Differential red blood cell age fractionation and Band 3 phosphorylation distinguish two different subtypes of warm autoimmune hemolytic anemia.

Warm autoimmune hemolytic anemia (wAIHA) is a blood disorder characterized by the increased destruction of autologous red blood cells (RBCs) due to the presence of opsonizing pathogenic autoantibodies. Preliminary reports published more than three decades ago proposed the presence of two wAIHA subtypes: Type I, in which autoantibodies preferentially recognize the oldest, most dense RBCs; and Type II, characterized by autoantibodies that show no preference. STUDY DESIGN AND METHODS: We evaluated patients having wAIHA for Type I and II subtype using discontinuous Percoll gradient age fractionation and direct antiglobulin test (DAT). We performed Western immunoblotting and mass spectrometry to show autoantibody specificity for Band 3. We investigated Band 3 tyrosine phosphorylation in different Percoll fractions to determine aging associated with oxidative stress. RESULTS: We confirm the existence of two subtypes of wAIHA, Type I and Type II, and that autoantibodies recognize Band 3. Type I patients were characterized by five Percoll fractions, with a DAT showing IgG opsonization F1 < F5 and elevated Band 3 phosphorylation compared to healthy controls (HCs). In contrast, Type II wAIHA patients were characterized by three to four Percoll fractions, where the DAT IgG opsonization shows F1 ≥ F3/4 and Band 3 phosphorylation was absent or significantly decreased compared to HC. CONCLUSIONS: Type I patients have increased Band 3 tyrosine phosphorylation that may represent accelerated aging of their RBCs resulting in exacerbation of a pathologic form of RBC senescence. Type II patients show decreased Band 3 tyrosine phosphorylation and lack the oldest, most dense RBCs suggesting premature RBC clearance and a more severe wAIHA.

Anemia hemolítica imunomediada primária em cães - Revisão de literatura / Primary immune-mediated hemolytic anemia in dogs - Literature review / Anemia hemolítica inmunomediada primaria en perros ­ Revisión de literatura

A anemia hemolítica imunomediada (AHIM) é o distúrbio imunológico de maior prevalência em cães. Caracteriza-se como uma hipersensibilidade do tipo II, que leva a destruição prematura de hemácias. Dentre as principais complicações, o estado de hipercoagulabilidade predispondo a coagulação intravascular disseminada e tromboembolismo pulmonar é a mais importante, sendo a causa de óbito em mais de 80% dos casos. O diagnóstico é realizado a partir da exclusão de outras causas para anemia e por meio da constatação de um ou mais desses sinais: anemia moderada a grave (hematócrito <25-35%), evidências de hemólise (hemoglobinemia, hemoglobinúria ou hiperbilirrubinemia) e presença de anticorpos na hemácia (caracterizado a partir da auto-aglutinação, esferocitose, teste de Coombs positivo ou citometria de fluxo). O tratamento é direcionado à supressão da resposta imune, sendo os corticosteroides e os imunossupressores, os fármacos de predileção.(AU)

Immune-mediated hemolytic anemia (IMHA) is the most prevalent immune disorder among dogs. It is characterized as type II hypersensitivity, leading to premature destruction of red blood cells. Among the main complications, hypercoagulability predisposing to disseminated intravascular coagulation and pulmonary thromboembolism is the most important, being the cause of death in more than 80% of the cases. The diagnosis is made by excluding other causes for anemia and the presence of one or more of these signs: moderate to severe anemia (hematocrit <25-35%), evidence of hemolysis (hemoglobinemia, hemoglobinuria or hyperbilirubinemia) and presence of antibodies in the erythrocyte (characterized by self-agglutination, spherocytosis, positive Coombs test, or flow cytometry). Treatment is directed to suppression of the immune response, with corticosteroids and immunosuppressants the drugs of predilection.(AU)

La anemia hemolítica inmunomediada (AHIM) es el disturbio inmunológico con mayor prevalencia en perros. Es definido como una hipersensibilidad tipo II, que lleva a destrucción prematura de hematíes. Dentro de las principales complicaciones, el estado de hipercoagulabilidad que predispone a coagulación intravascular diseminada y tromboembolismo pulmonar es el más importante, siendo la causa de muerte en más de 80% de los casos. El diagnóstico se realiza excluyendo otras causas de anemia y confirmando una o más de las siguientes alteraciones: anemia moderada a grave (hematocrito <25-35%), evidencias de hemolisis (hemoglobinemia, hemoglobinuria o hiperbilirrubinemia) y presencia de anticuerpos en hematíes (caracterizado a partir de autoaglutinación, esferocitosis, test de Coombs positivo o citometría de flujo). El tratamiento se basa en la supresión de la respuesta inmune, siendo los cortico esteroides y los inmunosupresores los fármacos de elección.(AU)

Diagnosis and treatment of cold agglutinin mediated autoimmune hemolytic anemia.

Exact diagnosis of the subtype has essential therapeutic consequences in autoimmune hemolytic anemia. Cold-antibody types include primary chronic cold agglutinin disease (CAD) and rare cases of cold agglutinin syndrome (CAS) secondary to cancer or acute infection. Primary CAD is a clonal lymphoproliferative disorder. Not all patients require pharmacological therapy, but treatment seems indicated more often than previously thought. Corticosteroids should not be used to treat primary CAD. Half of the patients respond to rituximab monotherapy; median response duration is 11 months. The most efficient treatment to date is fludarabine and rituximab in combination, resulting in responses in 75%, complete responses in 20% and median response duration of more than 66 months. Toxicity may be a concern, and an individualized approach is discussed. Erythrocyte transfusions can be given provided specific precautions are undertaken. No evidence-based therapy exists in secondary CAS, but optimal treatment of the underlying disorder is essential when feasible.

Classification and therapeutic approaches in autoimmune hemolytic anemia: an update.

Autoimmune hemolytic anemia (AIHA) is an uncommon autoantibody-mediated immune disorder that affects both children and adults. The diagnosis of AIHA relies mainly on the direct antiglobulin test, which is a highly sensitive and relatively specific test. The classification of AIHA is based on the pattern of the direct antiglobulin test and on the immunochemical properties of the autoantibody (warm or cold type), but also on the presence or absence of an underlying condition or disease (secondary vs primary AIHAs) that may have an impact on treatment and outcome. The distinction between AIHAs due to warm antibody (wAIHA) and AIHAs due to cold antibody is a crucial step of the diagnostic procedure as it influences the therapeutic strategy. Whereas corticosteroids are the cornerstone of treatment in wAIHA, they have no or little efficacy in cold AIHA. In wAIHA that is refractory or dependent to corticosteroids, splenectomy and rituximab are both good alternatives and the benefit?risk ratio of each option must be discussed on an individual basis. In chronic agglutinin disease, the most common variety of cold AIHA in adults, beyond supportive measures, rituximab given either alone or in combination with chemotherapy may be helpful. In this article, the classification of AIHA and the recent progress in therapeutics are discussed.

[Evaluation of different prognostic markers in dogs with primary immune-mediated hemolytic anemia]. / Untersuchung verschiedener prognostischer Marker bei Hunden mit primärer immunhämolytischer Anämie.

Canine primary immune-mediated hemolytic anemia (IMHA) is associated with a high mortality rate. Hypothesis of this study was that laboratory parameters not only determined initially but also in the course of the disease might be useful as prognostic markers. Included in the study were dogs with primary IMHA. Inclusion criteria were anemia (PCV < 0.30 L/L), a positive Coombs'test or persistent autoagglutination of erythrocytes, and the exclusion of underlying diseases. Dogs were divided into two groups based on survival: dogs that were still alive 14 days after start of treatment (group 1) and dogs that died or were euthanized before day 14 (group 2). Hematological and biochemical analyses as well as a coagulation profile were performed initially and on day 3. Out of 37 dogs with primary IMHA 28 belonged to group 1 and 9 to group 2. Significantly associated with mortality were thrombocytopenia (p = 0.001), lymphopenia (p = 0.026), a prolonged PT (p = 0.003) and aPTT (p = 0.005), hypofibrinogenemia (p = 0.028), disseminated intravascular coagulation (DIC) (p = 0.019), and high plasma ALT (p = 0.003) and AST (p = 0.004) plasma activities on initial presentation, as well as a decrease in hemoglobin (p = 0.034) and an increase in WBC count (p = 0.034), plasma bilirubin (p = 0.012) and urea concentration (p = 0.003) between day 0 and 3. In conclusion various laboratory parameters were useful as prognostic

[Characteristics of warm autoimmune hemolytic anemia and Evans syndrome in adults]. / Caractéristiques des anémies hémolytiques auto-immunes à anticorps "chauds" et du syndrome d'Evans de l'adulte.

The diagnosis of autoimmune hemolytic anemia (AIHA) relies mainly on the direct antiglobulin test (DAT), that is, Coombs' direct test, which has a sensitivity of about 95%. The classification of different forms of AIHA depends on the characteristics of the autoantibody and is an essential part of the diagnostic procedure. AIHAs mediated by warm-reactive autoantibodies (wAIHA) account for approximately 70% of all types: they may be either idiopathic or secondary to another autoimmune disorder (such as systemic lupus, lymphoma or primary immune deficiencies) or drug-induced. In adults, AIHA may also precede by many years the onset of non-Hodgkin lymphoma (NHL) or myelodysplastic syndrome. The management of wAIHA, based mainly on empirical data and uncontrolled studies, relies principally on corticosteroids as a first-line therapy. In cases of steroid resistance ( approximately 5-10% of the cases) and in cases of steroid-dependency, the most frequent second-line options are splenectomy or immunosuppressive agents. More recently, rituximab has shown promising results in small uncontrolled and retrospective studies and it is now widely used in refractory wAIHA. Future prospective studies should assess its efficacy earlier in the course of disease as a steroid-sparing strategy. Evans syndrome is an autoimmune disorder defined by the simultaneous or sequential combination of AIHA and immune thrombocytopenia or immune neutropenia. It may reveal an underlying condition (e.g., lupus, common variable immunodeficiency, etc.). Its management is usually extrapolated from the standard of care for isolated wAIHA. Before the availability of rituximab, the overall prognosis was relatively poor for adults with both wAIHA and ES, with a mortality rate of 15 to 20%.

[Autoimmune hemolytic anemia].

Diagnosis of autoimmune hemolytic anemia (AIHA) requires both serologic evidence of an autoantibody and hemolysis. Based on the characteristic temperature reactivity of the autoantibody to red cell membranes, AIHA is classified into warm AIHA or cold AIHA (cold agglutinin disease and paroxysmal cold hemoglobinuria). Sensitized RBCs are destructed by intravascular and/or extravascular hemolysis. On the basis of etiology, AIHA are classified as idiopathic or secondary. The common cause of secondary AIHA is lymphoproliferative disorders, autoimmune diseases, and infections. The first line therapy of patients with warm AIHA is glucocorticoids and primary treatment for cold AIHA is avoiding cold exposure. The other standard treatments include splenectomy and immunosuppressive drugs. Recently, rituximab, a monoclonal anti-CD20 antibody, has been used in refractory AIHA with excellent responses.

Autoimmune hemolytic anemia: classification and therapeutic approaches.

Autoimmune hemolytic anemia (AIHA) is a relatively uncommon cause of anemia. Classifications of AIHA include warm AIHA, cold AIHA (including mainly chronic cold agglutinin disease and paroxysmal cold hemoglobinuria), mixed-type AIHA and drug-induced AIHA. AIHA may also be further subdivided on the basis of etiology. Management of AIHA is based mainly on empirical data and on small, retrospective, uncontrolled studies. The therapeutic options for treating AIHA are increasing with monoclonal antibodies and, potentially, complement inhibitory drugs. Based on data available in the literature and our experience, we propose algorithms for the treatment of warm AIHA and cold agglutinin disease in adults. Therapeutic trials are needed in order to better stratify treatment, taking into account the promising efficacy of rituximab.

Anemia hemolítica autoinmune por anticuerpos calientes: reporte de un caso pediátrico / Autoimmune hemolytic anemia, warm antibody: a case report pediatric

Las anemias hemolíticas autoinmunitarias se caracterizan por la presencia de inmunoglobulinas en la superficie eritrocitaria dirigidas contra los determinantes antigénicos de los hematíes. La anemia hemolítica autoinmune por anticuerpos calientes se caracteriza porque los autoanticuerpos actúan a la temperatura del organismo (37°C), son de clase IgG y la hemólisis es predominantemente extravascular, siendo el tipo más frecuente de anemia hemolítica autoinmune en los niños de 2-12 años de edad. Sus manifestaciones clínicas son postración, palidez, ictericia, fiebre y hemoglobinuria. El diagnóstico de las AHAI se establece con la prueba de Coombs. La administración de corticoesteroides constituye el tratamiento inicial de elección. Se presenta el caso de una preescolar femenina de tres años de edad procedente del medio rural, quien exhibe las características clínicas, paraclínica y epidemiológicas de anemia hemolítica autoinmune por anticuerpos calientes, con respuesta satisfactoria a la terapia con esteroides.

Detection of serum hemolysins in autoimmune hemolytic anemia.

BACKGROUND: Some patients with autoimmune hemolytic anemia (AIHA) have in their sera autohemolysins able to hemolyze RBCs in vitro by activation of complement. We describe three autohemolysins in patients with AIHA and we study clinical correlations. STUDY DESIGN AND METHODS: Thirty-two patients with AIHA were explored by immuno-hematological investigations (DAT, elution and serum testing). RESULTS: Three autohemolysins were detected in three patients. All of these autoantibodies were likely IgM and reacted in vitro only with enzyme-treated RBCs. Two warm autohemolysins were detected in patients with warm-type AIHA. The first one was active at neutral pH with low title. The second, having a wide thermal amplitude reacting at 22 degrees C and a title of 16, was acid. The hemolysin detected in patient 3 with cold hemagglutinin disease, was active at 4 and 22 degrees C, at acid pH. The thermal optimum was 4 degrees C and the title 64. It was also detected at 37 degrees C with the same title, but only at neutral pH. CONCLUSION: Although these autohemolysins were incomplete, hemolyzing in vitro only enzyme-treated RBCs, they were associated for the three patients with severe hemolysis.

Warm autoimmune hemolytic anemia is an IgM-IgG immune complex disease.

Warm autoimmune hemolytic anemia (WAIHA) is characterized by polyclonal IgG autoantibodies binding to red blood cells (RBC). The characterization of the autoantigen in WAIHA has not yet led to definitive results, and the etiology of RBC autoantibodies remains unclear. An altered control of self-reactive IgG by autologous IgM has been proposed as the underlying mechanism of disease in WAIHA, suggesting that IgM-IgG immune complexes contribute to the pathophysiology of the disease. In the present study, we purified and characterized IgM from plasma of WAIHA patients and from healthy controls using FPLC-based protocols and optical biosensor technology, and investigated IgG present within the IgM fractions. We provide evidence that IgM-IgG immune complexes in plasma and associated with the RBC membrane are the characteristic feature of WAIHA, independent of the etiology of the disease. IgM-IgG immune complexes of WAIHA patients differ from IgM-IgG immune complexes of healthy individuals with regard to quantity and to structural composition. The data suggest that self-immunoglobulin is the original autoantigen underlying WAIHA. The molecular characterization of IgM-IgG immune complexes may define new targets for therapeutic intervention in WAIHA.

Anemia hemol¡tica autoinmune caliente. Estudio de 183 pacientes / Warm reactive autoimmune hemolytic anemia. Study of 183 cases

Se diagnosticaron 183 pacientes con AHAC, 125 adultos y 58 ni¤os, en el Instituto de Hematolog¡a e Inmunolog¡a de Ciudad de La Habana, Cuba, entre enero de 1990 y septiembre de 1999. La presencia de IgG y de C3 en los hemat¡es fue el patr¢n más com£n y en menores frecuencias se observaron autoanticuerpos de los isotipos IgA e IgM. Se encontr¢ un incremento significativo del patron C3 en los ni¤os en relaci¢n con los adultos y en los ni¤os con AHAC idiopática con respecto a los de AHAC secundaria. En los pacientes con hem¢lisis grave predominaron las combinaciones de IgG y C3 asociado a IgA e IgM. Se detect¢ un 15 por ciento y un 3 por ciento de aloinmunizaci¢n eritrocitaria en los pacientes adultos y pediátricos, respectivamente. No se demostraron diferencias en los patrones de inmunoprote¡nas en los hemat¡es entre los pacientes adultos y pediátricos, excepto un aumento de la frecuencia del patr¢n C3 en estos £ltimos. La hem¢lisis grave se asoci¢ a la presencia de m£ltiples clases de autoanticuerpos.

Anemia hemolítica autoinmune caliente. Estudio de 183 pacientes / Warm reactive autoimmune hemolytic anemia. Study of 183 cases

Se diagnosticaron 183 pacientes con AHAC, 125 adultos y 58 niños, en el Instituto de Hematología e Inmunología de Ciudad de La Habana, Cuba, entre enero de 1990 y septiembre de 1999. La presencia de IgG y de C3 en los hematíes fue el patrøn más común y en menores frecuencias se observaron autoanticuerpos de los isotipos IgA e IgM. Se encontrø un incremento significativo del patron C3 en los niños en relaciøn con los adultos y en los niños con AHAC idiopática con respecto a los de AHAC secundaria. En los pacientes con hemølisis grave predominaron las combinaciones de IgG y C3 asociado a IgA e IgM. Se detectø un 15 por ciento y un 3 por ciento de aloinmunizaciøn eritrocitaria en los pacientes adultos y pediátricos, respectivamente. No se demostraron diferencias en los patrones de inmunoproteínas en los hematíes entre los pacientes adultos y pediátricos, excepto un aumento de la frecuencia del patrøn C3 en estos últimos. La hemølisis grave se asociø a la presencia de múltiples clases de autoanticuerpos. (AU)

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.