• DOI:10.1002/cyto.a.22466
• Corpus ID: 205730283

Major pitfalls in BAT performance may be caused by gating protocols and CD63% cut off evaluation

@article{Chirumbolo2014MajorPI,
  title={Major pitfalls in BAT performance may be caused by gating protocols and CD63\% cut off evaluation},
  author={Salvatore Chirumbolo},
  journal={Cytometry Part A},
  year={2014},
  volume={85}
}

• S. Chirumbolo
• Published 1 May 2014
• Biology
• Cytometry Part A

A paper by Ariza et al., published in the current issue of Cytometry Part A, described the ability of basophil activation test (BAT) in diagnosing patients with cross-intolerance to NSAIDs (1). Ariza et al. concluded that BAT used in their study did not appear useful to evaluate patients with NSAIDderived allergy, despite the fact that their flow cytometry approach to investigate basophils by a CD193/CD203c/CD63 BAT represented a novelty in the field. The data reported by the authors (1), particularly as for Table 1 of their paper, indicated that a main pitfall of BAT to evaluate NSAIDs allergy was the many false-positive results, reflected in the low specificity obtained, which may be explained by NSAIDs capacity to interact pharmacologically with basophils, inducing cell activation in both NSAID-intolerant and NSAID-tolerant subjects, although they showed that only one single drug appeared to be in contradiction with a cross-reacting NSAIDhypersensitivity (1). The authors tried to address the problem by adding CD203c marker in a previously standardized CD193/CD63 gating protocol. Basophil phenotyping markers, such as CD193 (CCR3) and CD203c, deserved major attention in recent years (2–4). They are used to capture basophils from whole peripheral blood samples and allow to investigate cell response to allergens or other molecules in the in vitro assay by flow cytometry. Many of these molecules are also expressed on the surface of activated basophils, therefore, some concern raises when they are indifferently used either as phenotyping or activation markers (5–7). Ariza et al. used the eotaxin receptor CCR3 (or CD193) and the basophil specific marker CD203c to gate basophils and evaluate their CD63 expression on patients with NSAIDs-induced uticaria/angioedema (NIUA) (1). CD203c was fundamentally included in the protocol because is a highly specific marker expressed only by basophils and, therefore, it can be used to selectively gate CD193-expressing cells as basophils (1). The problem of CCR3 (eotaxin receptor or CD193) regards its coexpression on eosinophils (which anyway gate on a different SSC scale quite far from basophils, although few CD63-expressing basophils may be captured in the area) and on T-cells, for which adding a CD3 markers was recently recommended (3). Despite to their heavy endowment of granules, basophils gate on low SSC within the lympho-area; and therefore, it is quite difficult to find these cells on high SSC where eosinophils are located in a flow cytometry (FC) dot plot. On the contrary, T cells may be present within the CCR31 events indicated as basophils. Furthermore, CCR3-expressing and CD203c-bright cells change their fluorescence following an activation signal, as allergens do, causing possible bias in FC electronic capture. BAT remains a flow cytometry approach that may provide a reasonable alternative to traditional mediator release assays used to test cell participation in allergy mechanism, despite the many issues about suitability, sensitivity, and specificity, which are yet to be further elucidated (8). For certain aspects, practitioners may feel as staggering explorers over a fluttering icepack in the Arctic, when gaining full awareness of the many criticisms raised on the technical aspects with which most of BATs are performed and interpreted. The metaphor