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.