CCR3 for basophil activation test: a necessary but insufficient step

I have been particularly interested in the recent study by Eberlein et al. [1] comparing the use of two different markers for basophil recognition by flow cytometry (surface IgE vs. CCR3 stainings) in the context of antibiotic allergy diagnosis. Although some minor technical issues remain to be addressed [2], there is now accumulating evidence indicating that the basophil activation test (BAT), using either CD203c or CD63 as activation markers, has become a robust and reliable tool usable in clinical practice for the in vitro investigation of immediate allergy [3]. However, one important drawback was that basophil recognition through the detection of surface IgE expression was not supposed to represent an appropriate method to ensure acceptable cell purity. Indeed, in a previous article published in Clinical and Experimental Allergy [4], Abuaf et al. observed a major contamination of the basophil gate with monocytes (up to 50% of gated IgE cells). This drawback had been suspected for long (explaining some observed low sensitivities) but had never been appropriately investigated. Furthermore, another important drawback was that IgE expression on basophil surface is considered to be highly variable from one patient to another, rendering cell recognition (and thus recovery) difficult to standardize and in some cases virtually impossible. As an alternative and because of its high expression on basophils and eosinophils, Eberlein and colleagues appropriately used CCR3 staining to identify basophils in whole blood [1, 5]. It agrees with a similar approach based on CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells) expression for basophil recognition [6]. At a first sight, there is no doubt that this protocol is likely to improve the reliability of the BAT. However, in terms of the quality of flow cytometry analysis, a potential major drawback of Eberlein’s protocol has to be mentioned and kept in mind. When performing flow cytometry analysis, one constant challenge is to ensure the quality of the gating strategy regarding cells of interest. The crucial end-point is to provide excellent recovery and purity. Here, we need to include and monitor every basophil (recovery) but we also need to exclude all non-basophil cells (purity). If basophil recovery does not appear to be questionable after CCR3 staining, in that case, the purity still needs to be improved. Indeed, CCR3 is also expressed on T cells, mainly Th2 lymphocytes [7] and regulatory T cells [8]. As basophils and lymphocytes show similar light-scattering characteristics (forward or side scatters), Eberlein and colleagues, in their putative ‘basophil’ gate (R1 in fig. 1a), might have included T cells as well. It is unfortunate that the authors did not take this aspect into account as this could have falsely decreased the sensitivity of BAT. Indeed, some cells – supposedly identified as basophils – would have definitely not responded to allergens (i.e. no CD63 expression) because they do not contain histamine granules. At least, the authors should have demonstrated that a putative contamination by T cells did not significantly modify the clinical value of BAT. Such a demonstration remains to be established. That said, an alternative exists. Nowadays, flow cytometers easily allow for multiple colour stainings. Adding an additional labelled antibody to a dual-colour protocol (e.g. PE-CCR3/FITC-CD63) is thus not a limiting challenge. As an example, basophils could be readily distinguished from Th2 lymphocytes/regulatory T cells based on CD3 expression. This very simple improvement to the Eberlein’s protocol should lead to an unquestionable basophil gating strategy.