Red Cell Autoantibodies Research Articles

Red cell autoantibodies in patients with acquired immune deficiency syndrome : McGinniss MH, Macher AM, Rook AH, et al. Transfusion 26:405, 1986

Red cell autoantibodies in patients with acquired immune deficiency syndrome : McGinniss MH, Macher AM, Rook AH, et al. Transfusion 26:405, 1986

Novel V genes encode virtually identical variable regions of six murine monoclonal anti-bromelain-treated red blood cell autoantibodies.

The variable (V) region sequences of six immunoglobulin M (IgM, kappa) monoclonal autoantibodies that recognize bromelinized isologous red blood cells, obtained by fusions of peritoneal cells from NZB or CBA/J nonimmunized mice with BALB/c myeloma cells, were determined by direct mRNA sequencing. The V regions of the light chains (VL) are almost identical with one another, as are the V regions of the heavy chains (VH), which, however, differ by six linked-base substitutions, depending on the strain of mice producing the autoantibodies. Such variations may reflect allelic differences. The VH segments determined have no obvious correspondence to any VH genes identified so far. They may belong to the small VH group 4, where 73% homology, at the most, can be calculated at the protein level for codons 1 to 94. Alternatively, the VH regions may be members of a new group of VH sequences not previously found. The V kappa regions appear closely homologous to members of the V kappa-9 subgroup of myeloma proteins of unknown antigen-binding specificity. The joining segments, J kappa and JH, used by the autoantibodies investigated, originate from the J kappa 2 and JH1 germ-line gene segments, respectively. The nine base-long diversity segments, D, derive from one member of the germ-line D gene SP2 family.

Red cell autoantibodies in patients with acquired immune deficiency syndrome.

Mild-to-profound anemia, thrombocytopenia, and rarely neutropenia have been observed in patients with the acquired immune deficiency syndrome (AIDS). To investigate a possible immune mechanism, blood samples from 28 hospitalized AIDS patients, four asymptomatic homosexual men, four homosexual men with the AIDS-related lymphadenopathy syndrome, 30 hospitalized patients with diseases other than AIDS, and 60 blood donors were tested for the presence of atypical red cell antibodies. Eighteen AIDS patients (64%) had anti-i, nine (32%) had autoanti-U, and 12 (43%) had a positive direct antiglobulin test. One asymptomatic homosexual man and three homosexual men with lymphadenopathy also had anti-i. In contrast, of the 30 patients with diseases other than AIDS and 60 donors, none had anti-U or a positive direct antiglobulin test. One patient with sickle cell disease had anti-i. The mean hemoglobin level of AIDS patients with anti-i or anti-U was significantly lower than the mean hemoglobin level of patients who did not have those antibodies.

細管式等速電気泳動法による自己免疫性溶血性貧血患者の赤血球Ｄｉｓｐａｓｅ処理液ならびに血清の分析

On the red blood cell membrane in a patient with autoimmune hemolytic anemia (AIHA) several glycophorin class blood group antigens were assumed to be acquired in relation to the disease. When the red blood cells were treated with Dispase, all of the acquired antigens were released to the supernatant in company with the native glycophorin class antigens and disappeared on the red blood cell membrane. Then a trial was made to analyze the supernatant of the red blood cell suspension with Dispase and the serum which contained the red cell autoantibody by capillary isotachophoresis. In each of the isotachopherogram, a specific peak which was not detected in the sample from normal human was found in the acidic zone. The protein substances expressed by these peaks will be available as a marker to diagnose AIHA because they are thought to be formed in association with AIHA.

Drug induced red blood cell autoantibodies co-developed with drug specific antibodies causing haemolytic anaemias.

Four individuals with anti-glafenine, anti-latamoxef and anti-teniposide antibodies were found to have an associated red blood cell autoantibody. The two components could be separated by selective absorption and showed distinct time course patterns. In three patients a well-defined blood group antigen was recognized as the receptor for both auto- and drug specific antibodies. Similarities between this type of immune response to drugs and the well-known hapten and carrier specificities developed in animals immunized by hapten-carrier conjugates are discussed.

Increased IgG molecules bound to the surface of red blood cells of patients with sickle cell anemia

We have used the complement-fixing antibody consumption ( CFAC ) test to detect small concentrations of IgG on red blood cells from patients with hemolytic anemias that are not thought to be caused by an immune mechanism. Although patients with hereditary spherocytosis, pyruvate kinase deficiency, and mechanical hemolytic anemias generally had normal concentrations of IgG bound to their red cells (less than 25 molecules IgG per red cell), we found that 39/62 (63%) patients with sickle cell anemia had elevated values. These 39 patients had a mean of 195 and a maximum of 890 molecules of IgG per red cell. None of the patients had been transfused within the previous 90 days, and some had never been transfused. Direct antiglobulin tests were positive in only two instances and autoantibodies were not found in the serum of any patient. However, eluates from the red cells of 6 of 23 patients demonstrated antibody activity against all of a panel of normal red cells by the indirect antiglobulin test. There was no correlation between the number of IgG molecules on patients' red cells and the severity of their anemia, the incidence of painful sickle cell crises, the reticulocyte count, or with blood transfusion history. We conclude that further study of immunohematologic abnormalities in patients with sickle cell anemia is warranted, especially in view of previous reports in this population of patients with red cell autoantibodies, autoimmune hemolytic anemia, hemolytic transfusion reactions without detectable alloantibodies, and an association of some episodes of pain crises with immunologically mediated red cell destruction.

Increased IgG Molecules Bound to the Surface of Red Blood Cells of Patients With Sickle Cell Anemia

We have used the complement-fixing antibody consumption ( CFAC ) test to detect small concentrations of IgG on red blood cells from patients with hemolytic anemias that are not thought to be caused by an immune mechanism. Although patients with hereditary spherocytosis, pyruvate kinase deficiency, and mechanical hemolytic anemias generally had normal concentrations of IgG bound to their red cells (less than 25 molecules IgG per red cell), we found that 39/62 (63%) patients with sickle cell anemia had elevated values. These 39 patients had a mean of 195 and a maximum of 890 molecules of IgG per red cell. None of the patients had been transfused within the previous 90 days, and some had never been transfused. Direct antiglobulin tests were positive in only two instances and autoantibodies were not found in the serum of any patient. However, eluates from the red cells of 6 of 23 patients demonstrated antibody activity against all of a panel of normal red cells by the indirect antiglobulin test. There was no correlation between the number of IgG molecules on patients' red cells and the severity of their anemia, the incidence of painful sickle cell crises, the reticulocyte count, or with blood transfusion history. We conclude that further study of immunohematologic abnormalities in patients with sickle cell anemia is warranted, especially in view of previous reports in this population of patients with red cell autoantibodies, autoimmune hemolytic anemia, hemolytic transfusion reactions without detectable alloantibodies, and an association of some episodes of pain crises with immunologically mediated red cell destruction.

Use of intravenous immunoglobulin in chronic lymphocytic leukemia

Five patients with clinical stage III and IV chronic lymphocytic leukemia were treated with a five-day infusion of high-dose intravenous immunoglobulin for either autoimmune hemolytic anemia or immune thrombocytopenic purpura or by a dose of intravenous immunoglobulin G every three to four weeks for prevention and control of infections associated with hypogammaglobulinemia. The hematocrlt level stabilized with a decrease in red blood cell destruction by intravenous immunoglobulin G in chronic lymphocytic leukemia patients with AIHA without altering red blood cell autoantibody titers, but severe hemolysis recurred after 21 days. A long-term remission was observed when intravenous immunoglobulin G was combined with steroids, chemotherapy, plasmapheresis, and splenectomy. The platelet count increased and platelet transfusion requirement was eliminated in a chronic lymphocytic leukemia patient with immune thrombocytopenic purpura by the intravenous immunoglobulin G, and chemotherapy was administered and a complete remission resulted. An interesting observation was the lymphocytopenic effects observed in the five patients during a daily infusion of intravenous immunoglobulin for five days. Furthermore, the maintenance intravenous immunoglobulin G given every three weeks controlled both infections and lymphocyte counts with the chemotherapy. Changing the schedule to every four weeks resulted in poor control of the lymphocyte counts with chemotherapy. These observations indicate a direct macrophage blocking effect in autoimmune hemolytic anemia and immune thrombocytopenic purpura, but the lymphocytopenic effect suggests immunomodulating effects of intravenous immunoglobulin G, which may alter the response of chronic lymphocytic leukemia cells to chemotherapy.

Experimental erythrocyte autoantibodies: V. Induction and suppression of red blood cell autoantibodies in mice injected with rat bromelain-treated red blood cells

Mice injected with rat red blood cells (RBC), or rat bromelain-treated (brom) RBC, produce RBC autoantibodies and suppressor cells that specifically inhibit the autoimmune response without inhibiting the net production of antibodies against rat RBC. It has been investigated whether suppressor cells induced by injections of rat RBC are effective in preventing autoantibody production induced by rat brom RBC and vice versa. Autoantibodies were induced in C 3H mice by weekly ip injections, each 0.2 ml, of a 6% suspension of rat RBC or rat brom RBC. Autoantibody production was assayed using Coombs' test. Suppressor cells were present in the spleens of mice positive in Coombs' tests and were shown by intravenous injections of 40 × 10 6 viable cells per mouse into untreated syngeneic mice 18 hr before the first injection of rat RBC or rat brom RBC. Autoantibodies eluted from mice positive in Coombs' tests after injections of rat RBC or brom RBC were absorbed by either type of rat RBC but not by RBC from sheep. This suggests that rat RBC and rat brom RBC display antigens that are similar, if not identical, to autoantigens on the mouse RBC. Spleen cells from mice injected with rat RBC suppressed autoantibodies induced by both rat RBC and rat brom RBC. In contrast, spleen cells from mice injected with rat brom RBC suppressed autoantibodies induced by rat brom RBC but not those induced by unmodified rat RBC. This differential suppression may be due to the removal from rat RBC, by bromelain, of a suppressor site and/or autoantigens of some specificities. Thus rat brom RBC may not induce the total range of specificities of autoantibodies, and of suppressor cells, induced by rat RBC.

Deposition of the terminal C5b-9 complement complex on erythrocytes by human red cell autoantibodies.

A radioassay for the detection of complement activating autoantibodies (autohaemolysins) in patients with autoimmune haemolytic anaemia (AIHA) is described. The method is based on immunoradiometric quantitation of the cytolytic C5b-9 complement complex following its antibody-dependent deposition on red blood cells (RBC). The use of affinity-purified, radiolabelled antibodies directed against the neoantigens of the C5b-9 complex ensured specificity of the test which proved more sensitive than conventional haemolytic assays and was not subject to disturbances by haemolytic sera. The results obtained in 70 patients with various forms of AIHA (warm type N = 45); cold type (N = 22); Donath-Landsteiner type (N = 3] support the prevailing assumption that autohaemolysins can trigger the complement cascade to completion. Since intact RBC from patients with detectable autohaemolysins carried C3/C4 components but never C5b-9, it is inferred that regulatory mechanisms operate in vivo at the level of C4b/C3b inactivation to arrest the cascade and rescue the autologous cells.

Simplified method for recovery of autologous red blood cells from transfused patients.

Separation of autologous and transfused red blood cells from recently transfused patients is necessary for the proper identification of any red blood cell alloantibody or autoantibody. We compared two methods of separation: the standard technic of microhematocrit centrifugation with phthalate ester solution, and a simplified method of microhematocrit centrifugation without the use of esters. Autologous red blood cells were concentrated in the top layer of the capillary tube by both methods. Separation efficacy was comparable, as determined by blood group antigen reactivity. Good separation was achieved only in samples drawn three or more days post transfusion. Microhematocrit centrifugation without the use of phthalate esters is a simple method for the recovery of autologous red blood cells from recently transfused patients that can be performed by any standard clinical laboratory.

Labeling of Rh antibodies on solid-phase protein A

Rh 0(D) antibodies which retain immune specificity after radiolabeling were prepared by a procedure which does not require IgG isolation from serum, requires 10-fold less isotope than conventional techniques and yields antibody solutions of defined composition. The method involves radioiodination of IgG on immobilized protein A, depends on employing human red cells reduced in surface cytophilic IgG, and exploits the inability of goat IgG to interact with Staphylococcus aureus protein A. The technique concentrates IgG by affinity adsorption and should prove useful in preparing radiolabeled alloantibodies from dilute human antisera and for red cell autoantibodies.

Autoimmune haemolytic anaemia with monoclonal IgM (κ) anti-P cold autohaemolysins

A patient is described with immunoblastic non-Hodgkin's lymphoma and autoimmune haemolytic anaemia of the cold autoantibody type. The autoantibodies were monoclonal IgM-kappa cold haemagglutinins/haemolysins with blood-group specificity, anti-P. Red-cell autoantibodies directed against blood-group-P antigen have until now only been detected, as polyclonal IgG antibodies, in paroxysmal nocturnal haemoglobinuria.

Specific immunoadsorption of IgG antibody in a patient with chronic lymphocytic leukemia and autoimmune hemolytic anemia: A new form of therapy for the acute critical stage

Progressive and severe autoimmune hemolytic anemia developed in a patient with chronic lymphocytic leukemia (CLL) despite treatment with chlorambucil, high doses of corticosteroids and attempts to transfuse packed red blood cells. Splenectomy was not performed because of severe coronary artery disease. Direct antiglobulin tests revealed a warm red blood cell autoantibody of IgG-type with anti-e specificity. The patient was treated by extracorporeal immunoadsorption of plasma IgG using a cell separator and protein A as the immunoadsorbent. The patient responded by an increase in the hemoglobin levels and platelet counts after two treatments. Specificity of the procedure was shown by a decrease in the serum IgG and by the demonstration of the same reactivity to ficin-treated reagent red blood cell panel of the eluate from the protein A. Antibody titers of the patient's red blood cell eluate decreased from 1:128 to 1:64 and eventually the anti-e specificity was lost. This is a report of a novel approach to treatment of the acute phase of an autoimmune hemolytic anemia.

Assessment of red cell autoantibodies in autoimmune hemolytic anemia of warm type by a radioactive anti-IgG test.

To evaluate the applicability of a radioactive 125I-anti-IgG test (RIAT) for the detection of small amounts of IgG antibodies on red blood cells (RBC) of patients with autoimmune hemolytic anemia of warm type (AIHA), RBC of 125 patients were studied (AIHA, n = 53; Coombs'-negative AIHA, n = 6; chronic cold agglutinin disease, n = 7; non-immune anemias, n = 59). It was found that the RBC of all cases (33/33) with a positive direct IgG antiglobulin test (DAT-IgG), but also 13 out of 20 patients with a negative DAT-IgG, but detectable complement (C3/C4), and 4 out of 6 cases with Coombs'-negative AIHA gave positive results in the direct RIAT. RBC-associated IgG was higher in the DAT-IgG positive group (n = 33; -x = 8.1%) than in the DAT-IgG negative group (n = 26; -x3.4%). There was no correlation between hypergammaglobulinemia and RBC-associated IgG. The sensitivity of the indirect RIAT was not remarkable better as compared to the indirect antiglobulin test. The RIAT is valuable in the serology of borderline cases of AIHA.

Assessment of Red Cell Autoantibodies in Autoimmune Hemolytic Anemia of Warm Type by a Radioactive Anti-IgG Test

Assessment of Red Cell Autoantibodies in Autoimmune Hemolytic Anemia of Warm Type by a Radioactive Anti-IgG Test

Murine autoimmune hemolytic anemia induced via xenogeneic erythrocyte immunixation: IV. Immunosuppression of the response

The immunization of mice with rat erythrocytes has been demonstrated to induce a direct antiglobulin-positive hemolytic anemia. This study was undertaken to evaluate the immunosuppressive effect of passive antibody and/or lymphoid subpopulation cell transfer on this induced autoimmune response. The passive transfer of IgG mouse antirat erythrocyte antibody was extremely effective in protecting against induction of antimouse red cell autoantibodies, whereas IgM mouse anti-rat erythrocyte antibody was ineffective. In contrast, transfer of spleen cells, singly primed with rat red blood cell (Rrbc), to syngeneic recipients enhanced development of a direct antiglobulin-positive hemolytic anemia. However, transfer of hyperimmunized spleen cells suppressed the response. Although Rrbc-specific suppressor T cells appeared to play a role in this suppression, anti-Rrbc antibody appears to be the major mediator of the inhibition. Indeed, the suppressive capacity of donor cells, in all transfer experiments, was proportional to the transfer of inhibitory anti-Rrbc antibody and related to the kinetics of autoantibody formation.

Methyldopa Inhibition of Suppressor-Lymphocyte Function

To test the hypothesis that methyldopa induces red-cell autoantibodies by inhibiting the activity of suppressor lymphocytes, we studied its effect on several immune functions. Methyldopa inhibited T-lymphocyte suppression of IgG production by peripheral-blood mononuclear cells stimulated by poke-weed mitogens. This effect occurred in isolated T cells incubated with methyldopa and in T cells obtained from patients taking methyldopa. In addition, the drug caused a 30 to 80 per cent reduction in the proliferative response of peripheral-blood mononuclear cells to mitogens in vitro, and this reduction primarily involved the activation of T lymphocytes. Methyldopa also caused a persistent elevation of intracellular lymphocyte cyclic AMP in vitro and in vivo. We postulate that methyldopa alters the immune system by causing a persistent increase in lymphocyte cyclic AMP, which inhibits suppressor T-cell function. These effects may lead to unregulated autoantibody production by B cells in some patients.

Autoantibodies Mimicking Alloantibodies

A patient with myelofibrosis, who has produced many red blood cell autoantibodies, is described. Although the patient is phenotypically R1R1 (CDe/CDe), eluates made from his red blood cells have consistently contained what appeared to be anti-E, and more recently another antibody that appeared to be anti-c. In in vitro experiments we have shown that the "anti-E" and "anti-c" can be totally adsorbed by E-negative and c-negative red blood cells, respectively. We conclude that the two antibodies have quite different specificities from those indicated by simple antibody identification studies, and that both are more closely related to the anti-Hr series of antibodies than to anti-E or anti-c.

Drug-induced immune hemolytic anemia: Garratty G, Petz LD: Am J Med 58: 398–407, 1975

Drug-induced immune hemolytic anemia (DIIHA) is rare, and a specialized laboratory is often required to provide the optimal serological tests to confirm the diagnosis. The most common drugs associated with DIIHA and the hypotheses for the mechanisms thought to be involved have changed during the last few decades. The drugs most frequently associated with DIIHA at this time are cefotetan, ceftriaxone, and piperacillin. DIIHA is attributed most commonly to drug-dependent antibodies that can only be detected in the presence of drug (eg, cephalosporin antibodies). DIIHA can also be associated with drug-independent antibodies; such antibodies do not need drug to be present to obtain in vitro reactions (eg, fludarabine). In these latter cases, the drug affects the immune system, causing production of red cell (RBC) autoantibodies; the clinical and laboratory findings are identical to autoimmune hemolytic anemia (AIHA), other than the remission associated with discontinuing the drug. Some of the mechanisms involved in DIIHA are controversial. The most acceptable one involves drugs, like penicillin, that covalently bind to proteins (eg, RBC membrane proteins); RBCs become coated with drug in vivo and, a drug antibody (usually IgG) attaches to the drug-coated RBCs that are subsequently cleared by macrophages. The most controversial is the so-called immune complex mechanism, which has been revised to suggest that most drugs are capable of binding to RBC membrane proteins, but not covalently like penicillins. The combined membrane plus drug can create an immunogen; the antibodies formed can be IgM or IgG and often activate complement, leading to acute intravascular lysis and sometimes renal failure; fatalities are more common in this group. It is still unknown why and how some drugs induce RBC autoantibodies, sometimes causing AIHA.