ANALYSIS OF ANTI-IGE AND ALLERGEN-INDUCED HUMAN BASOPHIL ACTIVATION BY FLOW-CYTOMETRY - COMPARISON WITH HISTAMINE-RELEASE

Objective and Design: On the basis of flow cytometric methods previous ly described for the analysis of human basophil activation, we present here a bi-color anti-IgE FITC, anti CD63 PE method and the correlatio n with histamine release. Materials and Subjects: Subjects allergic to grass pollen were selected by their clinical history, skin tests and specific IgE. Methods: Basophils gated in the lymphocyte region of the side scatter (SSC)/forward scatter (FSC) pattern were selected by the ir high IgE epitope density. Percentage of cells expressing CD63 marke r, upregulated on activated basophil membrane, was calculated by the c ytometer. Histamine released into the supernatants was measured by RTA . Results: In these conditions, flow cytometric analysis of blood leuk ocytes showed that the selected cells had the phenotype CD14-, CD19-, CD45+, IgE++ and CD63- or + which is related to human basophil phenoty pe, the isotype controls being negative. The use of an anti-CD41 FITC antibody also showed the presence of aggregated platelets on the basop hil membrane, CD63 antigen being, however, expressed by basophils them selves and not by platelets. Moreover, no statistical difference was o bserved between histamine release and flow cytometry after passive sen sitization of blood donor leukocytes. Conclusion: Flow cytometry, as a popular method often used in the immunology and haematology departmen ts of clinical laboratories may represent a new alternative for allerg y diagnosis and basophil pharmacology.