The basophil activation test by flow cytometry: recent developments in clinical studies, standardization and emerging perspectives

Abstract

The diagnosis of immediate allergy is mainly based upon an evocative clinical history, positive skin tests (gold standard) and, if available, detection of specific IgE. In some complicated cases, functional in vitro tests are necessary. The general concept of those tests is to mimic in vitro the contact between allergens and circulating basophils. The first approach to basophil functional responses was the histamine release test but this has remained controversial due to insufficient sensitivity and specificity. During recent years an increasing number of studies have demonstrated that flow cytometry is a reliable tool for monitoring basophil activation upon allergen challenge by detecting surface expression of degranulation/activation markers (CD63 or CD203c). This article reviews the recent improvements to the basophil activation test made possible by flow cytometry, focusing on the use of anti-CRTH2/DP2 antibodies for basophil recognition. On the basis of a new triple staining protocol, the basophil activation test has become a standardized tool for in vitro diagnosis of immediate allergy. It is also suitable for pharmacological studies on non-purified human basophils. Multicenter studies are now required for its clinical assessment in large patient populations and to define the cut-off values for clinical decision-making.

Figure 1

Principle of the basophil activation test by flow cytometry (triple staining). Basophils are identified on the basis of CD45 expression (fluorescence 3 / Phyco-Cyanine 5) and the presence of IgE or CRTH2/DP2 on their surface (fluorescence 1 / Fluorescein isothiocyanate). Resting basophils do not express CD63 (anchored in the basophilic granule) and weakly express CD203c. The cross-linking of two FcεRI (induced by an allergen or anti-IgE antibodies) provokes the histamine release (and as a consequence the CD63 expression) and the upregulation of CD203c. The rise in CD63 or CD203c expression (measured by fluorescence 2 / Phycoerythrin) before and after allergen challenge reflects thus the basophil activation / degranulation in response to an allergen.

Figure 2

CD203c expression in whole blood before and after basophil activation. Ungated leukocytes are shown as a biparametric representation on the basis of side scatter characteristics (SSC, y-axis) and CD203c (x-axis). Left histogram depicts resting cells, basophils express low levels of CD203c (some of them are not distinguishable from lymphocytes and monocytes). Right histogram depicts cells after anti-IgE challenge, activated basophils are easily recognized on the basis of their high CD203c expression.

Figure 3

Identification of CRTH2 expressing cells by flow cytometry. Left histogram : ungated leukocytes biparametric representation on the basis of side scatter characteristics (SSC, Y axis) and FITC-CRTH2 (X axis). Two CRTH2 expressing cell populations are easily distinguishable: the one with high light scatterings corresponds to the eosinophil population; the second one (gating region: A) comprises Th2 lymphocytes and basophils. Right histogram: cells from the gating region (A) expressed on the basis of PE-CD203c (X axis) and PC5-CD3 (Y axis) characteristics. Th2 lymphocytes were readily separated from basophils based on their positive CD3 expression while activated basophils express high levels of CD203c without expressing CD3.

Figure 4

Representative increased expression of CD203c after allergen challenge in a patient allergic to Dermatophagoïdes pteronyssinus (d1). Gated CRTH2-positive basophils (after excluding Th2 lymphocytes as described in figure 3) are presented on the basis of CD203c-CRTH2 staining: before stimulation (negative control, upper left dot-plot), after anti-IgE challenge (positive control, upper right) and after allergen challenge at 3 different concentrations (dose-effect response, lower dot-plots). Activated basophils: percentage of basophils expressing CD203c.