Basic principles of BAT
BAT focuses on the basophil population at a single cell level using flow cytometry and allows the assessment of the activation state of these cells before and after stimulation with allergens or controls. Basophils have low side scatter, intermediate between lymphocytes and monocytes, and can be identified through a number of near-unique selection markers: CD193+ (also expressed on SSChigheosinophils), CD123+ (also expressed on HLA-DR+ plasmacytoid dendritic cells) and CD203c and FcεRI are also expressed on pluripotent progenitors of mast cells. Common methods of identifying basophils are as SSClowCD193+CD203c+, CD123+HLA-DR-, CD123+HLADR-, CD203c+ or CD193+CD123+ 2,3,4. FcεRI and IgE, when used as selection markers in isolation, have the disadvantages of varying with plasma concentration of IgE and of inducing activation of the IgE-mediated pathway leading to degranulation. Figure 2shows examples of gating strategies used in assays used clinically and for research purposes.
Activation of basophils can be detected through upregulation of selected surface proteins; of which CD63 is the most commonly used activation marker1 and CD203c is upregulated slightly earlier5,6. CD107a and CD107b co-localise with CD63 in secretory lysozymes, whereas CD164 and CD13 co-localise with CD203c in vesicles distinct from these. Further, upregulation of CD18/CD11b1 and CD45 can also be detected, but these are not nearly as dichotomous as the upregulation of CD63. The tetraspanin CD63 is located in the membrane of secretory lysosomes in basophil granulocytes1 and mast cells7. It is a 4-transmembrane protein that may be associated with reorganisation of the cell membrane8and with exosome formation9. Its role in these processes is not yet well understood, but it is very useful as a biomarker of basophil activation. The expression of CD63 on the surface of basophils is directly and strongly correlated with histamine released into the cell supernatant1,10,11.