Detection of allergen-induced basophil activation by expression of CD63 antigen using a tricolour flow cytometric method

Abstract

In the field of allergy diagnosis, most in vitro functional tests are focused on basophils. Nevertheless, the very small number of circulating basophils limits these experiments and their clinical benefit remains controversial. As flow cytometry is a valuable tool for identifying cell populations, even at low concentrations, we developed a tricolour flow cytometric method for the study of allergen-induced basophil activation. Identification of cells was based both on CD45 expression and on the presence of IgE on the cell surface, since basophils express high-affinity receptors for IgE (Fc epsilon RI). Cell activation upon allergen challenge was assessed by the expression of CD63 antigen on the plasma membrane. Basophil isolation and activation (with the chemotactic peptide formyl-methionyl-leucyl-phenylalanine) were validated in 32 non-allergic patients. In 12 allergic patients, basophil stimulation by a relevant allergen was in most cases positive (10/12). Furthermore a concentration-dependent hook effect was observed. Of the allergic and non-allergic patients, none showed non-specific activation with an irrelevant allergen (specificity 100%). Overall, our preliminary results, even in a small population, suggest that this is a reliable and valuable method for the diagnosis of allergies complementing specific allergen IgE and skin test results. Obviously, additional clinical studies are needed to validate these first results.

Indomethacin Causes Prostaglandin D2-like and Eotaxin-like Selective Responses in Eosinophils and Basophils

“…In keeping with these data, eosinophil respiratory burst (Fig. 3C) and CD63 up-regulation (a degranulation-associated marker (40) (Fig. 3D)) were strongly induced by C5a.…”

Use of Flow Cytometry to Assess Basophil Activation in Patients Allergic to Betalactam Antibiotics: Correlation between Flow-Cytometric Allergen Stimulation Test and Other in vivo and in vitro Tests

“…The sensitivity of the test was 56.10%, specificity 95.65%, positive predictive value 95.83% and negative predictive value 55%. This technique has been proved to be useful in the in vitro diagnosis against different antigens [4, 5, 6, 7]. …”

LAMP‐1 and LAMP‐2, but not LAMP‐3, are reliable markers for activation‐induced secretion of human mast cells

“…Structurally, the molecules differ because LAMP-1 and LAMP-2 contain a type 1 transmembrane and LAMP-3 contains a tetraspanin (transmembrane 4 superfamily) domain (2,3). Although the function of LAMPs is unknown, LAMP-3 is a reliable activation marker for basophils (4 -7) because, after stimulation of cells via the FceRI receptor, LAMP-3 is translocated from its intracellular localisation to the plasma membrane (4,8). This process can be quantified by flow cytometry (1).…”

“…This process can be quantified by flow cytometry (1). The translocation of LAMP-3 has been shown to be accompanied by the release of histamine and leukotriene C 4 (LTC 4 ) in these cells (4,8). In peripheral blood leukocytes, expressions of LAMP-1 and LAMP-2 were identified as activation-dependent processes, and the involvement of these molecules in the adhesion of cells to the vascular endothelium has been discussed (9).…”