Monitoring of CD63% in basophil activation test and suggested new parameters for allergy diagnosis

Abstract

The basophil activation test (BAT) is the only cellular test used to investigate basophil function for allergy diagnosis [1–4]. Most basophil activation tests (BATs) evaluate the percentage of basophils expressing the CD63 molecule on activation, in order to address a possible hypersensitivity reaction to an allergen; non-activated resting basophils do not express this tetraspanin on the cell surface but, on the contrary, cells up-regulate the molecule upon stimulation [5]. CD63 up-regulation can be related to basophil degranulation and histamine release [6], two main activation steps of basophils, which have made this molecule a good activation marker for providing a reliable test of cellular function for allergy diagnosis. Although evidence of other mechanisms able to change CD63 up-regulation on the membrane by histamine release has been also reported [7, 8], its association with degranulation events enabled CD63 to be included in BATs preferentially to other activation markers having comparably good performance. How reliable a parameter is CD63% for evaluating basophil response to an allergen? This is an important issue in assessing how far BATs are reliable tools for diagnosing allergy and hypersensitivity reactions to different allergens. The relatively high frequency of involvement of this marker in allergy cellular tests necessitates a review of CD63 performance in BATs, and so a closer look at how this molecule is expressed as a result of immunoglobulin (Ig) E-mediated activation has to be addressed. Normally, the extent to which CD63 is expressed is evaluated using the fluorescence pattern in a flow cytometry assay. In any case, very few papers seem to use the full potential of fluorescence information on expression of membrane markers, often limiting assessment to a fraction of cells which express the molecule and using only a percentage value to elaborate a possible diagnostic algorithm. However, the expression of CD63 related to basophil activation is a complex mechanism. CD63 displacement onto the membrane occurs in the first few minutes following stimulation, after 5–10 min reaching a plateau lasting throughout the whole 30-minute period of incubation with the allergen or anti-IgE [9]. Most CD63 is stored within cell lysosome-associated granules; it is not synthesized very rapidly, it is not recycled, and the whole amount of CD63 does not appear to be externalized when cells undergo the strongest activation available [8]. Mean fluorescence intensity (MFI) should describe an activated population in which basophils express heterogeneously different amounts of CD63 on their surface, resulting in different parametric distributions in a fluorescence histogram. Flow cytometers are used regularly for the determination of the density of specific molecules on the surface of cells in the population analyzed. Obviously, these measurements may be relative, semiquantitative, or quantitative depending upon the question asked and the reagents and methods applied. In most cases, when fluorescence signals derived from monoclonal antibody (mAb) binding are measured, the data are log-transformed to provide sufficient resolution of the cells. If the fluorescence intensity of the population appears normally (i.e., Gaussian) distributed (not bimodal), this procedure means that the population is a ‘‘log-normal’’ distribution, in which the mean fluorescence intensity will be skewed to the right, Responsible Editor: Liwu Li.