Detection of PNH cells by flow cytometry, using multiparameter analysis

Introduction:The laboratory diagnosis of paroxysmal nocturnal hemoglobinuria (PNH), disease that is categorized by reduced synthesis of glycosylphosphatidylinositol (GPI) anchor, is based on the detection of blood cells deficient in GPI-anchored proteins by flow cytometry. PNH clones have been detected in patients with aplastic anaemia (AA) and myelodysplastic syndrome (MDS), with therapeutic implications.Objectives:To validate a sensitive assay for detection of GPI-anchored protein-deficient cells, by flow cytometry, and to analyze the clone frequency in AA and MDS patients.Methods:Samples from 20 AA patients, 30 MDS patients and 20 adult volunteers (control group) were analyzed using monoclonal antibodies to CD16, CD24, CD55 and CD59 (neutrophils); CD14 and CD55 (monocytes); CD55 and CD59 (erythrocytes); besides fluorescent aerolysin reagent (FLAER) (neutrophils and monocytes) and lineage markers. The proportions of PNH cells detected in neutrophils and monocytes, using different reagent combinations, were compared by analysis of variance (ANOVA) and Pearson's correlation.Results:PNH cells were detected in five (25%) AA patients, and the proportions of PNH cells varied from 0.14% to 94.84% of the analyzed events. PNH cells were not detected in the MDS patients. However, by the analysis of these samples, it was possible to identify the technical challenges caused by the presence of immature and dysplastic circulating cells. FLAER showed clear distinction of GPI-deficient cells.Conclusion:Multiparameter flow cytometry analysis offers high sensitivity and accuracy in the detection of subclinical PNH clones. FLAER shows excellent performance in detection of PNH neutrophils and monocytes

Diagnóstico laboratorial da doença falciforme em neonatos e após o sexto mês de vida Laboratorial diagnosis of sickle cell disease in the neonate and after the sixth month of life

A doença falciforme é uma desordem genética da hemoglobina, com alta prevalência no Brasil. Apresenta elevada morbidade e mortalidade, necessitando de identificação e tratamento precoces. O reconhecimento disso tornou o seu diagnóstico obrigatório pelos testes de triagem neonatal, em todo o país. O diagnóstico laboratorial da doença no neonato baseia-se na detecção da hemoglobina S utilizando-se preferencialmente as técnicas de IEF e/ou HPLC que apresentam elevada sensibilidade e especificidade. Estes métodos permitem também a detecção dos portadores de traço e de outras hemoglobinas variantes. Na doença falciforme as principais possibilidades fenotípicas ao nascimento são: Hb FS, Hb FSC, Hb FSA e Hb FSD-Punjab. Qualquer que seja o fenótipo encontrado para a doença a criança deverá ser encaminhada à consulta médica e o exame repetido após o sexto mês de vida para confirmação do perfil hemoglobínico. Outros exames complementares podem ser necessários para o diagnóstico diferencial das diferentes formas da doença tais como a dosagem de Hb A2 e Hb Fetal como também a realização do estudo familiar.Sickle Cell disease (SCD) is a genetic disorder of the hemoglobin, with high prevalence in Brazil. It presents high morbidity and mortality with early identification and treatment being necessary. The recognition of this situation has made the diagnosis of SCD mandatory through neonatal screening in Brazil. SCD Laboratorial diagnosis in the neonate is based on the detection of S hemoglobin, in preference, using IEF and/or HPLC techniques, which present high sensitivity and specificity. These methods also allow the detection of carriers traits and other variant hemoglobins. The main phenotypic possibilities on SCD at birth are: Hb FS, Hb FSC, Hb FSA and Hb FSD-Punjab. The child should be referred for medical assessment independent of the phenotype found and the examination being repeated after six months of life to confirm of the hemoglobinic profile. Other complementary examinations may be necessary for differential diagnoses of the different forms of the disease, such as the measurement of Hb A2 and Fetal Hb, as well as a study of the family history

Traço falciforme: heterozigose para hemoglobina S Sickle cell trait: heterozygous for the hemoglobin S

A hemoglobina S (HbS) é uma das alterações hematológicas hereditárias de maior freqüência. No Brasil, a prevalência do traço falciforme (HbAS) varia de 2% a 8%. Com esta freqüência gênica, estima-se, no Brasil, a existência de mais de dois milhões de portadores do traço falciforme. Diferentemente dos portadores em homozigose para a Hb S (Hb SS), os indivíduos com traço falciforme não apresentam sintomas vaso-oclusivos sob condições fisiológicas. Alguns sinais clínicos associados ao traço falciforme somente ocorrem sob condições que propiciam o processo de falcização, como hipóxia, acidose e desidratação. A expectativa de vida é semelhante ao do resto da população. Assim, a condição de portador assintomático não deve ter nenhum impacto no estilo e qualidade de vida.Hemoglobin S (HbS) is one of the most common hereditary hematological alterations. In Brazil, the frequency of the sickle cell trait (HbAS) varies from 2% to 8%. Thus, there is an estimate of more than two million carriers of the HbAS sickle cell trait in Brazil. Different to homozygous carriers for Hb S (Hb SS), individuals with the sickle cell trait do not present with vaso-occlusive symptoms under physiologic conditions. Some clinical signals associated to the sickle cell trait only occur under conditions that favor the sickling process, including hypoxia, acidosis and dehydration. Life expectancy is similar to the general population. Hence, the condition of asymptomatic carrier should not affect the style and quality of life

Incidence and risk factors for central nervous system relapse in children and adolescents with acute lymphoblastic leukemia

BACKGROUND: Despite all the advances in the treatment of childhood acute lymphoblastic leukemia, central nervous system relapse remains an important obstacle to curing these patients. This study analyzed the incidence of central nervous system relapse and the risk factors for its occurrence in children and adolescents with acute lymphoblastic leukemia. METHODS: This study has a retrospective cohort design. The studied population comprised 199 children and adolescents with a diagnosis of acute lymphoblastic leukemia followed up at Hospital das Clinicas, Universidade Federal de Minas Gerais (HC-UFMG) between March 2001 and August 2009 and submitted to the Grupo Brasileiro de Tratamento de Leucemia da Infância - acute lymphoblastic leukemia (GBTLI-LLA-99) treatment protocol. RESULTS: The estimated probabilities of overall survival and event free survival at 5 years were 69.5% ( 3.6%) and 58.8% ( 4.0%), respectively. The cumulative incidence of central nervous system (isolated or combined) relapse was 11.0% at 8 years. The estimated rate of isolated central nervous system relapse at 8 years was 6.8%. In patients with a blood leukocyte count at diagnosis > 50 x 10(9)/L, the estimated rate of isolated or combined central nervous system relapse was higher than in the group with a count < 50 x 10(9)/L (p-value = 0.0008). There was no difference in cumulative central nervous system relapse (isolated or combined) for the other analyzed variables: immunophenotype, traumatic lumbar puncture, interval between diagnosis and first lumbar puncture and place where the procedure was performed. CONCLUSIONS: These results suggest that a leukocyte count > 50 x 10(9)/L at diagnosis seems to be a significant prognostic factor for a higher incidence of central nervous system relapse in childhood acute lymphoblastic leukemia

Deficiência de ferro em lactentes brasileiros com doença falciforme

Objective: To assess iron deficiency or overload in infants with sickle cell disease in order to support the decision to recommend (or not) iron prophylactic supplementation in this population.Methods: Cross-sectional and retrospective study with 135 infants below 2 years old (66 boys and 69 girls), 77 with SS and 58 with SC hemoglobin, born between 2005 and 2006 in Minas Gerais, Brazil. Indicators of possible iron deficiency were: mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), transferrin saturation (TS), and ferritin. Blood transfusions had been given to 17 infants (12.6%, 95% confidence interval [95% CI] 7.0-18.2%) before laboratory tests were done.Results: Ferritin and TS were significantly lower in SC infants (p < 0.001). When two indices were considered for the definition of iron deficiency (low MCV or MCH plus low ferritin or TS), 17.8% of children (95% CI 11.3-24.3%) presented iron deficiency, mainly those with SC hemoglobin (p = 0.003). An analysis of infants who were not given transfusions (n = 118) showed that 19.5% presented iron deficiency. Fifteen infants (11.3%, 95% CI 5.9-16.7%) presented increased ferritin; the majority had been transfused.Conclusions: Most infants with sickle cell disease do not develop iron deficiency, though some have a significant deficit. This study indicates that infants with sickle cell disease, mainly those with SC hemoglobin, may receive prophylactic iron; however, supplementation should be withdrawn after the first blood transfusion.Brazilian Ministry of Health, BrazilUniv Fed Minas Gerais, Dept Pediat, BR-30130100 Belo Horizonte, MG, BrazilFdn Hemominas, Belo Horizonte, MG, BrazilUniv Fed Minas Gerais, Dept Clin Med, BR-30130100 Belo Horizonte, MG, BrazilUniv Fed Minas Gerais, Nucleo Acoes & Pesquisa Apoio Diagnost Nupad, BR-30130100 Belo Horizonte, MG, BrazilUniversidade Federal de São Paulo, São Paulo, BrazilUniversidade Federal de São Paulo, São Paulo, BrazilWeb of Scienc