Flow cytometric basophil activation tests: Staining of exteriorized basophil granule matrix by fluorescent avidin versus appearance of CD63.

Didier G Ebo, Jessy Elst, Michel van Houdt, Isabel Pintelon, Jean-Pierre Timmermans, Tatsuo Horiuchi, Margaretha A Faber, Margo M Hagendorens, Christel M Mertens, Vito Sabato

Cytometry. Part B, Clinical cytometry 2020 Nov

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Staining of exteriorized basophil granule matrix by fluorescent avidin might be a reliable technique to monitor basophil degranulation. This study compares the avidin-based technique with the upregulation of CD203c and appearance of CD63 in response to various stimuli. Fourteen individuals responsive to anti-IgE, nine healthy controls, and five birch pollen-allergic patients, and five nonresponders were studied. Activation experiments included anti-IgE, fMLP, interleukin-(IL)-3, and birch pollen allergen. Basophil activation/degranulation was analyzed by flow cytometry and microscopy using anti-CD63, anti-CD203c, and avidin. Stimulation with anti-IgE, fMLP, and relevant allergen results in upregulation of CD203c, CD63 appearance, and an increase in avidin binding. In response to anti-IgE and allergen, upregulation of CD203c peaks within 10 min, CD63 and avidin binding reach a plateau after 10-20 min. CD63 staining leads to a bimodal distribution, avidin staining causes a unimodal shift with a less clear discrimination between degranulating and nondegranulating cells. In response to fMLP, upregulation of CD203c and CD63 and avidin binding are maximal after 2.5 min. Following incubation with anti-IgE and fMLP, percentages of CD203c+ cells are higher than those of CD63+ and avidin+ cells, pointing to a dissociation between activation and degranulation. Percentages of CD63+ cells are systemically higher than those of avidin+ cells. Incubated with IL-3 only upregulates CD203c, while no CD63 or avidin binding is observed. Staining of exteriorized proteoglycans by avidin is a reliable technique to quantify basophil degranulation but offers no added value when compared to traditional assays that use CD63 as a readout. © 2020 International Clinical Cytometry Society.

Citation

Didier G Ebo, Jessy Elst, Michel van Houdt, Isabel Pintelon, Jean-Pierre Timmermans, Tatsuo Horiuchi, Margaretha A Faber, Margo M Hagendorens, Christel M Mertens, Vito Sabato. Flow cytometric basophil activation tests: Staining of exteriorized basophil granule matrix by fluorescent avidin versus appearance of CD63. Cytometry. Part B, Clinical cytometry. 2020 Nov;98(6):483-490