How I investigate minimal residual disease in acute lymphoblastic leukemia.

Minimal Residual Disease (MRD) is the most important independent prognostic factor in acute lymphoblastic leukemia (ALL) and refers to the deep level of measurable disease in cases with complete remission by conventional pathologic analysis, especially by cytomorphology. MRD can be detected by multiparametric flow cytometry, molecular approaches such as quantitative polymerase chain reaction for immunoglobulin and T-cell receptor (IG/TR) gene rearrangements or fusion genes transcript, and high-throughput sequencing for IG/TR. Despite the proven clinical usefulness in detecting MRD, these methods have differences in sensitivity, specificity, applicability, turnaround time and cost. Knowing and understanding these differences, as well as the principles and limitations of each technology, is essential to laboratory standardization and correct interpretation of MRD results in line with treatment time points, therapeutic settings, and clinical trials. Here, we review the methodological approaches to measure MRD in ALL and discuss the advantages and limitations of the most commonly used techniques.

High-throughput sequencing of immunoglobulin heavy chain for minimal residual disease detection in B-lymphoblastic leukemia.

INTRODUCTION: Minimal residual disease (MRD) is a cornerstone for stratification of upfront B-lymphoblastic leukemia (B-ALL) treatment protocols to decrease relapse risk. Although its detection by flow cytometry (FC) and real-time quantitative polymerase has clinical usefulness, evidence suggests that methods with increased sensitivity could lead to improved outcomes. The aim of this study was to develop an amplicon-based assay followed by high-throughput sequencing of the immunoglobulin heavy chain variable region for MRD detection in B-ALL. METHODS: We analyzed 84 samples, 27 from diagnosis, 5 from relapse, 40 from post-treatment samples, and 12 from healthy controls. RESULTS: Our assay was able to identify more neoplastic clones at diagnosis than Sanger sequencing including incomplete DJ rearrangements. From the 40 MRD samples evaluated 21 were positive by our new approach on high-throughput sequencing assay, but only 15 of these were positive by FC. The remaining 19 were negative by the two techniques. CONCLUSION: We have developed a novel approach on high-sensitive assay for MRD detection in B-ALL, which could add clinical value in the management of patients, especially in cases negative for MRD by FC.

Aquios CL flow cytometer performance in the automated quantification of lymphocyte subpopulations / Desempenho do citômetro de fluxo Aquios CL na quantificação automatizada de subpopulações linfocitárias

ABSTRACT Flow cytometry (FC) is an essential tool for diagnosis, prognosis and therapeutic follow-up of several hematologic malignancies. In addition, it performs the quantification of lymphocytes subpopulations for diagnosis and monitoring of primary and acquired immunodeficiencies through the antigenic expressions of CD19 and CD20 for B lymphocytes; CD2, CD3, CD4, CD8 for T lymphocytes; and CD56 and CD16 for the identification of natural killer (NK) cells. The cytometry technique has revolutionized the way that the cells are identified, and over the years this platform has progressed with several advances in hardware and software that aim to improve workflow resulting in higher productivity, quality and cost savings. The Aquios CL - Beckman Coulter (BC) is an example of this advance because it is a complete automation instrument in flow cytometry called "Load & Go flow cytometer" for quantification of lymphocyte subpopulations in the routine diagnosis. In this study, the Aquios CL was validated, and quantification in frequency and absolute numbers of the lymphocyte subpopulations had an excellent correlation with the results obtained by the dual platform quantification performed in the Cytomics FC500 (BC) and automated Sysmex XE-2100 cell analyzer.

RESUMEN La citometría de flujo (CF) es una herramienta importante para diagnóstico, pronóstico y seguimiento terapéutico de diversas neoplasias hematológicas. Además, posibilita la cuantificación de las subpoblaciones linfocitarias (SPL) para asistencia diagnóstica y monitoreo de las inmunodeficiencias primarias y adquiridas a través de las expresiones antigénicas de CD19 y CD20 para linfocitos B; CD2, CD3, CD4 y CD8 para linfocitos T; y CD56 y CD16 para identificación de células natural killer (NK). La CF ha revolucionado la manera como las células son identificadas y, a lo largo de los años, esa plataforma ha progresado con diversos avances en hardware y software que aspiran a mejorar el flujo de trabajo resultando en mayor productividad, calidad y reducción de costos. El Aquios CL Beckman Coulter (BC) es un ejemplo de ese avance, pues es un instrumento de automación completa en CF (llamado Load & Go flow cytometer) para cuantificación de SPL en la rutina diagnóstica. En este estudio el Aquios CL fue validado, y la cuantificación en números relativos y absolutos de las subpoblaciones linfocitarias tuvo una excelente correlación con los resultados obtenidos por la cuantificación en plataforma dupla realizada en el Cytomics FC500 (BC) y en el analizador automatizado de células Sysmex XE-2100.

RESUMO A citometria de fluxo (CF) é uma ferramenta importante para diagnóstico, prognóstico e acompanhamento terapêutico de diversas neoplasias hematológicas. Além disso, possibilita a quantificação das subpopulações linfocitárias (SPL) para assistência diagnóstica e monitoramento das imunodeficiências primárias e adquiridas por meio das expressões antigênicas de CD19 e CD20 para linfócitos B; CD2, CD3, CD4 e CD8 para linfócitos T; e CD56 e CD16 para identificação de células natural killer (NK). A técnica de CF revolucionou a maneira como as células são identificadas e, ao longo dos anos, essa plataforma tem progredido com diversos avanços em hardware e software que visam melhorar o fluxo de trabalho, resultando em maior produtividade, qualidade e redução de custos. O Aquios CL - Beckman Coulter (BC) é um exemplo desse avanço, pois é um equipamento de automação completa em CF (denominado Load & Go flow cytometer) para quantificação de SPL na rotina diagnóstica. Neste estudo, o Aquios CL foi validado, e a quantificação em números relativos e absolutos das subpopulações linfocitárias teve uma excelente correlação com os resultados obtidos pela quantificação em plataforma dupla realizada no Cytomics FC500 (BC) e no analisador automatizado de células Sysmex XE-2100.

Recommendations for quality assurance in multiparametric flow cytometry: first consensus of the Brazilian Group of Flow Cytometry (GBCFLUX) / Recomendações para garantia da qualidade em citometria de fluxo multiparamétrica: primeiro consenso do Grupo Brasileiro de Citometria de Fluxo (GBCFLUX)

ABSTRACT The Brazilian Group of Flow Cytometry (Grupo Brasileiro de Citometria de Fluxo [GBCFLUX]), founded on April 24, 2010, is composed of experts in flow cytometry (FC) area who have the common objective of contributing to technical and scientific advances in Brazilian clinical and research laboratories. Among GBCFLUX working groups, the Quality Control (QC) subcommittee is responsible for discussing data in the literature and contributes to the quality assurance of the pre-analytical, analytical and post-analytical process in FC. The QC subcommittee's actions began through meetings and lectures, in which data from the literature were reviewed and discussed with all participating members of the GBCFLUX. In a second step, it was decided to draw up a text of technical and scientific consensus recommendations, informative and educative, for dissemination to all FC working groups in Brazil. To this effect, a questionnaire with objective responses was designed and sent to 35 recognized Brazilian institutions, in order to evaluate the QC profile of these institutions. Thus, the QC technical-scientific recommendations, which will be described in this updating article, are intended to ensure the process quality, technical standardization, and reproducibility of results in FC.

RESUMO O Grupo Brasileiro de Citometria de Fluxo (GBCFLUX), fundado em 24 de abril de 2010, é composto por especialistas da área de citometria de fluxo (CF) que possuem o objetivo comum de contribuir para avanços técnico-científicos em laboratórios clínicos e de pesquisa brasileiros. Entre os grupos de trabalho do GBCFLUX, o subcomitê de Controle de Qualidade (CQ) é responsável por discutir dados da literatura e contribuir para a garantia da qualidade do processo pré-analítico, analítico e pós-analítico em CF. As ações do subcomitê de CQ iniciaram-se por meio de reuniões e palestras, nas quais dados da literatura foram revisados e discutidos com todos os membros participantes do GBCFLUX. Em uma segunda etapa, definiu-se elaborar um texto de recomendações consensuais técnicocientíficas, informativas e educativas para divulgação a todos os grupos que trabalham com CF no Brasil. Para tanto, foi elaborado um questionário com respostas objetivas, sendo enviado para 35 instituições brasileiras cadastradas, com a finalidade de avaliar o perfil de CQ dessas instituições. Dessa forma, as recomendações técnico-científicas de CQ que serão descritas neste artigo de atualização têm o objetivo de contribuir para a garantia da qualidade do processo, a padronização técnica e a reprodutibilidade dos resultados em CF.