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

Abstract

Background: 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. Methods: 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. Results: 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. Conclusions: 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.

Principles, potential, and limitations of ex vivo basophil activation by flow cytometry in allergology: A narrative review

“…8 At present, different protocols allowing detection of surface marker alterations, intracellular changes, and exteriorization of granular content have been developed. [1][2][3] As shown in Figs 1 and 2 and Table E1 (in this article's Online Repository at www.jacionline. org), traditional FCM-based BATs use whole blood and rely on cellular identification and quantification of activation and/or FIG 1.…”

“…For more details on the identification and activation/degranulation markers see the text and Table E1 (available in this article's Online Repository at www.jacionline.org). For technical details, see Ebo et al, 1 Bridts et al, 2 and Elst et al 3 DAO, Diamino oxidase; pp38MAPK, phosphorylated p38 mitogen-activated protein kinase. degranulation markers on the surface membrane.…”

“…18,19 Degranulation of basophils can also be explored by measuring the exteriorization of granule matrix and decrease in intracellular histamine content. Briefly, anionic proteoglycans from exteriorized basophil granule matrix are stained by cationic fluorescent avidin probes, 1,20,21 whereas the quantification of the intracellular histamine relies on a histaminase affinity technique in which diamino oxidase is coupled to a fluorochrome. 22 Newly synthesized cytokines such as IL-4 and IL-13, or their mRNA, can be trapped and measured intracellularly.…”

“…Such exposure is not associated with an upregulation of CD63, but it might be demonstrable by an upregulated expression of CD69, CD203c, p38MAK, and STAT5. 1,18,19,26,27 Note that in whole blood settings the outcome of the test can be influenced by blocking antibodies (through CD32) 28 or stoichiometrically via interference with the IgE-allergen interaction. 29 These blocking antibodies can be deleted by washing leukocytes or using purified basophils.…”

“…Degranulation of basophils and mast cells (MCs) can be triggered by various processes (Fig 1 [1][2][3] ). IgE-dependent degranulation involves synthesis of allergen-specific IgE (sIgE) antibodies by plasma cells.…”