We are grateful to Dr. Chirumbolo for the letter in response to our paper describing a basophil activation test for chronic urticaria [12]. We agree with most of the issues raised by Dr. Chirumbolo, but some points need to be addressed.

The non-atopic donors in our study were adults, not infants. Among 3-5 healthy individuals with blood group O, one with a high basophil count was selected for each experiment. The average basophil count was 0.55% by flow cytometric analysis, 0.75% by manual cell counting on a peripheral blood smear, and 0.81% by the complete blood count analyzer (data not shown in the article). Therefore, the purity of basophils in gate 3 appeared to be sufficiently high for our indirect flow basophil activation test. In order to retrieve as many cellular events as possible during flow cytometry, data acquisition was continued until non-cellular events appeared in the time/side scatter plot (plot not shown in the article). The contaminating non-cellular events were excluded in the subsequent data analysis.

Gate 3 for purified basophils was the intersection area of the CD123^bright and CCR3^pos populations. These two combined basophil identification markers may reciprocally help to exclude T lymphocytes (CD123^neg) and monocytes (CCR3^neg), even without additional exclusion markers. The sufficiently high purity of basophils in gate 3 was confirmed by the CD63 FITC vs. CD203c APC plot, where most events were either CD203c^dim or CD203c^bright.