ROR1 expression in mature B lymphoid neoplasms by flow cytometry.

Immunophenotyping by flow cytometry is an integral part of the diagnosis and classification of leukemias/lymphomas. The expression of ROR1 associated with chronic B lymphocytic leukemia (CLL) is well described in the literature, both in its diagnosis and in the follow-up of minimal residual disease (MRD) research, however, there are few studies regarding the expression pattern of ROR1 in other subtypes of mature B lymphoid neoplasms. With the aim of evaluating the expression of ROR1 and associating it with the expression of other important markers for the differentiation of mature B lymphoid neoplasms (MBLN), 767 samples of cases that entered our laboratory for immunophenotyping with clinical suspicion of MBLN were studied. ROR1 expression is predominant in CD5+/CD10- neoplasms. Overall, positive ROR1 expression was observed in 461 (60.1%) cases. The CD5+/CD10- group had a significantly higher proportion of ROR1 positive samples (89.9%) and more brightly expressed ROR1 than the other groups. Our results highlight the importance of evaluating ROR1 expression in the diagnosis of MBLN to contribute to the differential diagnosis, and possibly therapy of mainly CLL, and indicate that this marker could be considered as a useful addition to immunophenotypic panels, particularly for more challenging cases.

Performance of eight-color dry antibody reagent in the detection of minimal residual disease in chronic lymphocytic leukemia samples.

BACKGROUND: Minimal residual disease (MRD) in chronic lymphocytic leukemia (CLL) has prognostic and predictive significance. One of the approaches to detect MRD by flow cytometry (FC) is the use of dry antibody reagents such as DuraClone® RE CLB (Beckman Coulter-BC). The aim of this study was to evaluate the performance of the DuraClone® RE CLB in detecting MRD in CLL compared to liquid reagents. METHODS: DuraClone® RE CLB is composed by CD81FITC, ROR1PE, CD79bPC5.5, CD19PC7, CD5APC, CD43APCA750, CD20PB, and CD45KrO. For the liquid reagent assay, we used CD43FITC, ROR1PE, CD3ECD, CD5PC5.5, CD20PC7, CD79bAPC, CD19APC750, CD81 APCH7, and CD45KrO. The liquid and dry tubes were used to detect 20 MRD-positive CLL samples. The samples were analyzed using Radar Plots Kaluza Software (BC). RESULTS: The statistical correlation between the liquid and dry reagents was acceptable (R2 = .9583) and no discrepancy was observed in MRD percentages. The average of the total number of acquired events in DuraClone® RE CLB was 758.583 (362.632-2.290.387), which allowed accurate sensitivity for the FC assay. The lowest MRD frequency detected by DuraClone® RE CLB was 0.01%, corresponding to a cluster with 106 events in a total of 737.030. The radar plots allowed the discrimination between normal B-cell population and CLL cells. CONCLUSION: The DuraClone® RE CLB method allowed the accurate detection of MRD in clinical and interlaboratorial CLL samples, thereby supporting the use of this method to potentially increase productivity, reduce pipetting-associated errors and cost, and allow better standardization.

Myelodysplastic syndrome: validation of flow cytometry multilineage score system.

OBJECTIVE: To validate multilineage score system correlating results of flow cytometry, cytogenetics, cytomorphology and histology from samples of patients with suspected myelodysplastic syndrome or cytopenia of unknown origin. METHODS: A retrospective study analyzing laboratory data of 49 patients with suspected myelodysplastic syndrome or cytopenia of unknown origin, carried out between May and September 2017. The inclusion criteria were availability of flow cytometry results, and at least one more method, such as morphology, histology or cytogenetics. Thirty-eight patients were classified as diagnosis of myelodysplastic syndromes, whereas 11 were classified as normal. Patients were evaluated based on score systems, Ogata score and flow cytometry multilineage score. RESULTS: Comparing the scores obtained in the Ogata score and the multilineage score, it was observed that in four cases the Ogata score was zero or 1 point, while the multilineage score was higher than 3 points. In addition, in 12 cases with Ogata score of 2, the multilineage score was greater than 3. CONCLUSION: The flow cytometry multilineage score system demonstrated to be more effective in dysplasia analysis, by assessing the erythroid, monocytic, granulocytic and precursor cell lineages, apart from the parameters evaluated by the Ogata score.

Myelodysplastic syndrome: validation of flow cytometry multilineage score system / Síndrome mielodisplásica: validação de ficha de escore multilinhagem por citometria de fluxo

ABSTRACT Objective To validate multilineage score system correlating results of flow cytometry, cytogenetics, cytomorphology and histology from samples of patients with suspected myelodysplastic syndrome or cytopenia of unknown origin. Methods A retrospective study analyzing laboratory data of 49 patients with suspected myelodysplastic syndrome or cytopenia of unknown origin, carried out between May and September 2017. The inclusion criteria were availability of flow cytometry results, and at least one more method, such as morphology, histology or cytogenetics. Thirty-eight patients were classified as diagnosis of myelodysplastic syndromes, whereas 11 were classified as normal. Patients were evaluated based on score systems, Ogata score and flow cytometry multilineage score. Results Comparing the scores obtained in the Ogata score and the multilineage score, it was observed that in four cases the Ogata score was zero or 1 point, while the multilineage score was higher than 3 points. In addition, in 12 cases with Ogata score of 2, the multilineage score was greater than 3. Conclusion The flow cytometry multilineage score system demonstrated to be more effective in dysplasia analysis, by assessing the erythroid, monocytic, granulocytic and precursor cell lineages, apart from the parameters evaluated by the Ogata score.

RESUMO Objetivo Validar ficha de escore multilinhagem correlacionando resultados obtidos de citometria de fluxo, citogenética, citomorfologia e histologia de amostras de pacientes com suspeita de síndrome mielodisplásica ou citopenias a esclarecer. Métodos Estudo retrospectivo de análise de dados laboratoriais de 49 pacientes com suspeita clínica de síndrome mielodisplásica ou citopenias a esclarecer realizado entre maio e setembro de 2017. Os critérios de inclusão foram a disponibilidade de resultados de citometria de fluxo e de, pelo menos, outra metodologia, entre morfologia, histologia, ou citogenética. Trinta e oito pacientes foram classificados como diagnosticados com síndromes mielodisplásicas enquanto 11 foram classificados como normais. Os pacientes foram avaliados utilizando sistemas de escore, escore de Ogata e ficha multilinhagem. Resultados Comparando as pontuações obtidas no escore de Ogata e na ficha multilinhagem, observou-se que, em quatro casos, o score de Ogata foi zero ou 1 ponto, enquanto, pela ficha multilinhagem, a pontuação foi superior a 3 pontos. Além disso, em 12 casos com escore de Ogata 2, a pontuação pela ficha multilinhagem foi superior a 3. Conclusão A ficha multilinhagem demonstrou ser mais eficaz na análise de displasia por avaliar as linhagens eritroide, monocítica, granulocítica e células precursoras, além dos parâmetros avaliados no escore de Ogata.

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.

Detection of Central Nervous System Infiltration by Myeloid and Lymphoid Hematologic Neoplasms Using Flow Cytometry Analysis: Diagnostic Accuracy Study.

INTRODUCTION: Infiltration of the central nervous system (CNS) by hematologic or lymphoid malignant cells can cause extensive neurological damage, be progressive and fatal. However, usually, the cerebrospinal fluid (CSF) has low cellularity and rapid cell degeneration, which can impair cytometry analysis. Storage and transport measures, sample preparation, and staining protocols can interfere with diagnostic accuracy. OBJECTIVE: To calculate the diagnostic performance of flow cytometry (FC) using a cell stabilizer for sample preservation compared to cytomorphology in the detection of CNS infiltration by lymphoid and hematologic neoplasms. METHODS: Cell samples from all consecutive patients with suspected infiltration by hematological malignancies evaluated between January 2014 and December 2016 were included. Cases were analyzed by FC using a cell preservation medium and cytomorphology. Sensitivity and specificity were calculated. RESULTS: From 414 CSF samples, 72 had a phenotype compatible with characteristics of infiltration by hematological disease, whereas cytology was positive for 35 cases. FC showed higher sensitivity and specificity when compared to cytomorphology, particularly in cases with cellularity under 5 leukocytes/mm3. CONCLUSION: We demonstrated that collecting CSF in a medium that preserves the stability of the sample improves accuracy when compared to cytomorphology, particularly in low-volume and low-cellularity samples.

Technical advances in flow cytometry-based diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria.

OBJECTIVE:: To discuss the implementation of technical advances in laboratory diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria for validation of high-sensitivity flow cytometry protocols. METHODS:: A retrospective study based on analysis of laboratory data from 745 patient samples submitted to flow cytometry for diagnosis and/or monitoring of paroxysmal nocturnal hemoglobinuria. RESULTS:: Implementation of technical advances reduced test costs and improved flow cytometry resolution for paroxysmal nocturnal hemoglobinuria clone detection. CONCLUSION:: High-sensitivity flow cytometry allowed more sensitive determination of paroxysmal nocturnal hemoglobinuria clone type and size, particularly in samples with small clones. OBJETIVO:: Discutir as melhorias técnicas no diagnóstico e no acompanhamento laboratorial de hemoglobinúria paroxística noturna para a validação da técnica de citometria de fluxo de alta sensibilidade. MÉTODOS:: Estudo retrospectivo, que envolveu a análise de dados laboratoriais de 745 pacientes com hipótese diagnóstica e/ou acompanhamento de hemoglobinúria paroxística noturna por citometria de fluxo. RESULTADOS:: Os avanços técnicos não só reduziram o custo do ensaio, mas também melhoraram a identificação e a resolução da citometria de fluxo para a detecção de clone hemoglobinúria paroxística noturna. CONCLUSÃO:: A citometria de fluxo de alta sensibilidade possibilitou a identificação do tipo e do tamanho de clone de hemoglobinúria paroxística noturna, especialmente em amostras com pequeno clone.

Technical advances in flow cytometry-based diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria / Avanços técnicos no diagnóstico e no monitoramento de hemoglobinúria paroxística noturna por citometria de fluxo

ABSTRACT Objective: To discuss the implementation of technical advances in laboratory diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria for validation of high-sensitivity flow cytometry protocols. Methods: A retrospective study based on analysis of laboratory data from 745 patient samples submitted to flow cytometry for diagnosis and/or monitoring of paroxysmal nocturnal hemoglobinuria. Results: Implementation of technical advances reduced test costs and improved flow cytometry resolution for paroxysmal nocturnal hemoglobinuria clone detection. Conclusion: High-sensitivity flow cytometry allowed more sensitive determination of paroxysmal nocturnal hemoglobinuria clone type and size, particularly in samples with small clones.

RESUMO Objetivo: Discutir as melhorias técnicas no diagnóstico e no acompanhamento laboratorial de hemoglobinúria paroxística noturna para a validação da técnica de citometria de fluxo de alta sensibilidade. Métodos: Estudo retrospectivo, que envolveu a análise de dados laboratoriais de 745 pacientes com hipótese diagnóstica e/ou acompanhamento de hemoglobinúria paroxística noturna por citometria de fluxo. Resultados: Os avanços técnicos não só reduziram o custo do ensaio, mas também melhoraram a identificação e a resolução da citometria de fluxo para a detecção de clone hemoglobinúria paroxística noturna. Conclusão: A citometria de fluxo de alta sensibilidade possibilitou a identificação do tipo e do tamanho de clone de hemoglobinúria paroxística noturna, especialmente em amostras com pequeno clone.