BACKGROUND: The possible occurrence of an erroneous immunophenotyping due to interference between monoclonal antibodies (MoAbs) is often overlooked when the epitopes are assumed to be not close to each other. This is particularly important when exploring immune cell populations whose identification is still investigational. The commonly held view is that myeloid derived suppressor cells can be identified as either HLA-DR(neg/dim) cells or interleukin-4 receptor-α (CD124)(+) cells among peripheral blood monocytes. We made the serendipitous observation that the fluorescence signal provided by the PE-CD124 MoAb was attenuated when the PE-CF594-HLA-DR MoAb was added to the staining tube. METHODS: Peripheral blood mononuclear cells from healthy donors were stained with the PE-CD124 MoAb and, as control, PE -CD40, -CD4 and -CD14, and either the PE-CF594-HLA-DR MoAb or its unlabeled form. B cells, which also express CD124, were analyzed for comparison. RESULTS: The PE-CF594-HLA-DR MoAb but not its unlabeled form reduced PE-CD124 MoAb staining on monocytes and B cells. No other monocyte and B cell surface marker staining was affected by the PE-CF594-HLA-DR MoAb. The PE-CF594-HLA-DR MoAb interfered with the PE-CD124 MoAb likely because of steric hindrance by bulky fluorochromes, although a quenching due to fluorescence resonance energy transfer might also cooperate to the PE-CD124 MoAb staining attenuation. CONCLUSIONS: Present observations highlight the importance of interference between MoAbs as a source of error when analyzing multicolor flow cytometry data.
BACKGROUND: The possible occurrence of an erroneous immunophenotyping due to interference between monoclonal antibodies (MoAbs) is often overlooked when the epitopes are assumed to be not close to each other. This is particularly important when exploring immune cell populations whose identification is still investigational. The commonly held view is that myeloid derived suppressor cells can be identified as either HLA-DR(neg/dim) cells or interleukin-4 receptor-α (CD124)(+) cells among peripheral blood monocytes. We made the serendipitous observation that the fluorescence signal provided by the PE-CD124 MoAb was attenuated when the PE-CF594-HLA-DR MoAb was added to the staining tube. METHODS: Peripheral blood mononuclear cells from healthy donors were stained with the PE-CD124 MoAb and, as control, PE -CD40, -CD4 and -CD14, and either the PE-CF594-HLA-DR MoAb or its unlabeled form. B cells, which also express CD124, were analyzed for comparison. RESULTS: The PE-CF594-HLA-DR MoAb but not its unlabeled form reduced PE-CD124 MoAb staining on monocytes and B cells. No other monocyte and B cell surface marker staining was affected by the PE-CF594-HLA-DR MoAb. The PE-CF594-HLA-DR MoAb interfered with the PE-CD124 MoAb likely because of steric hindrance by bulky fluorochromes, although a quenching due to fluorescence resonance energy transfer might also cooperate to the PE-CD124 MoAb staining attenuation. CONCLUSIONS: Present observations highlight the importance of interference between MoAbs as a source of error when analyzing multicolor flow cytometry data.
Authors: Fabian Junker; Pratiksha Gulati; Uwe Wessels; Stefan Seeber; Kay-Gunnar Stubenrauch; Laura Codarri-Deak; Christoph Markert; Christian Klein; Priscila Camillo Teixeira; Henry Kao Journal: Cytometry A Date: 2021-03-18 Impact factor: 4.355