Quantificação de blastos por citometria de fluxo no oitavo dia de prednisona e avaliação do perfil de polimorfismos do gene receptor de glicocorticoide NR3C1 em crianças com Leucemia Linfoblástica Aguda
Arquivos
Data
2014
Autores
Sousa, Ana Virginia Lopes de 
Orientadores
Petrilli, Antonio Sérgio
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Objetivos: Avaliar em pacientes com leucemia linfoide aguda (LLA), tratados de acordo com o protocolo do Grupo Cooperativo Brasileiro de Tratamento de Leucemia Linfoide Aguda na Infancia (GBTLI LLA-2009) no oitavo dia da inducao, a reprodutibilidade da contagem de blastos perifericos intra e interobservadores; a concordancia entre citometria de fluxo e morfologia e descrever a distribuicao genotipica dos SNP intron 2 +646C/G e 9βT/C do gene receptor de glicocorticoide NR3C1. Metodos: Foram analisadas prospectivamente, amostras de sangue periferico e/ou medula ossea de 54 pacientes, dos quais 53 foram genotipados para os polimorfismos estudados. Em 31 pacientes, no oitavo dia da inducao, foi realizada a contagem de blastos no SP, em 200 e 100 celulas, com e sem EDTA, por tres observadores fixos, diferentes, com contagens independentes. A imunofenotipagem foi realizada com a tecnica de lise de hemacias e as amostras foram adquiridas e analisadas em citometro de fluxo BD FACSCalibur, com 15.000 eventos totais. Resultados: 59,3% dos pacientes foram do sexo masculino, com idade media de 7,9 anos, leucometria inferior a 50.000/ mm³ em 79,6% e fenotipo LLA comum em 48,1%. Na avaliacao isolada de cada observador, nao houve diferenca na contagem percentual de blastos em 100 ou 200 celulas, com ICC proximo de 1 (p<0,001), independente do anticoagulante. Houve uma concordancia excelente entre as contagens de blastos no SP interobservadores com e sem EDTA, ICC=0,889 (p<0,001) e ICC=0,960 (p<0,001), respectivamente. Nas 27/31(87%) amostras avaliaveis, houve concordancia de 100% entre morfologia e citometria de fluxo quando considerada a categorizacao em maior ou igual a 1.000 ou inferior a 1000 blastos/mm³. Em 4/31(13%) dos pacientes, nao foi possivel essa avaliacao por leucometria inferior a 1.000/mm3 no D8. Observou-se concordancia satisfatoria (rs=0,465; p=0,014) entre morfologia e citometria de fluxo na quantificacao dos blastos. Em 14,8% dos casos ocorreu discordancia entre os metodos; com ausencia de blastos pela CF, e pela morfologia com contagens que variaram de 41 a 925 blastos/mm³. Nos 53 pacientes genotipados para o SNP intron 2 +646C/G, 62,3% apresentaram o genotipo CC, 30,2% CG e 7,5% genotipo GG e, para SNP 9βT/C, 71,7% dos pacientes apresentaram genotipo TT e 28,3% CT. Nao houve associacao entre os perfis genotipicos e sexo, idade, leucometria inicial, linhagem celular e contagem de blastos no D8. Conclusoes: A contagem de linfoblastos, no D8 da inducao, foi altamente reprodutivel entre os diferentes observadores independente da presenca do EDTA; com concordancia satisfatoria entre morfologia e citometria de fluxo na quantificacao dos blastos. Esses dados sugerem a aplicabilidade desse metodo como uma ferramenta livre de subjetividade na avaliacao da contagem de blastos. Em nossa amostra o alelo polimorfico G foi menos prevalente para o SNP intron 2 +646C/G; e o genotipo TT para o SNP 9βT/C foi o mais prevalente, inclusive nos pacientes que apresentaram recaida e obito.
Objective: To assess patients with acute lymphoblastic leukemia (ALL) treated according to the protocol of The Cooperative Brazilian Group of Acute Lymphoblastic Leukemia treatment in childhood (GBTLI LLA-2009) on the eighth day of induction, the reproducitibility of the intra and interobserver peripheral blasts count; concordance between the flow cytometry (FC) and morphology; to describe the genotypes of SNP íntron 2 +646C/G and 9βT/C of the glucocorticoid receptor gene NR3C1. Methods: Peripheral blood samples (PB) and/or bone marrow (BM) of 54 patients were analyzed prospectively from which 53 were genotyped for the research of the studied polymorphisms. In 31 patients, on the eighth day of induction, the blast count in the PB was performed, in 200 and 100 cells, with and without EDTA, by three steady observers, different, with independent counts. The immunophenotyping was carried out with the lysis of erythrocytes technique and samples acquired and analyzed in the flow cytometer BD FACSCalibur, with 15.000 events in total.Results: 59,3% of the patients belonged to male gender at the average age of 7,9 years, leucometry lower than 50.000/ mm³ in 79,6% and common ALL phenotype in 48,1%. In the isolated assessment of each observer, there was no difference in the percentage count of blasts in 100 or 200 cells, with ICC close to 1 (p<0,001), independent of the anticoagulant. There was a great concordance between the counts of blasts in PB interobserver with and without EDTA, ICC=0,889 (p<0,001) and ICC=0,960 (p<0,001) respectively. In the 27/31(87%) available samples, there was a 100% concordance between morphology and FC when considering the categorization higher or equal to 1.000 or lower than 1.000 blasts/mm³.In 4/31(13%) of the patients, it was not possible to perform the assessment for leucometry lower than 1.000/mm3 on D8. The satisfying concordance was observed (rs=0,465; p=0,014) between morphology and FC in the quantification of the blasts. In 14,8% of the cases, there was discordance among the methods; with the absence of the blasts by FC, and by morphology with count that ranged from 41 up to 925 blasts/mm³. In the 53 patients genotyped for the SNP intron 2 +646C/G, the genotype CC occurred in 62,3%, CG in 30,2 % and CG in 7,5 % and, for SNP 9βT/C, 71,7% of the patients presented genotype TT and 28,3% CT. There was no association among the genotypic profiles and gender, age, initial leucometry, cell lineage and the count of blasts on D8. Conclusions: The count of lymphoblasts on D8 of induction, was highly reproducible among the different observers independent of the presence of EDTA, with satisfying concordance between morphology and flow cytometry in the quantification of the blasts. These data suggest the applicability of this method as a tool free from the subjectivity in the assessment of the blasts count. In our sample, the polymorphic allele G was less prevalent for the SNP íntron 2+646C/G; and the genotype TT in SNP 9βT/C was the most prevalent, mainly in the patients who presented relapse and death.
Objective: To assess patients with acute lymphoblastic leukemia (ALL) treated according to the protocol of The Cooperative Brazilian Group of Acute Lymphoblastic Leukemia treatment in childhood (GBTLI LLA-2009) on the eighth day of induction, the reproducitibility of the intra and interobserver peripheral blasts count; concordance between the flow cytometry (FC) and morphology; to describe the genotypes of SNP íntron 2 +646C/G and 9βT/C of the glucocorticoid receptor gene NR3C1. Methods: Peripheral blood samples (PB) and/or bone marrow (BM) of 54 patients were analyzed prospectively from which 53 were genotyped for the research of the studied polymorphisms. In 31 patients, on the eighth day of induction, the blast count in the PB was performed, in 200 and 100 cells, with and without EDTA, by three steady observers, different, with independent counts. The immunophenotyping was carried out with the lysis of erythrocytes technique and samples acquired and analyzed in the flow cytometer BD FACSCalibur, with 15.000 events in total.Results: 59,3% of the patients belonged to male gender at the average age of 7,9 years, leucometry lower than 50.000/ mm³ in 79,6% and common ALL phenotype in 48,1%. In the isolated assessment of each observer, there was no difference in the percentage count of blasts in 100 or 200 cells, with ICC close to 1 (p<0,001), independent of the anticoagulant. There was a great concordance between the counts of blasts in PB interobserver with and without EDTA, ICC=0,889 (p<0,001) and ICC=0,960 (p<0,001) respectively. In the 27/31(87%) available samples, there was a 100% concordance between morphology and FC when considering the categorization higher or equal to 1.000 or lower than 1.000 blasts/mm³.In 4/31(13%) of the patients, it was not possible to perform the assessment for leucometry lower than 1.000/mm3 on D8. The satisfying concordance was observed (rs=0,465; p=0,014) between morphology and FC in the quantification of the blasts. In 14,8% of the cases, there was discordance among the methods; with the absence of the blasts by FC, and by morphology with count that ranged from 41 up to 925 blasts/mm³. In the 53 patients genotyped for the SNP intron 2 +646C/G, the genotype CC occurred in 62,3%, CG in 30,2 % and CG in 7,5 % and, for SNP 9βT/C, 71,7% of the patients presented genotype TT and 28,3% CT. There was no association among the genotypic profiles and gender, age, initial leucometry, cell lineage and the count of blasts on D8. Conclusions: The count of lymphoblasts on D8 of induction, was highly reproducible among the different observers independent of the presence of EDTA, with satisfying concordance between morphology and flow cytometry in the quantification of the blasts. These data suggest the applicability of this method as a tool free from the subjectivity in the assessment of the blasts count. In our sample, the polymorphic allele G was less prevalent for the SNP íntron 2+646C/G; and the genotype TT in SNP 9βT/C was the most prevalent, mainly in the patients who presented relapse and death.
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Citação
SOUSA, Ana Virginia Lopes de. Quantificação de blastos por citometria de fluxo no oitavo dia de prednisona e avaliação do perfil de polimorfismos do gene receptor de glicocorticoide NR3C1 em crianças com Leucemia Linfoblástica Aguda. Dissertação (Mestrado em Ciências) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2014.