Basophil Activation Test for Remibrutinib

To study basophil activation, Flow CAST^® BAT was used following manufacturer's instructions. In these experiments, blood from healthy donors was incubated with or without 1 μM remibrutinib for 1 h at 37°C before the addition of controls' and patients' sera (10%). In each experiment, a negative control consisting of unstimulated blood and a positive control (blood stimulated with anti‐FcɛR1 monoclonal antibodies [mAb] to crosslink the receptor) were included. After an additional 30 min of incubation with the sera at 37°C, blood cells were stained with anti‐CD193 (chemokine receptor 3, CCR3)‐PE and the anti‐CD63‐fluorescein (FITC) included in the staining reagent of the kit. Finally, red blood cells were lysed, and samples were analyzed by flow cytometry.