Portal de Periódicos da CAPES

Eight color immunophenotyping of T-, B- and NK-cell subpopulations for characterization of chronic immunodeficiencies

2014; Wiley; Linguagem: Inglês

10.1002/cytob.21162

1552-4957

Andreas Boldt, Stephan Borte, Stephan Fricke, Karim Kentouche, Frank Emmrich, Michael Borte, Franka Kahlenberg, Ulrich Sack,

Immunodeficiency and Autoimmune Disorders

Background: The heterogeneity of primary and secondary immunodeficiencies demands for the development of a comprehensive flow cytometric screening system, based on reference values that support a standardized immunophenotypic characterization of most lymphocyte subpopulations. Methods: Peripheral blood samples from healthy adult volunteers (n=25) were collected and split into eight panel fractions (100µl each). Subsequently, pre-mixed 8-color antibody cocktails were incubated per specific panel of whole blood to detect and differentiate cell subsets of: (i) a general lymphocyte overviews, (ii) B-cell subpopulations, (iii) CD4+ subpopulations, (iv) CD8+ subpopulations, (v) regulatory T-cells, (vi) recent thymic emigrants, (vii) NK-cell subpopulations, (viii) NK-cell activation markers. All samples were lysed, washed and measured by flow cytometry. FACS DIVA software was used for data analysis and calculation of quadrant statistics (mean values, standard error of mean, percentile ranges). Results: Whole blood staining of lymphocytes provided the analysis of: (i) CD3+, 4+, 8+, 19+, 16/56+, and activated CD4/8 cells; (ii) immature, naïve, non-switched/switched, memory, (activated) CD21