: We describe the third and fourth examples of immune haemolytic anaemia caused by anti-piperacillin; one was associated with fatal haemolytic anaemia. As piperacillin is commonly used in the treatment of cystic fibrosis, anti-piperacillin should be considered whenever patients with cystic fibrosis develop haemolytic anaemia and/or positive DATs.