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.

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.

Preparation of Internal Quality Control Material for Lymphocyte Subset Analysis

Lymphocyte subset analysis is widely used in clinical laboratories, and more than two levels of daily QC materials are required for reliable results. Commercially available, expensive QC materials have short shelf lives and may not be suitable in resource-poor settings. We compared different methods for preparing homemade QC material, including fixation with 1%, 2%, or 4% paraformaldehyde (PFA); freezing with 10% dimethylsulfoxide (DMSO), 0.1% bovine serum albumin-phosphate buffered saline, or after ethanolic dehydration; and using cryopreservation temperatures of -20℃, -80℃, or -196℃. We found an optimal experimental condition, which is 'fixation with 4% PFA, freezing with 10% DMSO, and storage at 80℃'. To evaluate long-term stability of QC materials prepared in this optimal condition, two levels of QC materials (QM1 and QM2) were thawed after 30, 33, 35, 37, 60, 62, 64, and 67 days of cryopreservation. Lymphocyte subset was analyzed with BD Multitest IMK kit (BD Biosciences, USA). QM1 and QM2 were stable after 1-2 months of cryopreservation (CV <3% for CD3, CD4, and CD8 and 5-7% for CD16/56 and CD19). We propose this method as an alternative cost-effective protocol for preparing homemade internal QC materials for lymphocyte subset analysis in resource-poor settings.

The New Sysmex XN-2000 Automated Blood Cell Analyzer More Accurately Measures the Absolute Number and the Proportion of Hematopoietic Stem and Progenitor Cells Than XE-2100 When Compared to Flow Cytometric Enumeration of CD34+ Cells

No abstract available.

Trypan blue exclusion assay by flow cytometry

Dye exclusion tests are used to determine the number of live and dead cells. These assays are based on the principle that intact plasma membranes in live cells exclude specific dyes, whereas dead cells do not. Although widely used, the trypan blue (TB) exclusion assay has limitations. The dye can be incorporated by live cells after a short exposure time, and personal reliability, related to the expertise of the analyst, can affect the results. We propose an alternative assay for evaluating cell viability that combines the TB exclusion test and the high sensitivity of the flow cytometry technique. Previous studies have demonstrated the ability of TB to emit fluorescence when complexed with proteins. According to our results, TB/bovine serum albumin and TB/cytoplasmic protein complexes emit fluorescence at 660 nm, which is detectable by flow cytometry using a 650-nm low-pass band filter. TB at 0.002% (w/v) was defined as the optimum concentration for distinguishing unstained living cells from fluorescent dead cells, and fluorescence emission was stable for 30 min after cell treatment. Although previous studies have shown that TB promotes green fluorescence quenching, TB at 0.002% did not interfere with green fluorescence in human live T-cells stained with anti-CD3/fluorescein isothiocyanate (FITC) monoclonal antibody. We observed a high correlation between the percentage of propidium iodide+CD3/FITC+ and TB+CD3/FITC+ cells, as well as similar double-stained cell profiles in flow cytometry dot-plot graphs. Taken together, the results indicate that a TB exclusion assay by flow cytometry can be employed as an alternative tool for quick and reliable cell viability analysis.

Establecimiento de intervalos de referencia en subpoblaciones linfocitarias por citometría de flujo / Reference values in lymphocyte subpopulations using flow cytometry

La determinación de Subpoblaciones Linfocitarias Humanas (SLH) por Citometría de Flujo, Linfocitos CD3+ (LT), LTCD4+, LTCD8+ y la relación LTCD4+/LTCD8+, permiten monitorear pacientes infectados con virus de la inmunodeficiencia humana (VIH), los cuales son comparados con Valores de Referencia (VR). A su vez los VR informados por la bibliografía son variables debido a la falta de validación analítica y partición estadística de los datos de VR, entre otros. En este trabajo, utilizando un ensayo validado (ISO15189), se establecieron VR para SLH con criterios estadísticos de partición en individuos con serología negativa para VIH (397 mujeres (F) y 279 varones (M), edad: 17-83 años). Las SLH fueron medidas en un Citómetro de Flujo (Coulter Epics XL-MCL). Los datos fueron procesados empleando como criterio estadístico de partición el algoritmo de Martin Gellerstedt (AMG). Del análisis estadístico y del AMG se determinó la partición de los datos en 6 subpoblaciones definidas por edad (intervalo de años) y género (F y M): grupos I y IV: 17-35 (F y M); grupos II y V: 36-50 (F y M); y grupos III y VI: >50 años (F y M). Para los 6 grupos se definieron VR de linfocitos totales, LT, LTCD4+, LTCD8+ y LTCD4+/LTCD8+. Para cada límite inferior y superior de VR se estableció el intervalo de confianza al 90%. En conclusión, este estudio permitió establecer VR para SLH en un ensayo validado empleando un criterio estadístico de partición, lo cual permitiría incrementar la confiabilidad de los resultados y uniformar a nivel internacional los VR en SLH en diferentes poblaciones.

The determination of Human Lymphocyte Subpopulations (HLS) including Lymphocytes CD3+ (LT), LTCD4+, LTCD8+ and LTCD4+/LTCD8+ ratio by flow cytometry, makes it possible to monitor patients infected who HIV, which are compared against reference values (RV). However, RV reported by the international bibliography are variable due to the absence of two main factors: analytical validation procedures and partitioning statistical criteria. This study, using a validated method (ISO15189), RV were established for HLS in individuals with negative serology for HIV (397 females (F) and 279 males (M), age: 17-83 years) applying partitioning statistical criteria. The values of HLS were obtained by flow cytometry (Coulter Epics XL-MCL). The data were processed using partitioning statistical criteria based on Martin Gellerstedt's algorithm (MGA). From the statistical analysis and MGA the partition of the data was determined in 6 subpopulations defined by age (interval of years) and gender (F and M): groups I and IV: 17-35 (F and M); groups II and V: 36-50 (F and M); and groups III and VI: >50 years (F and M). Hence, RV of total lymphocytes, LT, LTCD4+, LTCD8+ and LTCD4+/LTCD8+ ratio were defined. For each limit (lower and upper) of reference values, the 90% confidence interval was determined. In conclusion, this study allowed establishing RV for HLS in a validated method using a partitioning statistical criterion, which would allow increasing the assurance results and establishing an international criterion for reference values in HLS in different populations.

External quality assurance schemes in flow cytometry.

Quality assurance schemes were devised to monitor the performance of laboratory tests commonly utilized in routine clinical practice. In the area of Flow Cytometry, external quality assurance schemes are very much in their infancy. There are 3 major applications of Flow Cytometry in the clinical laboratory: lymphocyte subset enumeration, CD34 enumeration and leukemia immuno-phenotyping. Pilot schemes for CD34 enumeration and leukemia immuno-phenotyping have revealed a considerable lack of consensus with a wide divergence in practices. The coefficient of variation in reported results from these assessments have been very large, ranging from 20-200%. Results for lymphocyte subset enumeration surveys are sometimes not much better despite having been around for a longer period of time. Quality assurance programs in Flow Cytometry are difficult to conduct and 3 or 4 concurrent factors are often cited as reasons for the large variability. The need for viable, functionally intact cells as suitable assay material is probably the biggest contributor to the large variability. This difficulty in providing stable cells for inter-laboratory comparison is compounded by the lack of consensus in testing methodology. Participation in a distant external quality assurance scheme is consequently not very helpful to improving the practices in the laboratory. Currently, there are more problems than solutions. An awareness of these issues will hopefully contribute to the development of future consensus and offer up solutions to some of these problems.

Flow cytometric analysis of T-lymphocytes subsets in adult Thais.

Flow cytometer (FACScan) was used to determine the range of T lymphocyte subpopulations in normal Thai blood donors at Maharaj Nakorn Chiang Mai Hospital, Chiang Mai. Reference population consisted of 150 healthy HIV seronegative blood donors. T lymphocyte subsets were analysed using two-color immunophenotyping of peripheral blood lymphocytes with a lysed whole blood technique and enumerated. The study showed that the normal values for CD3+ lymphocytes (percent), CD4+ lymphocytes (percent), CD8+ lymphocytes (percent), CD4/CD8 ratio, absolute CD3+ lymphocyte count, absolute CD4+ lymphocyte count and absolute CD8+ lymphocyte count were 64 +/- 8.8, 36.1 +/- 6.4, 25.7 +/- 7.3, 1.5 +/- 0.6, 1,630 +/- 600 cells/microl, 910 +/- 300 cells/microl and 670 +/- 350 cells/microl, respectively. We found that the values of CD3, CD4 and CD4/CD8 ratio were significantly lower than those in the Caucasians but those of CD8 was not significantly different. This observations have important clinical implication for the use of T lymphocyte subsets measurement, especially in the management of HIV infection in Thais. These normal ranges can be used as a reference for the decisions in clinical practice.