Analysis of anti-IgE and allergen induced human basophil activation by flow cytometry. Comparison with histamine release.
OBJECTIVE
On the basis of flow cytometric methods previously described for the analysis of human basophil activation, we present here a bi-color anti-IgE FITC, anti CD63 PE method and the correlation with histamine release.


METHODS
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 their high IgE epitope density. Percentage of cells expressing CD63 marker, upregulated on activated basophil membrane, was calculated by the cytometer. Histamine released into the supernatants was measured by RIA.


RESULTS
In these conditions, flow cytometric analysis of blood leukocytes showed that the selected cells had the phenotype CD14-, CD19-, CD45+, IgE++ and CD63- or + which is related to human basophil phenotype, the isotype controls being negative. The use of an anti-CD41 FITC antibody also showed the presence of aggregated platelets on the basophil membrane, CD63 antigen being, however, expressed by basophils themselves and not by platelets. Moreover, no statistical difference was observed between histamine release and flow cytometry after passive sensitization of blood donor leukocytes.


CONCLUSIONS
Flow cytometry, as a popular method often used in the immunology and haematology departments of clinical laboratories may represent a new alternative for allergy diagnosis and basophil pharmacology.
Sainte-Laudy
J
J
Unité d'immuno-allergologie, Laboratoire Cerbra, Val d'Oise, France.
Sabbah
A
A
Vallon
C
C
Guerin
J C
JC
D000485:Allergens / Q000276:immunology*; D000888:Antibodies, Anti-Idiotypic; D015703:Antigens, CD / Q000032:analysis; D001491:Basophils / Q000276:immunology* / Q000502:physiology; D001792:Blood Platelets / Q000276:immunology; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006636:Histamine Release; D006801:Humans; D007073:Immunoglobulin E / Q000032:analysis / Q000276:immunology*; D016130:Immunophenotyping; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000032:analysis; D010980:Platelet Membrane Glycoproteins / Q000032:analysis; D012680:Sensitivity and Specificity; D060149:Tetraspanin 30
D000485:Allergens; D000888:Antibodies, Anti-Idiotypic; D015703:Antigens, CD; C554586:CD63 protein, human; D005456:Fluorescent Dyes; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D010980:Platelet Membrane Glycoproteins; D060149:Tetraspanin 30; D007073:Immunoglobulin E; D016650:Fluorescein-5-isothiocyanate