Silent red blood cell autoantibodies: Are they naturally occurring or an effect of tolerance loss for a subsequent autoimmune process?

Unexpected anti-red blood cell (RBC) alloantibodies are routinely investigated in immunohematology and blood banking since their existence in pregnant women may induce haemolytic disease of the foetus and newborn, and their presence in donors may induce haemolytic transfusion reactions or hyperacute rejection in solid organ transplantation. Unexpected anti-RBC alloantibodies may target antigens of the most blood types excluding the expected antibodies targeting the ABO antigens. Their incidence in humans was originally linked to alloimmunization events such as blood transfusions, transplants, or pregnancies. But later, many findings revealed their existence in pathogenic processes such as malignancies, infections, and autoimmune diseases; and usually (but not always) associated to autoimmune haemolytic anaemia (AIHA). Nevertheless, unexpected anti-RBC autoantibodies are also occasionally found in healthy individuals in the absence of AIHA and with no history of alloimmunization or the associated pathologic processes. Hence, they are generally known as non-clinically significant, are excluded for typification and called "silent red blood cell autoantibodies (SRBCAA)". This review highlights evidence related to genetic predisposition, molecular mimicry, immune dysregulation, and immune tolerance loss surrounding the existence of anti-RBC antibodies, describing the presence of SRBCAA as possible early witnesses of the development of autoimmune diseases.

Anticuerpos contra la cardiolipina en glomerulonefritis postestreptocócica concurrente con anemia hemolítica autoinmunitaria: a propósito de un caso / Anticardiolipin antibodies in concurrent poststreptococcal glomerulonephritis and autoimmune hemolytic anemia: A case report

En este artículo, presentamos el caso de una paciente con glomerulonefritis aguda postestreptocócica (GNAPE) y anemia hemolítica autoinmunitaria (AHAI). Además de los signos típicos de la GNAPE, la paciente tuvo un resultado positivo en la prueba de antiglobulina directa y anticuerpos contra la cardiolipina sin que presentara las manifestaciones clínicas típicas del síndrome antifosfolipídico. Este caso genera dudas respecto de la relación entre el estreptococo y el desarrollo de anemia hemolítica autoinmunitaria en los niños. Este caso destaca la posibilidad de que las infecciones estreptocócicas de nuestra paciente podrían haber causado la anemia, ya sea en el contexto de anticuerpos antifosfolipídicos preexistentes o por haber desencadenado el desarrollo de anticuerpos patogénicos, que luego lleva a la presentación clínica de hemólisis. Se presume que, en la paciente, los anticuerpos contra la cardiolipina inducidos por la infección estreptocócica podrían tener una función directa en la presentación clínica de AHAI.

We present a case of acute post-streptococcal glomerulonephritis (APSGN) with autoimmune hemolytic anemia (AIHA). Along with the classic findings of APSGN, the patient had a positive direct antiglobulin test and an anticardiolipin antibody without any typical clinical manifestations of antiphospholipid syndrome (APS). This case raises questions of the relationship between Streptococcus and the development of autoimmune hemolytic anemia in children. Our case highlights the possibility that the streptococcal infections in this patient might be responsible for her anemia, either in setting of underlying antiphospholipid antibodies, or in having triggered the development of pathogenic antibodies, which subsequently leads to the clinical evolution of hemolysis. It is presumed that in our case, the anticardiolipin antibody induced by streptococcal infection may play a direct role in the clinical evolution of AIHA.

Anticardiolipin antibodies in concurrent poststreptococcal glomerulonephritis and autoimmune hemolytic anemia: A case report. / Anticuerpos contra la cardiolipina en glomerulonefritis postestreptocócica concurrente con anemia hemolítica autoinmunitaria: a propósito de un caso.

We present a case of acute post-streptococcal glomerulonephritis (APSGN) with autoimmune hemolytic anemia (AIHA). Along with the classic findings of APSGN, the patient had a positive direct antiglobulin test and an anticardiolipin antibody without any typical clinical manifestations of antiphospholipid syndrome (APS). This case raises questions of the relationship between Streptococcus and the development of autoimmune hemolytic anemia in children. Our case highlights the possibility that the streptococcal infections in this patient might be responsible for her anemia, either in setting of underlying antiphospholipid antibodies, or in having triggered the development of pathogenic antibodies, which subsequently leads to the clinical evolution of hemolysis. It is presumed that in our case, the anticardiolipin antibody induced by streptococcal infection may play a direct role in the clinical evolution of AIHA.

En este artículo, presentamos el caso de una paciente con glomerulonefritis aguda postestreptocócica (GNAPE) y anemia hemolítica autoinmunitaria (AHAI). Además de los signos típicos de la GNAPE, la paciente tuvo un resultado positivo en la prueba de antiglobulina directa y anticuerpos contra la cardiolipina sin que presentara las manifestaciones clínicas típicas del síndrome antifosfolipídico. Este caso genera dudas respecto de la relación entre el estreptococo y el desarrollo de anemia hemolítica autoinmunitaria en los niños. Este caso destaca la posibilidad de que las infecciones estreptocócicas de nuestra paciente podrían haber causado la anemia, ya sea en el contexto de anticuerpos antifosfolipídicos preexistentes o por haber desencadenado el desarrollo de anticuerpos patogénicos, que luego lleva a la presentación clínica de hemólisis. Se presume que, en la paciente, los anticuerpos contra la cardiolipina inducidos por la infección estreptocócica podrían tener una función directa en la presentación clínica de AHAI.

Red blood cell aquaporin-1 expression is decreased in hereditary spherocytosis.

Aquaporin-1 (AQP1) is the membrane water channel responsible for changes in erythrocyte volume in response to the tonicity of the medium. As the aberrant distribution of proteins in hereditary spherocytosis (HS) generates deficiencies of proteins other than those codified by the mutated gene, we postulated that AQP1 expression might be impaired in spherocytes. AQP1 expression was evaluated through flow cytometry in 5 normal controls, 1 autoimmune hemolytic anemia, 10 HS (2 mild, 3 moderate, 2 severe, and 3 splenectomized), and 3 silent carriers. The effect of AQP1 inhibitors was evaluated through water flow-based tests: osmotic fragility and hypertonic cryohemolysis. Serum osmolality was measured in 20 normal controls and 13 HS. The effect of erythropoietin (Epo) on AQP1 expression was determined in cultures of erythroleukemia UT-7 cells, dependent on Epo to survive. Independent of erythrocyte size, HS patients showed a lower content of AQP1 in erythrocyte membranes which correlated with the severity of the disease. Accordingly, red blood cells from HS subjects were less sensitive to cryohemolysis than normal erythrocytes after inhibition of the AQP1 water channel. A lower serum osmolality in HS with respect to normal controls suggests alterations during reticulocyte remodeling. The decreased AQP1 expression could contribute to explain variable degrees of anemia in hereditary spherocytosis. The finding of AQP1 expression induced by Epo in a model of erythroid cells may be interpreted as a mechanism to restore the balance of red cell water fluxes.

Successful rituximab therapy in refractory autoimmune hepatitis and Evans syndrome / Tratamiento exitoso de hepatitis autoinmune y síndrome de Evans con rituximab

A 44-year-old woman was found to have elevated aminotransferases, twice the upper limit of normal. Liver biopsy demonstrated a mixed inflammatory process suggestive of both primary biliary cirrhosis and autoimmune hepatitis (AIH). Prednisone and azathioprine were started, with normalization of aminotransferases. Six months later, she returned with worsening pruritus and re-evaluation demonstrated probable reactivation of AIH with acute elevation of liver injury tests. Repeat liver biopsy was suggestive of a flare of AIH which did not respond to prednisone, azathioprine, or mycophenolate mofetil. One month later the patient was hospitalized for sudden onset of anemia and thrombocytopenia, suggestive of autoimmune hemolytic anemia and idiopathic thrombocytopenic purpura consistent with Evans syndrome. Rituximab was initiated and mycophenolate mofetil discontinued. After one infusion of rituximab, liver injury tests significantly improved. Within four weeks of rituximab infusion (4 doses) the patient's Evans syndrome completely resolved with normal hemoglobin and platelet levels; aminotransferases also significantly improved to less than twice the upper limit of normal.

Successful rituximab therapy in refractory autoimmune hepatitis and Evans syndrome.

A 44-year-old woman was found to have elevated aminotransferases, twice the upper limit of normal. Liver biopsy demonstrated a mixed inflammatory process suggestive of both primary biliary cirrhosis and autoimmune hepatitis (AIH). Prednisone and azathioprine were started, with normalization of aminotransferases. Six months later, she returned with worsening pruritus and re-evaluation demonstrated probable reactivation of AIH with acute elevation of liver injury tests. Repeat liver biopsy was suggestive of a flare of AIH which did not respond to prednisone, azathioprine, or mycophenolate mofetil. One month later the patient was hospitalized for sudden onset of anemia and thrombocytopenia, suggestive of autoimmune hemolytic anemia and idiopathic thrombocytopenic purpura consistent with Evans syndrome. Rituximab was initiated and mycophenolate mofetil discontinued. After one infusion of rituximab, liver injury tests significantly improved. Within four weeks of rituximab infusion (4 doses) the patient's Evans syndrome completely resolved with normal hemoglobin and platelet levels; aminotransferases also significantly improved to less than twice the upper limit of normal.

Adult Evans syndrome: complete hematologic recovery with steroids and rituximab: a case report.

Evans syndrome is an autoimmune disorder characterized by the simultaneous or sequential development of autoimmune hemolytic anemia and immune thrombocytopenia. It may be primary (idiopathic, or associated with other diseases. First line therapy is immunosupression. A second line therapy includes danazol and splenectomy. Rituximab was approved by the Federal Drug Administration since 1998 for the treatment of lymphomas. We report a 46-year-old-male Hispanic with Evans syndrome. He presented with severe life threatening autoimmune hemolytic anemia and subsequently developed autoimmune thrombocytopenia. After treatment with steroids and rituximab he remains in remission. This case report supports the use of rituximab in an adult patient with Evans syndrome.

Expression of CD55 and CD59 on peripheral blood cells from systemic lupus erythematosus (SLE) patients.

CD55 and CD59 are glycosylphosphatidylinositol-anchored proteins with complement inhibitory properties. CD55 inhibits the formation of C3 convertases, and CD59 prevents the terminal polymerisation of the membrane attack complex. It has been reported that SLE patients seems to have an acquired deficiency of these proteins associated with secondary autoimmune haemolytic anaemia and lymphopenia. The aim of this study was to evaluate the presence of altered CD55 and CD59 expression on peripheral blood cells from SLE patients. Flow cytometric analyses were performed on red and white blood cells from 23 SLE patients and 23 healthy controls. We observed more CD55- and CD59-lymphocytes (p=0.005 and p=0.019, respectively), and CD59-granulocytes (p=0.045) in SLE patients than in controls. These results suggest there is an altered pattern of CD55 and CD59 expression on the peripheral blood cells of SLE patients, and it may play a role in the cytopenias in these patients.

Autoimmune hemolytic anemia in systemic lupus erythematosus: association with thrombocytopenia.

Hematological disturbances are common in systemic lupus erythematous (SLE). Specifically, autoimmune hemolytic anemia (AHA) may manifest in SLE patients at the time of diagnosis or within the first year of the disease. AHA is often associated with thrombocytopenia, lupus nephritis, and central nervous system activity. In this study we investigated these associations in Brazilian patients with SLE. Forty-four consecutive SLE patients who had a history of AHA were age, gender, and disease duration matched with 318 SLE patients without AHA who formed the control group. All patients fulfilled the revised American College of Rheumatology criteria for SLE and were followed-up within our Service. Clinical and laboratorial manifestations were similar in both groups, except for the predominance of leukopenia, thrombocytopenia, and anti-dsDNA on univariate analysis in the AHA group. The multivariate logistic regression model revealed risk only for thrombocytopenia in the AHA group compared to the control group (odds ratio, 2.70; 95% confidence interval, 1.32-5.50). Our results corroborate previous data that AHA in SLE increases the risk of thrombocytopenia in individuals with SLE. This association suggests a common mechanism in AHA and SLE pathophysiologies.

Abnormalities in the expression of CD55 and CD59 surface molecules on peripheral blood cells are not specific to paroxysmal nocturnal hemoglobinuria.

The regulatory proteins CD55 and CD59 are glycolsylphosphatidylinositol-anchored, type I cell surface proteins, which inhibit formation of the C3 convertases and prevent the terminal polymerization of the membrane attack complexes, respectively. Paroxysmal nocturnal hemoglobinuria (PNH) is a genetic disorder due to the impaired conformation of the glycolsylphosphatidylinositol anchor, which results in the deficient expression of CD55 and CD59 leading to excessive destruction of red cells and leukocytes. We have studied the expression of these two molecules in red blood cells, granulocytes and platelets in patients with PNH (two patients), autoimmune hemolytic anemia (AIHA) (seven patients), autoimmune thrombocytopenia (ATP) (22 patients), systemic lupus erythematosus (SLE) (19 patients), aplastic anemia (AA) (eight patients), and Evans syndrome (ES) (two patients). A diminished expression of CD55 and CD59 was found in the three cell lines of the two patients with PNH. In the seven patients with AIHA two were found with CD59 diminished expression in red blood cells and one with CD59 diminished expression in granulocytes. In the patients with ATP one was found with CD55 diminished expression in red blood cells, one with CD59 diminished expression granulocytes and one with a CD59 diminished expression in the platelets. In the subset of patients with SLE only one was found with a CD55 diminished expression in the red blood cells. In the patients with AA, a diminished expression in red blood cells was not found; however, one patient was found with a diminished expression of CD59 in granulocytes, and one patient with a diminished expression of CD55 in the platelets. In the two patients with ES we did not found changes in the expression of CD55 and CD59. We conclude that the diminished expression of the glycolsylphosphatidylinositol-anchored type I cell surface proteins CD55 and CD59 is not specific to PNH and that it can be found in patients with a variety of autoimmune disorders. Additional studies are needed to define the role of the deficiencies of CD55 and CD59 in the pathogenesis of autoimmune hemocytopenias.

Autoimmune thrombocytopenia related to chronic hepatitis C virus infection.

Since the identification of hepatitis C virus (HCV) in 1989 as a causative agent for a number of the extrahepatic alterations related to HCV infection an underlying immune mediated pathogenetic mechanism has been postulated. HCV-associated thrombocytopenia may be considered complex and multifactorial in origin, since different mechanisms have been implicated in its pathophysiology. With respect to autoimmune thrombocytopenia in chronic HCV infection, the detection of specific antibodies against platelet glycoproteins have been reported only in a few studies, but no systematic study has been carried out. We examined the clinical, laboratory, and virological characteristics of a case series of 10 patients with autoimmune thrombocytopenia (platelet count <150.0 x 10(9)/L) related to chronic HCV infection. Cases, six males and four females, aged 57.1 +/- 12.6 years, presented high titers of antibodies against platelet glycoprotein (GP) IIb/IIIa, GP Ia/IIa, and/or GP Ib/IX, and no other mechanism involved in the pathogenesis of HCV-associated thrombocytopenia was identified. Furthermore, cases were not associated with particular HCV genotype. Complete platelet response was observed in two patients treated with pegylated interferon plus ribavirin, and partial platelet response was seen in two patients treated with anti-D Ig and one patient treated with corticosteroids. These findings indicate that an autoimmune mechanism may play a role in the pathogenesis of HCV-associated thrombocytopenia in a proportion of these patients.

Deficiency of red cell bound CD55 and CD59 in patients with systemic lupus erythematosus.

CD55 and CD59 are glycosylphosphatidylinositol-anchored proteins with complement inhibitory properties. Autoimmune hemolytic anemia (AIHA) has been associated with antiphospholipid antibodies (APLA). The aim of this study was to evaluate the presence of APLA and its possible correlation with diminished CD55 and CD59 in red blood cells from patients with primary AIHA or secondary to systemic lupus erythematosus (SLE). Flow cytometric analyses were performed on CD55 and CD59 stained erythrocytes from 24 patients (primary AIHA, n=8; AIHA plus SLE, n=11; and SLE without AIHA, n=5) and 20 healthy controls. Antibodies to several phospholipids were detected in the sera by ELISA. Most patients with AIHA plus SLE and few with primary AIHA showed deficiency of either or both CD55 and CD59 expression and was not associated to the presence of APLA, while SLE patients exhibited a normal expression of these molecules. Although our findings showed CD55 and CD59 deficiency in primary or secondary AIHA, it appears that this defect plays a facilitator rather than a triggering role for the hemolytic process. Additionally, a role of anti-phospholipid antibodies as causative of this acquired defect is questionable.

RBC-associated IgG in patients with visceral leishmaniasis (kala-azar): a prospective analysis.

BACKGROUND: Despite the fact that anemia is one of the most striking clinical features of visceral leishmaniasis (kala-azar), the factors involved in its pathogenesis are not fully understood. Although the cause of the anemia seen in these patients is often multifactorial, sequestration and destruction of the RBCs in the enlarged spleen, immune mechanisms, and alterations in RBC membrane permeability have been implicated. STUDY DESIGN AND METHODS: To investigate whether RBCs of patients with kala-azar were coated with IgG, blood samples of 67 patients were tested, prospectively, before (Day 1), during (Day 30), and after (Day 90) antimonial therapy, to examine the presence of RBC-associated IgG, circulating immune complexes (CICs), and rheumatoid factor (RF). RESULTS: The prevalence of a positive DAT on Day 1 was significantly greater than the prevalence of a positive DAT performed on Day 30 and on Day 90 (32.8 vs. 4 vs. 0%, p < 0.001). With an enzyme-linked antiglobulin test (ELAT) to measure the number of IgG molecules per RBC more accurately, it was found that the amount of IgG molecules per RBC was increased (mean, 298 molecules of IgG per RBC) in the group of patients with kala-azar tested before antimonial therapy, but was considered normal (<50 molecules of IgG per RBC) in all patients tested 90 days after treatment. The prevalence of a positive eluate test was low (15.0%) in DAT (anti-IgG)-positive patients and the positivity of DATs and ELATs correlated with the presence of either RF or CICs, respectively. CONCLUSIONS: These data suggest that a nonspecific adsorption of CICs on the RBC surface is probably the most important factor involved in the increased amount of RBC-associated IgG in patients with untreated visceral leishmaniasis; however, further prospective studies are required to establish the exact role of the RBC-associated antibodies, CICs, and RF as contributing factors of the anemia seen in these patients.

Are there "Attenuated" Forms of Evans Syndrome?

Indirect evidence of hemolysis has been described in patients with autoimmune thrombocytopenic purpura. In order to collect more data, another method to assess red blood cell destruction in these patients was employed: measurement of free haptoglobin levels. In 17 individuals with autoimmune thrombocytopenic purpura the levels of free haptoglobins were assessed after carefully ruling out microangiopathic hemolysis, systemic lupus erythematosus or overt Evans syndrome. Abnormally low levels of free haptoglobins were found in five individuals (29%) as indirect evidence of hemolysis. The long-term thrombocytopenia-free status was lower in patients with low haptoglobin levels than in those with normal levels (40 versus 58%). These data, added to previous evidence, support the observation of Evans made 50 years ago: "there is a spectrum-like relationship between primary thrombocytopenia and haemolytic anemia".". Accordingly, the concept of "attenuated" variants of the Evans syndrome could be entertained.

Molecular determination of RhD phenotype by DNA typing: clinical applications.

Rhesus D (RhD) typing is performed by agglutination methods; however, in clinical situations where these techniques cannot be performed, RhD DNA typing is an alternative approach. The Rh antigens are encoded by the RHD and RHCE genes. In RhD-negative individuals the RHD gene is absent or grossly deleted, but variations in the arrangement of the RH locus in different populations are emerging. The aim of this study was to analyse the gross organization of the RH genes in our population using a previously described multiplex polymerase chain reaction (PCR) method with some modifications. We studied 253 DNA samples from Argentinian blood donors, 15 samples with a reduced expression of the D antigen and 1 Dc- phenotype. We evaluated the clinical utility of this method to ascertain the RhD antigen in 10 patients with warm-type autoimmune haemolytic anaemia (AIHA) and 14 samples of amniotic fluids. All Rh phenotypes were properly characterized and no discrepancies with serological typing were found. Analyses performed in the Dc- phenotype suggest the presence of a hybrid RHCE-RHD gene. DNA typing confirmed the RhD-negative type of one AIHA sample in which serological tests were inconclusive. Foetal DNA typing correctly indicated the RhD in every foetus. VNTR (variable number of tandem repeats) and STR (short tandem repeats) analysis detected maternal contamination in two amniocentesis samples and confirmed the foetal origin of 12. This multiplex PCR strategy is suitable for RhD determination in clinical situations in which serological typing cannot be accomplished with its usual ease.