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.     

Two distinct categories of warm autoantibody reactivity with age-fractionated red cells

Using age-fractionated erythrocytes, warm autoantibodies can be classified into two distinct categories, depending on their reactivity with reticulocyte-enriched (younger) or reticulocyte-poor (older) red cell fractions. The strength of the direct antiglobulin test (DAT) on the age-fractionated red cells of 24 patients indicated that 19 (79%) had an IgG warm autoantibody that reacted preferentially with older red blood cells. In 7 of these 19 patients (37%), the DAT was negative using reticulocyte-enriched red cell fractions. We have termed this preferential reactivity of warm autoantibodies with older red cells as type I. Five of the 24 patients studied (21%) had an IgG warm autoantibody that demonstrated no preference for young or older red cells. We have termed this pattern of warm autoantibody reactivity as type II. All 5 patients having type II warm autoantibodies had severe anemia. In contrast, 6 of 19 patients having type I warm autoantibody did not have clinical evidence of anemia when tested, and 11 of the 19 had only slight to moderate anemia. Additionally, our results using type I warm autoantibody raise questions regarding the blood group specificity of warm autoantibodies. The antigen recognized by type I warm autoantibody may be a cryptantigen. Rh specificity or relative Rh specificity, often associated with warm autoantibodies, may simply be a coincidental finding.     

Use of Preserved Autologous Red Blood Cells to Absorb Warm Autoantibodies from the Serum of Patients Receiving Blood Transfusion Therapy

This paper describes two practical methods for the preservation of pretransfusion patient red blood cells for antigen typing and autoabsorption during a course of transfusion therapy. Blood samples from patients who had serum warm autoantibodies and a positive direct antiglobulin test were collected, the serum frozen, and the red cell aliquots separately preserved by PVP-methanol or formaldehyde fixation. After storage and recovery, the IgG antibodies were dissociated and the cells used for absorption of the warm autoantibodies. The preserved red cells removed the warm autoantibodies as effectively as fresh red blood cells from the same patient. Preservation of autologous red cells prior to the onset of transfusion therapy provides an extension of the autoabsorption procedure and a simple alternative to differential absorption.     

Serologic abnormalities associated with L-dopa therapy

Abstract. Eighty patients with Parkinson's disease participating in a drug trial of L-dopa were studied prospectively for the development of antierythrocyte, antinuclear, and antiglobulin activities. The median age of the group was 65 years; and the median dose of L-dopa was 4 g/day. Five of the 80 patients developed positive red cell antiglobulin tests after 8 to 11 months of therapy. Three of these had IgG only on their red cells; eluates prepared from these cells reacted strongly with all except Rh_null erythrocytes, suggesting Rh specificity. Complement only was detected on the cells of the other 2 patients. One of the 5 individuals with positive erythrocyte antiglobulin tests became anemic. Significant titers of antinuclear antibody appeared in 9 patients and significant increases in titers of serum antiglobulins were found in 9 patients, 2 of whom also had a positive ANA test. These results indicate that administration of L-dopa is associated with induction of a variety of autoantibodies. The mechanism responsible for this phenomenon is unknown.     

Hemolytic anemia associated with multiple autoantibodies and low serum complement

A 37 year old woman with extravascular hemolytic anemia had a positive Monospot test associated with positive antiglobulin and anticomplement Coombs' tests, cold agglutinins and warm autoantibodies. IgG-kappa (κ) antibodies, which reacted with all panel red cells at 37 °C, were eluted from her circulating red cells. However, neither immunoglobulins nor C3 was detected after her serum was adsorbed with heterologous red cell stroma at 37 °C and eluted at the same temperature in glycine buffer. In contrast, IgM-κ and IgM-Iambda (λ), IgG3-κ, IgG4-λ, IgA-λ and C3 were eluted at 37 °C from heterologous red cell stroma after adsorption with her serum at 0 °C. Thus, antibodies of several types, which were present in the patient's serum, reacted optimally with red cell antigens at low temperature. Cold-reactive IgG3-κ antibodies, which were also capable of interacting with red cells at 37 °C, probably accounted for the IgG-κ antibodies eluted from the patient's circulating red cells.The patient's serum C4 titers were decreased, with low normal to moderately depressed C3 and low normal C5, indicating that the anti-red cell IgM and/or IgG3-κ antibodies probably fixed complement.A localized cold stress test resulted in a transient increase in plasma hemoglobin and a decrease in serum C3 titer. These findings, and the beneficial clinical response obtained with small doses of prednisone, suggest that both the cold-reactive antibodies and the IgG-κ on circulating red cells were pathophysiologically significant.This is the first report of a patient with multiple red cell autoantibodies in whom serum complement component titers were determined in conjunction with characterization of the anti-red cell immunoglobulins. Subclinical infectious mononucleosis may have preceded the prolonged hemolytic episode. Clinical evidence of systemic lupus erythematosus has not appeared.     

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.     

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.     

Positive Antiglobulin Test in Association with Sulindac: Involvement of the Rh Factor

We investigated the development of a positive direct antiglobulin test associated with the use of the nonsteroid anti-inflammatory drug sulindac. The drug was shown to be the cause of the positive direct antiglobulin test using red cells treated in vitro with solutions of native sulindac and its two major metabolites. Serum from the patient contained sulindac-dependent red cell antibodies which could not be demonstrated when red cells having the Rh phenotype D– were employed in the test procedure. An eluate prepared from the patients' red cells reacted against untreated red cells having common Rh phenotypes, but not against target red cells with the Rh phenotypes D– or Rh null. The eluate showed stronger reactivity against the cells having common Rh phenotypes when they were treated with solutions of a metabolite of sulindac, but failed to react against treated red cells having the Rh phenotype D– or Rh null. The results of our investigations point to an interaction between sulindac and/or its metabolites and Rh structures on the red cell membrane as the initial step in the production of drug dependent and autoantibodies leading to the positive direct antiglobulin test.     

Amoxicillin-induced immune hemolysis

Severe hemolysis occurred in a 51-year-old female after a 17-day course of intravenous amoxicillin. A strongly positive direct antiglobulin test (anti-IgG titer 1:2,000) ensued which disappeared after withdrawal of the drug. Both the patient's serum and eluate obtained from the patient's red cells contained an IgG antibody which reacted with red blood cells coated in vitro with amoxicillin, but not with uncoated cells. In addition, high-titer antipenicillin, antiampicillin and antiamoxicillin IgG antibodies could be demonstrated in her serum by a RAST-based solid-phase radioimmunoassay. The patient's hemolysis gradually subsided within 1 week after discontinuing the drug. This is the first report of amoxicillin-induced immune hemolytic anemia.     

Autoanti-Ge Associated with Severe Autoimmune Hemolytic Anemia

Severe autoimmune hemolytic anemia due to anti-Ge is described. The patient's red cells had a positive direct antiglobulin test, and they typed as Ge+ using saline reactive reagents. Anti-Ge was eluted from her RBCs, and her serum had an IgG and IgM anti-Ge2,3.     

Significance of multiple types of antibodies on red blood cells of patients with positive direct antiglobulin test: a study of monospecific antiglobulin reactions in 85 patients

Blood samples from 85 patients with a positive direct antiglobulin test were tested with monospecific antiglobulin reagents: anti-IgG, anti-IgM, anti-IgA, and anti-C3. No typical pattern of antiglobulin reaction could be correlated with specific diseases except for the patients with methyldopa-induced positive direct antiglobulin test, all of whom had only IgG on their red blood cells. The presence of more than 1 type of antibody on red blood cells was associated with severe haemolysis. These patients responded less frequently to steroids, and in most of them no underlying disease could be found. Most patients with complement alone on red blood cells had no evidence of haemolysis, and when present it was never severe.     

Anti-Wrb, and Other Autoantibodies Responsible for Positive Direct Antiglobulin Tests in 150 Individuals

SUMMARY Eluates from the red blood cells (and sera whenever free autoantibody was present) of 150 individuals with positive direct antiglobulin tests, have been studied for antibody specificity. Of 87 patients with AIHA, 64 had autoantibodies reacting with all red cell samples including Rh_null. Of these 64 anti-dl autoantibodies, two were, and 32 contained, auto-anti-Wr^b. Of 33 patients being treated with alphamethyldopa, who had developed positive direct antiglobulin tests, 23 had anti-dl autoantibodies four of which contained auto-anti-Wr^b. Of 30 haematologically normal donors with positive direct antiglobulin tests, 23 had anti-dl autoantibodies, two of which were, and six of which contained, auto-anti-Wr^b. The full specificities of autoantibodies, other than anti-Wr^b and anti-dl, in the 150 patients are described, as are the natures of the protein red cell coatings that caused the positive direct antiglobulin tests. The presence of free serum autoantibody as a correlate of the three clinical conditions is reported.
Several observations on auto-anti-Wr^b are documented. The antibody can cause gross red cell destruction in vivo , but can be benign on other occasions; it occurs with approximately the same frequency in AIHA patients and 'normal'donors with positive direct antiglobulin tests, but in fewer patients with alpha-methyldopa induced positive direct antiglobulin tests; it does not activate complement in vivo; , and finally it may eventually provide a clue to the aetiology of AIHA.     

An original method to study autoantibody specificity in haemoglobin stained eluates by the column agglutination techniques

Summary When studying autoantibody specificity by the indirect antiglobulin test with column agglutination techniques ether and xylene elution techniques result in haemoglobin stained eluates which give a red colouration to the gel or glass beads and do not allow the identification of positive reactions. Xylene eluates were incubated with commercially available group 0-test red cell panels at 37°C for 45 min in the wells of a microtitre plate in a 3:1 eluate:red cell ratio. After washing with normal saline, sensitized red cells, resuspended in low ionic strength solution (LISS), were applied onto the microtubes containing the antiglobulin serum and positive reactions were recorded after centrifugation. We studied the specificity of 35 autoantibody containing eluates from 12 patients with lymphoproliferative disorders (six having autoimmune haemolysis) and 23 HIV patients without autoimmune haemolysis. All patients had a gel or column positive (IgG) direct antiglobulin test while the tube direct antiglobulin test failed to show red cell bound IgG. We found a reactive indirect antiglobulin test in 20/23 eluates from HIV infected patients (with a panreactive specificity), in all patients with autoimmune haemolysis (one with anti-C, two with anti-E, one with anti-K and two with a panreactive specificity) and in all patients with positive direct antiglobulin test but without immune mediate haemolysis (in all cases with panreactive specificity). The method proposed is a promising tool for the study of the specificity of antibody containing haemoglobin stained eluates; in this study it allowed us to confirm that some HIV patients have specific binding of IgG on their RBC and to identify the specificity of tube test non-reactive eluates.     

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)     

The Titre of IgG Anti-A/B in the Serum of Group O Mothers of Incompatible Infants with Early Neonatal Jaundice

The titre of IgG anti-A/B was determined by means of a 2-mercaptoethanol test in the serum of 80 blood group O mothers, who gave birth at term to A/B incompatible infants, who developed early neonatal jaundice with a serum bilirubin level of over 10 mg/100 ml. The titres were compared with the results of the direct antiglobulin test performed on the red cells from the cord blood and the necessity for exchange transfusion. Only in mothers of group A infants, a significant correlation was found between the titre of IgG anti-A and the occurrence of a positive antiglobulin test. In no case the need for an exchange transfusion was correlated with the IgG anti-A/B titre.     

The value of gel test and ELAT in autoimmune haemolytic anaemia

Summary Three methods for detection of warm type IgG autoantibody were evaluated using 400 blood samples from 147 patients suspected of autoimmune haemolytic anaemia (AIHA). Three direct antiglobulin techniques (DAT) were used: conventional tube DAT, gel-DAT by micro-method and gel-DAT enzyme linked antiglobulin test (ELAT). Eluate examinations confirmed the presence of autoantibodies on red cells. These tests were compared directly using 126 selected blood samples from 85 patients with IgG molecules on their red cells detected by the gel test. In 106 of these samples, collected from 65 patients with clinical symptoms of AIHA, the presence of autoantibody was confirmed by acid elution. The ELAT was positive in 100 samples (94%), 87 samples for tube DAT (82%). The ELAT as well as the tube DAT was negative in 20 samples with non-reactive eluates by gel test. The gel-DAT was therefore not fully specific and detected IgG on red cells of patients with hypergammaglobulinaemia. However, due its higher sensitivity it proved useful as a screening test. The ELAT allowed changes in the number of IgG molecules per red cell to be monitored quantitatively. Both methods play a part in the diagnosis and monitoring patients with warm type IgG autoantibody.     

Diclofenac-induced immune haemolytic anaemia: simultaneous occurrence of red blood cell autoantibodies and drug-dependent antibodies

During the last 5 years we have identified a total of 17 patients (nine females and eight males aged between 53 and 85 years) with immune haemolytic anaemia related to diclofenac (a nonsteroidal anti-inflammatory drug). All patients developed acute intravascular haemolysis. Two patients died, and eight patients developed temporary renal failure that required haemodialysis. The direct antiglobulin test was positive with anti-IgG and anti-C3d in all cases, with anti-IgA in 4/10 cases tested, and negative with anti-IgM. The indirect antiglobulin test was moderately or weakly positive in 11 cases, and IgG autoantibodies could be eluted from the red blood cells (RBCs) of all patients. Initially, the diagnosis of autoimmune haemolytic anaemia of warm type was suggested in all cases. All patients had simultaneously developed autoantibodies and drug-dependent antibodies. The majority of drug-dependent antibodies ( n =13) reacted with urine containing the drug and its metabolites ( ex vivo antigen), the native drug, and diclofenac-treated RBCs. The antibodies in the remaining four cases were detectable only in the presence of ex vivo antigen. Diclofenac appears to bind only weakly to RBCs in the absence of the drug-dependent antibodies.
We conclude that diclofenac forms neoantigens with RBCs that may stimulate the production of autoantibodies and drug-dependent antibodies. The resulting haemolytic syndrome is very similar to autoimmune haemolytic anaemia of warm type.     

Auto-anti-Jka in Evans''syndrome with negative direct antiglobulin test

A patient presented with haemolytic anaemia and a negative direct antiglobulin test (DAT), and was found to have an IgG antibody with anti-Jka specificity in his serum. His red cells were typed as Jk(a-b+). Later he developed idiopathic thrombocytopenic purpura (ITP), and had a positive DAT due to anti-Jka bound to his red cells, which now typed as Jk(a+b+). Family studies suggested that the patient's true type was Jk(a+b+). Splenectomy and immunosuppression were required to treat the thrombocytopenia. The autoanti-Jka was no longer detectable following therapy.     

Positive direct antiglobulin test associated with echinococcosis: a case report

A patient with hydatid cyst of the liver was found to have a positive direct antiglobulin test, with IgG and complement on his red blood cells (RBCs), but with no evidence of haemolysis. The positive direct antiglobulin test disappeared following surgical removal of the cyst. To the best of our knowledge no such case has been reported in the literature.     

Complement is not activated in ABO-haemolytic disease of the newborn

We studied the lysis in vitro of group A red cells by IgG anti-A. IgG anti-A, which strongly lysed A red cells from adults, did not lyse A red cells from cord blood, if fresh cord serum from a child with blood group AB was used as a source of complement. In cases of haemolytic disease of the newborn due to A-O or B-O antagonism with a positive direct antiglobulin test with anti-IgG serum, the red cells did not react with anti-complement sera. Apparently, complement is also not activated in vivo in case of A-O haemolytic disease of the newborn.