Mechanism of autoimmune hemolytic anemia in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is a malignant clonal expansion of CD5+B lymphocytes. The CD5+B lymphocytes have been postulated to produce autoantibodies. CLL patients may demonstrate features of autoimmunity including autoimmune hemolytic anemia. However, the origin of the autoantibodies causing the hemolysis is not clear. The present studies were performed to determine whether these autoantibodies are the products of the neoplastic B-CLL clones. Immunoglobulins (Ig) were eluted from washed red blood cells (RBC) obtained from two CLL patients at the time they had autoimmune (DAT-direct antiglobulin test - positive) hemolytic anemia. The light chain phenotypes of these eluted autoantibodies were determined and found to be monotypic with exact correlation to the light chain expressed on the surface of the B-CLL clones. Elutions from RBC of DAT negative patients or normal volunteers failed to demonstrate measurable amounts of Ig. In contrast, Ig eluted from RBC obtained from SLE patients with DAT positive hemolytic anemia found to be polyclonal autoantibodies exhibiting both light chain types. Furthermore, CD5+B lymphocytes obtained from the same two CLL patients (DAT+) produce, in vitro understimulation with phorbal myristate acetate (PMA), monoclonal antibodies which react and bind to RBC. Thus these studies provide direct evidence demonstrating that the antibodies causing the autoimmune hemolytic anemia in our two CLL patients are the products of the B-CLL neoplastic clones.     

Autoimmune hemolytic anemia and periodic pure red cell aplasia in systemic lupus erythematosus

A patient with systemic lupus erythematosus and autoimmune hemolytic anemia complicated by periodic episodes of red cell hypoplasia is described. Using a plasma clot culture system a serum inhibitor of erythropoiesis was detected. In addition, heat eluates of the red cells of this patient were capable of impairing erythroid colony formation. The possibility that the autoantibodies of acquired autoimmune hemolytic anemia might influence the proliferation and/or maturation of erythroid progenitor cells is raised by these findings.     

Febrile transfusion reaction following initial transfusion in a man with immunoblastic lymphadenopathy and granulocyte autoantibodies

A 69-year-old man with immunoblastic lymphadenopathy and autoimmune hemolytic anemia who had no previous exposure to blood products developed a severe febrile nonhemolytic transfusion reaction following the initial infusion of packed red blood cells. The reaction recurred with transfusion of packed red blood cells, but not when freeze-thawed red blood cells were used. Immunofluorescence techniques demonstrated granulocyte antibodies in his serum and on the surface of his granulocytes. Circulating immune complex, HLA, and platelet antibodies were not present. The granulocyte antibodies fluctuated in titers with disease activities, and could be completely removed from the serum by autologous granulocyte absorption. We conclude that our patient had granulocyte autoantibodies which probably produced febrile transfusion reactions.     

Autoimmune hemolytic anemia by coexisting Anti-I and Anti-Fl cold agglutinins

Summary In association with atypical pneumonia, a patient developed acute severe autoimmune hemolytic anemia. Hemoglobin temporarily was only 7.0 g/100 ml, so that the patient needed red blood cell (RBC) transfusion. Hemolysis was found to be caused by high titer cold agglutinins (CA), which occurred transiently during the acute period of the disease. CA of two different specificities, anti-I and anti-Fl, were demonstrated in the patient's serum. Antibodies of the two specificities were clearly separated by absorption/elution experiments using neuraminidase (RDE)-treated RBC. They were distinguished by serologic means: Both anti-I and anti-Fl react more strongly with adult RBC than with newborn and i adult RBC; in contrast to anti-I, anti-Fl does not agglutinate RDE-treated cells. Inhibition experiments showed that I-active substances prepared from papainized RBC exhibited both I and Fl antigenic activity. By RDE-treatment of I-active substances, Fl-activity was markedly reduced, while I-activity was increased.     

Aortic stenosis and mitral regurgitation complicated by hemolytic anemia and positive Direct Coombs test: a case report

A 83-year-old man was admitted because of heart failure due to severe aortic stenosis and mitral regurgitation secondary to chordal rupture of the anterior leaflet. Mild anemia and elevated serum lactate dehydrogenase were present with reticulocytosis and haptoglobinemia. Direct Coombs test was positive. Coexistence of autoimmune hemolytic anemia was identified, but the main cause of his hemolysis was thought to be mechanical hemolysis due to stenotic valve and/or ruptured chordae because of the presence of red cell fragmentation. The patient successfully underwent double valve replacement. Improvement of anemia was coupled with reduction of the serum lactate dehydrogenase level. Valvular shear stress on the red cells and reduction of red cell deformability secondary to autoimmune hemolytic anemia were thought to be responsible for his hemolysis.     

Autoimmune hemolytic anemia

Red blood cell (RBC) autoantibodies are a relatively uncommon cause of anemia. However, autoimmune hemolytic anemia (AIHA) must be considered in the differential diagnosis of hemolytic anemias, especially if the patient has a concomitant lymphoproliferative disorder, autoimmune disease, or viral or mycoplasmal infection. Classifications of AIHA include warm AIHA, cold agglutinin syndrome, paroxysmal cold hemoglobinuria, mixed-type AIHA, and drug-induced AIHA. Characteristics of the autoantibodies are responsible for the various clinical entities. As a result, diagnosis is based on the clinical presentation and a serologic work-up. For each classification of AIHA, this review discusses the demographics, etiology, clinical presentation, laboratory evaluation, and treatment options.     

Reactivity of human gamma G erythrocyte autoantibodies with fetal, autologous and maternal red cells

Summary. Human erythrocyte autoantibodies of the γG (‘warm’) type, representing both ‘Rh-related’ and ‘non-Rh’ specificities, have been found to react strongly with (a) the red cells of 10–12 week human fetuses, (b) Coombs-negative autologous red cells obtained during subsequent remission, and (c) the red cells of the patient's own mother. These findings are interpreted to mean that the erythrocyte antigenic determinants with which such autoantibodies react (1) have an early appearance in ontogeny and, therefore, would have an opportunity to promote natural immunologic tolerance; (2) do not undergo a gross, serologically detectable change as the patient recovers from active hemolytic anemia and enters remission. The observations with maternal red cells indicate that the lymphoid cell population responsible for autoantibody synthesis in these patients does not exhibit selective tolerance toward maternal erythrocyte antigens, as might be expected if hemolytic anemia were caused by activation of a previously dormant colony of maternal lymphoid cells.     

On the mechanisms of sensitization and attachment of antibodies to RBC in drug-induced immune hemolytic anemia

The mechanisms of sensitization and attachment of drug-dependent antibodies to RBC in drug-induced immune hemolytic anemias are largely speculative. Nomifensine has been incriminated in causing immune hemolysis in a large number of patients. The hemolysis was usually of the so-called immune complex type, less commonly of the autoimmune type, and more surprisingly, few patients had developed both types of hemolysis. To determine whether nomifensine (metabolite)-dependent antibodies (ndab) exhibit specificity for antigenic structures of RBC membranes, 30 ndab were tested against large panels of RBC with common and rare antigens. We found that only 14 out of 30 ndab were invariably reactive with all cells tested. Nine antibodies were, similar to the majority of idiopathic or drug-induced autoantibodies, not or only weakly reactive with Rhnull RBC. Three antibodies did not react with cord RBC and could be inhibited by soluble I antigen. The remaining four antibodies gave inhomogeneous reaction patterns or were even negative with selected RBC; their specificity could not be identified. On a Scatchard plot analysis of one ndab, a maximum of 173,000 drug-dependent antibodies of the IgG class can specifically bind per RBC in the presence of the drug. Although nomifensine and its metabolites do not attach tightly onto RBC, our results clearly indicate that RBC do not act as "innocent bystanders," but rather serve as a surface for a loose attachment of drugs that possibly cause a subtle structural change in the cell antigens and, by this means, allow in vivo sensitization; and a specific binding of the resultant antibodies. This concept would explain why these antibodies can be directed against drug-cell complexes, against cell antigens alone (autoantibodies), or against both in the same patient.     

Partial tolerance of autoreactive B and T cells to erythrocyte-specific self-antigens in mice

Background

Breakdown of humoral tolerance to RBC antigens may lead to autoimmune hemolytic anemia, a severe and sometimes fatal disease. The underlying mechanisms behind the breakdown of humoral tolerance to RBC antigens are poorly understood.

Design and Methods

In order to study the pathogenesis of autoimmune hemolytic anemia, we developed a murine model with RBC-specific expression of a model antigen carrying epitopes from hen egg lysozyme and ovalbumin.

Results

Humoral tolerance was observed; this was not broken even by strong immunogenic stimulation (lysozyme or ovalbumin with adjuvant). Autoreactive CD4⁺ T cells were detected by tetramer enrichment assays, but failed to activate or expand despite repeat stimulation, indicating a nonresponsive population rather than deletion. Adoptive transfer of autoreactive CD4⁺ T cells (OT-II mice) led to autoantibody (anti-lysozyme) production by B cells in multiple anatomic compartments, including the bone marrow.

Conclusions

These data demonstrate that B cells autoreactive to RBC antigens survive in healthy mice with normal immune systems. Furthermore, autoreactive B cells are not centrally tolerized and are receptive to T-cell help. As the autoreactive T cells are present but non-responsive, these data indicate that factors that reverse T-cell non-responsiveness may be central to the pathogenesis of autoimmune hemolytic anemia.Key words: tolerance, B cells, T cells, erythrocyte-specific, self-antigen     

Cefotetan-induced immune hemolytic anemia.

Immune hemolytic anemia due to a drug-adsorption mechanism has been described primarily in patients receiving penicillins and first-generation cephalosporins. We describe a patient who developed anemia while receiving intravenous cefotetan. Cefotetan-dependent antibodies were detected in the patient's serum and in an eluate prepared from his red blood cells. The eluate also reacted weakly with red blood cells in the absence of cefotetan, suggesting the concomitant formation of warm-reactive autoantibodies. These observations, in conjunction with clinical and laboratory evidence of extravascular hemolysis, are consistent with drug-induced hemolytic anemia, possibly involving both drug-adsorption and autoantibody formation mechanisms. This case emphasizes the need for increased awareness of hemolytic reactions to all cephalosporins.     

Immunochemical specificity of autoanti-Gerbich from two patients with autoimmune haemolytic anaemia and concomitant alteration in the red cell membrane sialoglycoprotein β

Blood samples from two patients with autoimmune haemolytic anaemia, whose autoantibodies failed to agglutinate certain examples of red cells that lack Gerbich blood group antigens, were studied using immunochemical analyses. One of these autoantibodies differed from all other anti-Ge in that it showed a unique beta sialoglycoprotein (SGP) specificity. It reacted with normal beta but not with the abnormal beta-related SGPs associated with Gerbich-negative red cells of the Gerbich and Yus types. Red cells from this patient had an alteration of beta SGP, while the alpha, gamma and delta SGPs appeared to be normal. The autoantibody from the other patient did not show this unique characteristic. Its immunochemical specificity was similar to alloanti-Ge3 in that it reacted with both beta and gamma SGPs from normal red cell membranes and with the abnormal beta-related SGPs found in red cell membranes from individuals with Gerbich-negative red cells of the Yus type. Red cells from this patient could not be analysed because she had recently received a massive transfusion of red cells.     

Hemolytic anemia due to warm autoantibodies

The diagnosis of autoimmune hemolytic anemia (AHA) requires evidence of shortened red blood cell (RBC) survival mediated by autoantibodies directed against autologous RBCs. About 80 percent of patients with AHA have warm-reactive antibodies of the IgG isotype; the remainder exhibit cold-reactive autoantibodies. Typical patients exhibit anemia, reticulocytosis, spherocytes and polychromasia on the blood film and a positive direct antiglobulin test (DAT). Increased indirect serum bilirubin, urinary urobilinogen and serum lactate dehydrogenase (LDH), and decreased serum haptoglobin are not required for the diagnosis, but are frequently present. Patients with AHA and no underlying associated disease are said to have primary or idiopathic AHA. AHA in patients with associated autoimmune disease and certain malignant or infectious diseases is classified as secondary. The etiology of AHA is unknown. Patients with symptomatic anemia require transfusion of RBCs. Prednisone and splenectomy may provide long term remission. Rituximab, intravenous immunoglobulin, immunosuppressive drugs and danazol have been effective in refractory cases and for patients who are poor candidates for surgery.     

Studies on the specificity of autoantibodies in acquired hemolytic anemia

A significant proportion of patients with autoimmune hemolytic anemia formautoantibodies of blood group specificity, rather than nonspecific autoantibodies.

Ashby survival studies suggest that in such cases properly selected blood,lacking the antigens against which autoantibody has been formed, can be transfused successfully.

These concepts are important not only for a better understanding of thenature of autoantibodies, but also can be applied to the practical management ofpatients with autoimmune hemolytic anemia.

Submitted on November 21, 1955 Accepted on January 26, 1956     

An unusual autoimmune hemolytic anemia in a patient with immunoblastic sarcoma

This report describes a patient with immunoblastic sarcoma and autoimmune hemolytic anemia with reticulocytopenia and red cell hypoplasia. Both IgG and IgM red cell autoantibodies were present in the patient's serum, and both antibodies had the uncommon specificity of anti-I^T. This report appears to be the first to describe IgG auto anti-I^T and autoimmune hemolytic anemia in a patient who did not have Hodgkin's disease; the laboratory findings and clinical course are described. The reticulocytopenia and red cell hypoplasia may have been immunologically mediated. The patient responded to plasma exchange, corticosteroids, and chemotherapy.     

An in vitro method to study the participation of various components in autoimmune hemolytic anemia.

A method is described which allows for the in vitro evaluation of various etiological components in autoimmune hemolytic anemia. As an example of the applicability of the technique, erythrocytes and serum from a patient with an autoimmune hemolytic anemia suspected to be due to poliovirus III were tested with poliovirus III and complement. Incubation of the patient's cells with complement, poliovirus and autologous serum was required for optimal in vitro fragility. The method is proposed as a practical and convenient in vitro technique for evaluating the participation of individual components in the pathogenesis of autoimmune hemolytic anemias.     

Multiple autoantibody production in a patient with splenic lymphoma

Summary We report on a patient with splenic lymphoma of B-cell origin who developed autoimmune hemolytic anemia (AIHA). IgM M-protein, IgM anticardiolipin antibody (ACA), and lupus anticoagulant (LA) were detected in the serum, and direct Coombs' test showed autoantibodies of the IgG₁ and IgG₂ subclasses on red blood cells (RBC). In in vitro culture, tumor cells isolated from the spleen produced only IgM ACA, which was enhanced by IL-6 but not by IL-4 or IL-5. The levels of ACA and LA decreased after splenectomy and chemotherapy; the strength of the direct Coombs' test, however, did not change. These findings indicated that in this patient the lymphoma cells produced IgM. ACA, but not autoantibodies of the IgG₁ and IgG₂ subclasses against RBC. It was also suggested that IL-6 might at least partially stimulate the production of ACA.     

Characterization of Autoantibodies in Mixed-Type Autoimmune Hemolytic Anemia

Three of 46 patients with autoimmune hemolytic anemia (AIHA) satisfied the diagnostic criteria for mixed-type AIHA in which both warm-type and cold-type autoantibodies against red blood cells (RBCs) are present. The specificities of these autoantibodies were analyzed. All of the warm-type autoantibodies were IgG-chi, and the specificities were not serologically classified. The autoantibody of patient 1 reacted to the 41- and 80-kD peptides on immunoblotting, and the epitope corresponding to it was papain sensitive. Two warm-type autoantibodies from patients 2 and 3 resembled each other in serological analyses and reacted with a protease- and neuraminidase-resistant antigen. However, the antigen corresponding to the autoantibody of patient 3 was located on the 37-kD peptide by immunoprecipitation. All of the cold-type autoantibodies were IgM-cha and showed high titer and high thermal amplitude. According to the reaction pattern with untreated and enzyme-treated RBCs, the cold-type autoantibodies of patients 2 and 3 were revealed to be anti-Om and anti-I, respectively. In patient 1, the cold-type autoantibody was characterized as having high affinity for autologous RBCs, but its specificity was unclassified. The antigens corresponding to the cold-type autoantibodies were not located by immunoblotting and immunoprecipitation. These serological and immunochemical approaches to autoantibodies in mixed-type AIHA revealed that the warm and cold components recognized the different antigens.     

Deletion of the dominant autoantigen in NZB mice with autoimmune hemolytic anemia: effects on autoantibody and T-helper responses

The mechanisms underlying apparently spontaneous autoimmune diseases, such as autoimmune hemolytic anemia (AIHA) in New Zealand Black (NZB) mice, are unknown. Here, we determine the contribution of the dominant red blood cell (RBC) autoantigen, the anion exchanger protein Band 3, to the development of NZB autoimmune responses. The approach was to prevent Band 3 expression in NZB mice by disrupting the AE1 gene. AE1(-/-) NZB mice produced RBC autoantibodies at the same levels as the wild-type strain, but they differed in recognizing antigens that correspond to glycophorins, rather than Band 3. Splenic T-helper (Th) cells from wild-type NZB mice proliferated strongly against multiple Band 3 peptides, particularly the dominant epitope within aa861-874. This helper response was severely attenuated in AE1(-/-) animals, leaving only weak proliferation to peptide aa861-874. The results demonstrate that the defect in self-tolerance in NZB AIHA is directed to the RBC type, and is not specific for, or dependent on, Band 3. However, the predisposition to RBC autoimmunity may be focused onto Band 3 by weak Th cell cross-reactivity between the helper dominant epitope and an exogenous antigen. The redundancy of the major autoantigen illustrates the requirement for specific therapy to induce dominant forms of tolerance, such as T-cell regulation.     

Complement inhibitors to treat IgM-mediated autoimmune hemolysis

Complement activation in autoimmune hemolytic anemia may exacerbate extravascular hemolysis and may occasionally result in intravascular hemolysis. IgM autoantibodies as characteristically found in cold autoantibody autoimmune hemolytic anemia, in cold agglutinin disease but also in a considerable percentage of patients with warm autoantibodies are very likely to activate complement in vivo. Therapy of IgM-mediated autoimmune hemolytic anemia mainly aims to decrease autoantibody production. However, most of these treatments require time to become effective and will not stop immediate ongoing complement-mediated hemolysis nor prevent hemolysis of transfused red blood cells. Therefore pharmacological inhibition of the complement system might be a suitable approach to halt or at least attenuate ongoing hemolysis and improve the recovery of red blood cell transfusion in autoimmune hemolytic anemia. In recent years, several complement inhibitors have become available in the clinic, some of them with proven efficacy in autoimmune hemolytic anemia. In the present review, we give a short introduction on the pathogenesis of autoimmune hemolytic anemia, followed by an overview on the complement system with a special focus on its regulation. Finally, we will discuss complement inhibitors with regard to their potential efficacy to halt or attenuate hemolysis in complement-mediated autoimmune hemolytic anemia.     

Autoimmune hemolytic anemia in association with monoclonal IgM(kappa) with anti-i activity.

A patient with a warm autoimmune hemolytic anemia with an immunoglobulin G (IgG) panagglutinin, also had monocional IgM(kappa) cold agglutinin with anti-i activity. Ninety per cent of the peripheral blood lymphocytes had surface immunoglobulin and the number of T cells was diminished. A subpopulation of the patient's lymphocytes formed rosettes with cord (i) erythrocytes and not with adult (l) erythrocytes. The finding of increased lymphocytes bearing i-binding sites and a monoclonal antibody with anti-i activity could be related to shared idiotypic determinants between antigen-binding sites and serum antibody. The occurrence of two autoantibodies in this patient suggests an immune regulatory disorder.