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.

Use of capillary blood to diagnose hereditary spherocytosis.

We studied 31 children with hemolytic anemia, or with positive family history for hereditary spherocytosis (HS), to assess the reliability of capillary blood samples for the diagnosis. HS was diagnosed in 20 patients. Cryohemolysis (CH) was positive in 94% and eosin-5'-maleimide flow cytometry in 90% of them, whereas flow cytometric osmotic fragility was positive in 94%. Capillary blood sampling showed to be useful for the diagnosis. Simultaneous use of these three tests allows confirming diagnosis in 100% of patients. The use of very small blood volumes (300 µl) allows an earlier diagnosis in neonates and small infants.

A prospective study to assess the predictive value for hereditary spherocytosis using five laboratory tests (cryohemolysis test, eosin-5'-maleimide flow cytometry, osmotic fragility test, autohemolysis test, and SDS-PAGE) on 50 hereditary spherocytosis families in Argentina.

This prospective study was carried out to assess the usefulness of five laboratory tests in the diagnosis of hereditary spherocytosis (HS), based on the correlation of erythrocyte membrane protein defects with clinical and laboratory features, and also to determine the membrane protein deficiencies detected in Argentina. Of 116 patients and their family members tested, 62 of them were diagnosed to have HS. The specificity of cryohemolysis (CH) test was 95.2%, and its cut-off value to distinguish HS from normal was 2.8%. For flow cytometry, cut-off points of 17% for mean channel fluorescence (MCF) decrease and 14% coefficient of variation (CV) increase showed 95.9% and 92.2% specificity, respectively. Both tests showed the highest percentages of positive results for diagnosis. Either CH or flow cytometry was positive in 93.5% of patients. In eight patients, flow cytometry was positive only through CV increase. Protein defects were detected in 72.3% of patients; ankyrin and spectrin were the most frequently found deficiencies. The CV of the fluorescence showed significantly higher increases in moderate and severe anemia than in mild anemia (p = 0.003). Severity of anemia showed no other correlation with tests results, type of deficient protein, inheritance pattern, or neonatal jaundice. CH and flow cytometry are easy methods with the highest diagnostic accuracy. Simultaneous reading of mean channel fluorescence (MCF) decrease and CV increase improve diagnostic usefulness of flow cytometry. This test seems to be a reliable predictor of severity. The type of detected protein deficiency has no predictive value for outcome. Predominant ankyrin and spectrin deficiencies agree with reports from other Latin American countries.

[Hemolytic anemia due to hemoglobin Evans in an Argentinean family]. / Anemia hemolítica causada por hemoglobina Evans en una familia argentina.

Unstable hemoglobins are structural variants of the hemoglobin molecule, mostly originated by single amino-acid replacement in some globin chains. These changes affect molecule stability, leading to loss of solubility, precipitation, and cellular lysis. Patients carrying these unstable hemoglobins may present mild to severe chronic hemolytic anemia. Hemoglobin Evans is an unstable variant originated by replacement of valine with methionine at position 62 of the α-globin chain. We have identified this variant in a girl with an acute hemolytic crisis associated to pharyngitis, as well as in two of her family members. This is the third case of hemolytic anemia due to hemoglobin Evans reported in the literature.

Anemia hemolítica causada por hemoglobina evans en una familia argentina / Hemolytic anemia due to hemoglobin evans in an Argentinean family

Las hemoglobinas inestables son variantes estructurales de lamolécula de hemoglobina originadas, en su mayoría, por sustitucionespuntuales de aminoácidos en alguna de las cadenas de globina. Estos cambios afectan la estabilidad de la molécula,causan pérdida de la solubilidad y precipitación dentro del eritrocito, lo cual provoca su destrucción acelerada. Desde el punto de vista clínico, las hemoglobinas inestables puedenpresentar anemia hemolítica crónica de gravedad variable.La hemoglobina Evans es una hemoglobina inestable causadapor la sustitución de valina por metionina en la posición 62 de la cadena de alfa globina. Hemos identificado esta variante en una niña con crisis hemolítica aguda asociada a faringitis y en dos miembros de su grupo familiar. Éste es el tercer caso de anemia hemolítica congénita causada por hemoglobina Evans comunicado en la bibliografía mundial.

Unstable hemoglobins are structural variants of the hemoglobin molecule, mostly originated by single amino-acid replacement in some globin chains. These changes affect molecule stability, leading to loss of solubility, precipitation, and cellular lysis. Patients carrying these unstable hemoglobins may present mild to severe chronic hemolytic anemia. Hemoglobin Evans is an unstable variant originated by replacement of valine with methionine at position 62 of the αa-globin chain. We have identified this variant in a girl with an acute hemolytic crisis associated to pharyngitis, as well as in two of her family members. This is the third case of hemolytic anemia due to hemoglobin Evans reported in the literature.

[Recombinant erythropoietin as treatment for hyporegenerative anemia following hemolytic disease of the newborn]. / Tratamiento de la anemia hiporregenerativa tardía de la enfermedad hemolítica del recién nacido con eritropoyetina recombinante.

INTRODUCTION: The aim of the study is to report results of erythropoietin treatment for late hyporegenerative anemia in the hemolytic disease of the newborn (HDN). Reports previously published concern only a few cases, with controversial results. METHODS: Case series report concerning 50 neonates with HDN due to Rh, ABO or KpA antigens, aged more than 7 days. Erythropoietin treatment started when hematocrit dropped to levels requiring transfusion, with an inappropriate reticulocyte response (Reticulocyte Production Index <1). RESULTS: At start of treatment mean age was 24.3 +/- 12.0 days (range 8-65 days), hematocrit 24.1 +/- 2.8% (range 18-30%), and Reticulocyte Production Index 0.34 +/- 0.25 (range 0.05-0.98). Hematocrit and Reticulocyte Production Index showed significant increases after 7 and 14 days of treatment (p <0.001). No difference was observed either between infants with Rh-HDN and ABO-HDN or between Rh-HDN patients with or without intrauterine transfusions. Seven infants (14%) required one packed RBC transfusion during erythropoietin therapy, 2 of them within 72 hours from starting treatment. The percentage of transfused infants showed no difference either between ABO-HDN and Rh-HDN or between Rh-HDN with and without intrauterine transfusions. Moderate, short-lasting neutropenia, not associated to infections, was observed in 11 patients. No other adverse effect was observed. CONCLUSIONS: The administration of erythropoietin appears to be a safe and useful therapy. Its efficacy should be confirmed by randomized studies.

Tratamiento de la anemia hiporregenerativa tardía de la enfermedad hemolítica del recién nacido con eritropoyetina recombinante / Recombinant erythropoietin as treatment for hyporegenerative anemia following hemolytic disease of the newborn

Introducción. La experiencia previamente publicada sobre el tema se limita a pocos casos, con resultadoscontrovertidos. Objetivo. Comunicar los resultados del tratamiento con eritropoyetina en la anemia hiporregenerativa tardía de la enfermedad hemolítica del recién nacido (EHRN). Población, material y métodos. Estudio observacional prospectivo sobre 50 neonatos mayores de 7 días con EHRN secundaria a incompatibilidadesRh, ABO o KpA. Se comenzó tratamiento con eritropoyetina cuando el hematócrito descendía a valores que requerían transfusión, acompañado de una respuesta reticulocitaria inadecuada (Indice de Producción Reticulocitaria <1). Resultados. Al comienzo del tratamiento la edad fue 24,3 ± 12,0 días (intervalo 8-65 días), el hematócrito24,1 ± 2,8% (intervalo 18-30%) y el Índice de Producción Reticulocitaria 0,34 ± 0,25 (intervalo 0,05-0,98). Se observaron aumentos significativosdel hematócrito y del Indice de Producción Reticulocitaria a los 7 y 14 días de tratamiento (p <0,001). No hubo diferencias entre los niños con EHRN-Rh y con EHRN-ABO, o entre los pacientes con EHRNRh que habían recibido o no transfusiones intrauterinas.Durante el tratamiento con eritropoyetina fueron transfundidos 7 niños (14%), 2 de ellos durante las primeras 72 h de su comienzo. No hubodiferencias en el porcentaje de pacientes transfundidos entre aquellos con EHRN-Rh o con EHRNABO, o entre los pacientes con EHRN-Rh que habíanrecibido o no transfusiones intrauterinas. Se observó neutropenia moderada de corta duración, no asociada a infecciones, en 11 pacientes. No se registró ningún otro efecto adverso. Conclusiones. La eritropoyetina parece ser un tratamiento útil y seguro. Su eficacia deberá ser confirmada por futuros estudios aleatorizados.

Introduction. The aim of the study is to report results of erythropoietin treatment for late hyporegenerativeanemia in the hemolytic disease of thenewborn (HDN). Reports previously published concern only a few cases, with controversial results. Methods. Case series report concerning 50 neonates with HDN due to Rh, ABO or KpA antigens, aged more than 7 days. Erythropoietin treatment started when hematocrit dropped to levels requiring transfusion, with an inappropriate reticulocyte response (Reticulocyte Production Index <1). Results. At start of treatment mean age was 24.3 ± 12.0 days (range 8-65 days), hematocrit 24.1 ± 2.8% (range 18-30%), and Reticulocyte Production Index 0.34 ± 0.25 (range 0.05-0.98). Hematocrit and Reticulocyte Production Index showed significant increases after 7 and 14 days of treatment (p <0.001). No difference was observed either between infantswith Rh-HDN and ABO-HDN or between Rh-HDN patients with or without intrauterine transfusions. Seven infants (14%) required one packed RBC transfusion during erythropoietin therapy, 2 of them within 72 hours from starting treatment. The percentage of transfused infants showed no differenceeither between ABO-HDN and Rh-HDN or between Rh-HDN with and without intrauterine transfusions. Moderate, short-lasting neutropenia, not associatedto infections, was observed in 11 patients. No other adverse effect was observed. Conclusions. The administration of erythropoietin appears to be a safe and useful therapy. Its efficacy should be confirmed by randomized studies.

Tratamiento de la anemia hiporregenerativa tardía de la enfermedad hemolítica del recién nacido con eritropoyetina recombinante / Recombinant erythropoietin as treatment for hyporegenerative anemia following hemolytic disease of the newborn

Introducción. La experiencia previamente publicada sobre el tema se limita a pocos casos, con resultadoscontrovertidos. Objetivo. Comunicar los resultados del tratamiento con eritropoyetina en la anemia hiporregenerativa tardía de la enfermedad hemolítica del recién nacido (EHRN). Población, material y métodos. Estudio observacional prospectivo sobre 50 neonatos mayores de 7 días con EHRN secundaria a incompatibilidadesRh, ABO o KpA. Se comenzó tratamiento con eritropoyetina cuando el hematócrito descendía a valores que requerían transfusión, acompañado de una respuesta reticulocitaria inadecuada (Indice de Producción Reticulocitaria <1). Resultados. Al comienzo del tratamiento la edad fue 24,3 ± 12,0 días (intervalo 8-65 días), el hematócrito24,1 ± 2,8% (intervalo 18-30%) y el Indice de Producción Reticulocitaria 0,34 ± 0,25 (intervalo 0,05-0,98). Se observaron aumentos significativosdel hematócrito y del Indice de Producción Reticulocitaria a los 7 y 14 días de tratamiento (p <0,001). No hubo diferencias entre los niños con EHRN-Rh y con EHRN-ABO, o entre los pacientes con EHRNRh que habían recibido o no transfusiones intrauterinas.Durante el tratamiento con eritropoyetina fueron transfundidos 7 niños (14%), 2 de ellos durante las primeras 72 h de su comienzo. No hubodiferencias en el porcentaje de pacientes transfundidos entre aquellos con EHRN-Rh o con EHRNABO, o entre los pacientes con EHRN-Rh que habíanrecibido o no transfusiones intrauterinas. Se observó neutropenia moderada de corta duración, no asociada a infecciones, en 11 pacientes. No se registró ningún otro efecto adverso. Conclusiones. La eritropoyetina parece ser un tratamiento útil y seguro. Su eficacia deberá ser confirmada por futuros estudios aleatorizados.(AU)

Introduction. The aim of the study is to report results of erythropoietin treatment for late hyporegenerativeanemia in the hemolytic disease of thenewborn (HDN). Reports previously published concern only a few cases, with controversial results. Methods. Case series report concerning 50 neonates with HDN due to Rh, ABO or KpA antigens, aged more than 7 days. Erythropoietin treatment started when hematocrit dropped to levels requiring transfusion, with an inappropriate reticulocyte response (Reticulocyte Production Index <1). Results. At start of treatment mean age was 24.3 ± 12.0 days (range 8-65 days), hematocrit 24.1 ± 2.8% (range 18-30%), and Reticulocyte Production Index 0.34 ± 0.25 (range 0.05-0.98). Hematocrit and Reticulocyte Production Index showed significant increases after 7 and 14 days of treatment (p <0.001). No difference was observed either between infantswith Rh-HDN and ABO-HDN or between Rh-HDN patients with or without intrauterine transfusions. Seven infants (14%) required one packed RBC transfusion during erythropoietin therapy, 2 of them within 72 hours from starting treatment. The percentage of transfused infants showed no differenceeither between ABO-HDN and Rh-HDN or between Rh-HDN with and without intrauterine transfusions. Moderate, short-lasting neutropenia, not associatedto infections, was observed in 11 patients. No other adverse effect was observed. Conclusions. The administration of erythropoietin appears to be a safe and useful therapy. Its efficacy should be confirmed by randomized studies.(AU)

Demographic data, natural history, and prognostic factors of idiopathic thrombocytopenic purpura in children: a multicentered study from Argentina.

BACKGROUND: Demographics, outcome, and management of idiopathic thrombocytopenic purpura (ITP) in children present differences between countries. Although several factors influence outcome, it is impossible to predict at diagnosis which patients will have acute or chronic disease. High rates of spontaneous remission in chronic ITP have been reported. PROCEDURE: Data concerning 1,683 patients with ITP diagnosed from 1981 to date are presented; outcome was evaluated in 1,418 children. RESULTS: Remarkable presenting features were an incidence peak in the first 2 years of age and male predominance in patients <24 months of age. Three age groups with different recovery rates (P < 0.001) were established (2-12 months: 89.8%; 1-8 years: 71.3%; 9-18 years: 49.0%). Platelet count <10 x 10(9)/L and history of previous illness were associated with higher remission rates only in patients >12 months of age. The score developed by the NOPHO Group showed a predictive value of 83.9% for acute ITP. Spontaneous remission between 6 months and 11 years from diagnosis was achieved by 107 of 325 (32.9%) non-splenectomized children with chronic ITP, and in 44.9% of them between 6 and 12 months from diagnosis. CONCLUSIONS: Age and score were main prognostic factors. Infants <1 year of age are a special group with a brief course and very high recovery rate that are not influenced by other prognostic factors. Definition of groups based on age and scoring could be useful to establish differential management guidelines. The cut-off value to define chronic ITP should be changed to 12 months.

Splenectomy and spontaneous remission in children with chronic idiopathic thrombocytopenic purpura.

Two hundred thirty of 696 evaluable children were identified as having chronic idiopathic thrombocytopenic purpura (ITP). Splenectomy was performed in 30 (13%), achieving remission in 22 (73%). Favorable response was associated to higher initial platelet count. Spontaneous remission was achieved by 53/200 non-splenectomized patients (26.5%), up to 10 years from diagnosis. More than half of them recovered between 6th and 12th month from diagnosis. The recovery rate was significantly higher (P=0.03) in children aged<9 years (31.2%) than in older children (14.6%). No reliable factor predictive of response in individual cases is still available.