2.9. Basophil activation test (BAT)

BAT was performed at BML Inc. (Saitama, Japan) using an allergenicity kit (Beckman Coulter, CA, USA) according to the manufacturer's instructions. Briefly, after incubating heparinized whole blood with the antigen at 37°C for 15 min, basophil activation was assessed using antibodies against CD3, chemoattractant receptor-homologous molecule on Th2 cells (CRTH2), and CD203c. PBS and anti-IgE antibodies were used as the negative and positive controls, respectively. Serially diluted formula samples were used to assess basophil activation for formulas. CD203c expression in at least 300 basophils (CD3⁻CRTH2⁺ cells with FSC/SSC characteristics of lymphocytes) was analyzed using flow cytometry. Basophil activation was expressed as the net percentage of CD203c positive basophils above the threshold defined by the negative control. Basophils were defined as non-responders when the percentage of basophils activated by anti-IgE was less than 10% (13). The area under the curve (AUC) of the activated basophils with a log-formula concentration axis was calculated for each participant.