Análise de marcadores de hemólise em portadores de anemia falciforme

Hemolysis in Sickle Cell Anemia (SCA) is predominantly extravascular. The mechanism of removing abnormal or senescent erythrocytes, recently named eryptosis, depends on the exposure of classic erythrocyte phagocytosis signals (signals "eat me"), or signs that may inhibit their clearance by the reticuloendothelial system. Hemolysis also occurs by intravascular mechanismand is associated with the release of free Hemoglobin (Hb) in to the plasma. Haptoglobin and hemopexin proteins are needed to clear free Hb from the plasma. Hydroxyurea (HU), a drug that is used in the treatment of SCA, has among its effects ability to reduce hemolysis. However, little is known about how HU perform this important action. OBJECTIVES: To evaluate the importance of markers of hemolysis in patients with SCA in steady state, with or without the use of HU and those PATIENTS: 40 adult patients with SCA (36 diagnosed with SS and 4 S?0-thalassemia) who were being followed at the Hereditary Anemia Outpatient Clinic of EPM/UNIFESP were evaluated. They were categorized into three groups: a) SS - patients without HU in steady state (N = 20); b) SS-HU -who were using maximum tolerated dose of HU (N = 20). In addition, the control group (CG, N = 22) consisted of apparently healthy hemoglobin AA individuals. The study was approved by the Institution Research Ethics Committee and all patients gave written informed consent. Pregnant women and those on chronic transfusion were excluded from the study. METHODS: Laboratory evaluation: performed included complete blood count, reticulocyte count (Ret), estimation of fetal hemoglobin, indirect serum bilirubin, lactate dehydrogenase (LDH) isoforms and free plasma Hb (free-Hb), haptoglobin (Hp). Expression of band-3, phosphatidylserine, CD47, microvesicles, CD59, were evaluated by flow cytometry (FACSCalibur®, CellQuest Software, Becton Dickinson Biosciences®). Statistical analysis: It was performed by Kruskal-Wallis test and Spearman correlation, with a significance level of 5%. RESULTS/DISCUSSION: As expected, patients groups (SS and SS-HU) had markers of hemolysis significantly different from the control group. LDH showed strong negative correlation with Hp positive and free-Hb, confirming its role as a marker of intravascular hemolysis. The LDH-1, -2 and -3 isoforms showed strong positive correlation with free-Hb, suggesting that LDH-3, though still poorly understood, may also be a useful marker of hemolysis. The expression of band-3 was significantly increased in SS-HU when compared to the other groups analyzed. CD59 expression on RBCs was different among the groups studied, SS-HU showed the highest value and CG the lowest one.A hemólise na Anemia Falciforme (AF) é um dos mecanismos associados às manifestações clínicas da doença. Ela é predominantemente extravascular, a partir da exposição de sinais clássicos de fagocitose no eritrócito (sinais "eat me") contrabalançados por sinais de inibição (?don?t eat me?). O restante da hemólise ocorre por via intravascular, o que resulta na liberação de Hb livre no plasma que, para ser depurada, necessita das proteínas haptoglobina e hemopexina. A hidroxicarbamida (HU), utilizada no tratamento da AF, apresenta entre seus efeitos redução da hemólise. No entanto, pouco se sabe sobre a ação da HU nestes mecanismos. Objetivos: Avaliar a importância de marcadores relacionados à hemólise em indivíduos com AF no estado basal, com e sem uso de HU. CASUÍSTICA: Foram avaliados 40 indivíduos adultos com AF (36 com diagnóstico SS e 4 com S?0-talassemia) acompanhados no Ambulatório de Anemias Hereditárias da EPM/UNIFESP. Estes foram distribuídos nos grupos: a) SS ?pacientes em estado basal sem HU (N=20); b) SS-HU - em uso de dose máxima tolerada de HU (N=20). O grupo controle (N=22) foi constituído por indivíduos saudáveis. O trabalho foi aprovado pelo Comitê de Ética em Pesquisa e todos os pacientes concordaram em participar, assinaram TCLE. Critérios de exclusão:gestantes e indivíduos em transfusão crônica. MÉTODOS: Avaliação laboratorial: hemograma, contagem de reticulóctios (Ret), hemoglobina fetal (HbF), bilirrubina indireta, desidrogenase lática (DHL) e isoformas, Hb livre plasmática (HbL), haptoglobina (Hp). Foram avaliados por citometria de fluxo: expressão de banda-3, fosfatidilserina), CD47, microvesículas (MCV), CD59, (FACSCalibur®, Software CellQuest, Becton Dickinson Biosciences®). Análise estatística: Kruskal-Wallis e Correlação de Spearman, com nível descritivo de 5%. Resultados/DISCUSSÃO: Como esperado, os grupos de pacientes (SS e SS-HU) apresentaram marcadores de hemólise significativamente diferentes do grupo Controle. A DHL apresentou forte correlação negativa com Hp e positiva com HbL, confirmando seu papel como marcador de hemólise intravascular. As isoformas DHL-1, -2 e -3 apresentaram forte correlação positiva com HbL, sugerindo que a DHL-3, com origem ainda pouco esclarecida, possa ser também marcador útil na hemólise. Dos marcadores de eriptose avaliados, observou-se aumento da expressão da banda-3 no grupo SS-HU em relação aos outros grupos. Houve diferença entre os três grupos estudados em relação a expressão do CD59. Além disso, a expressão do CD59 nas MCVs estava aumentada no grupo SS-HU e isso foi diferente em relação aos outros grupos. Conclusões: Apesar da importância do CD47 na eriptose, não foi observada diferença em sua expressão entre pacientes e controles. No entanto, a possibilidade de utilização da isoformas de DHL-3, tanto como marcador de hemólise quanto no monitoramento do uso de HU, necessita ser melhor explorado. O aumento da expressão da banda-3 na presença de HU parece ter um papel importante na reologia do eritrócito. A expressão elevada do CD59 no grupo SS-HU sugere que esta droga possa ter papel protetor na lise do eritrócito.Dados abertos - Sucupira - Teses e dissertações (2013 a 2016

Haptoglobin gene polymorphisms and interleukin-6 and -8 levels in patients with sickle cell anemia

BACKGROUND: Haptoglobin genotypes, and interleukin-6 and -8 participate in the pathophysiology of sickle cell anemia. The expression of cytokines is regulated by genetic mechanisms however the effect of haptoglobin polymorphisms on these cytokines is not fully understood. This study aimed to compare the frequency of haptoglobin genotypes and the interleukin-6 and -8 concentrations in sickle cell anemia patients and controls to investigate the association between haptoglobin genotypes and cytokine levels.METHODS: Sixty sickle cell anemia patients and 74 healthy individuals were analyzed. Haptoglobin genotypes were determined by multiplex polymerase chain reaction, and the interleukin-6 and -8 levels by enzyme linked immunosorbent assay. The association between haptoglobin genotypes and cytokines was investigated by statistical tests.RESULTS:Hp2-1 was the most common genotype in both the cases and controls while Hp1-1 was less frequent among sickle cell anemia patients. Interleukin-6 and -8 levels were higher in patients than controls (p-value 0.05). A similar trend was observed among the controls.CONCLUSION: Although, levels of interleukin-6 and -8 were higher in the sickle cell anemia patients, they appeared not to be related to the haptoglobin genotypes. Further investigations are necessary to identify factors responsible for increased secretion of the interleukin-6 and -8 pro-inflammatory cytokines in patients with sickle cell anemia

Duffy-negative is associated with hemolytic phenotype of sickle cell anemia

Universidade Federal de São Paulo, Disciplina Hematol & Hemoterapia, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Disciplina Hematol & Hemoterapia, BR-04023900 São Paulo, BrazilWeb of Scienc