The expression of CD123 can decrease with basophil activation: Implications for the gating strategy of the basophil activation test

Background: Basophil activation test (BAT) reproduces IgE-mediated allergic reactions in vitro and has been used as a diagnostic test. Different markers can be used to identify basophils in whole blood and have implications for the outcome of the test. We aimed to assess changes in the expression of CD123 and HLA-DR following basophil activation and to select the best gating strategy for BAT using these markers. Methods: BAT was performed in whole blood from 116 children. Peanut extract, anti-IgE, anti-FcϵRI or formyl-methionyl-leucyl-phenylalanin (fMLP) was used for stimulation. Surface expression of CD123, HLA-DR, CD63 and CD203c was evaluated by flow cytometry. Results: In some cases, gating on CD123+/HLA-DR- led to the loss-to-analysis of basophils in conditions where basophils were activated. Adding CD203c as an identification marker restored the cell number. Basophils remained HLA-DR-negative with activation. CD123 expression decreased following stimulation with fMLP (n = 116, p < 0.001), anti-IgE (n = 104, p < 0.001) and peanut (n = 42, p < 0.001). The decrease in the mean fluorescence intensity of CD123 correlated with the up-regulation of basophil activation markers, CD63 (r_s = -0.31, p < 0.001) and CD203c (r_s = -0.35, p < 0.001). BAT to peanut gating basophils on CD203c+/CD123+/HLA-DR- reduced the false-negatives (1 vs. 5 %) and showed a higher diagnostic accuracy compared to using CD123+/HLA-DR- (97 vs. 91 %). CD203c+ appeared as an alternative gating strategy allowing two-colour BAT. Conclusions: Basophils of a subset of patients down-regulate CD123 with activation. The use of CD203c before gating on CD123+/HLA-DR- cells or in isolation ensures the identification of the entire basophil population and accurate assessment of basophil activation, with important diagnostic implications.