Using a CD45dim/CD123bright/HLA-DRneg phenotyping protocol to gate basophils in FC for airway allergy. CD123 does not decrease

Physicians in the field of respiratory medicine are particularly concerned about the availability of a reliable diagnostic tool to investigate respiratory allergy. Usually, basophils are easy to obtain from peripheral blood and therefore they represent a reproducible model to assess allergy in individuals. Cell assays called basophil activation tests (BATs) are widespread tools for allergy diagnosis and are easily available in most of the medical labs. The strategy by which basophils are captured in a flow cytometry protocol has met many suggestions, recommendations and experimental novelties in recent years, yet the debate needs to be further expanded. Concerns still remain about the suitability of the many approaches to make the basophil activation test (BAT) an excellent and practical tool to diagnose allergy, while improving its analytical performance. This technical report describes the methodological aspects of the use of the protocol adopting the panel CD45dim/CD123bright/HLA-DRneg to gate basophils in flow cytometry, trying to highlight the main biases related to an incorrect use of this protocol.