Differential response of human basophil activation markers: a multi-parameter flow cytometry approach

Abstract

Background: Basophils are circulating cells involved in hypersensitivity reactions and allergy but many aspects of their activation, including the sensitivity to external triggering factors and the molecular aspects of cell responses, are still to be focused. In this context, polychromatic flow cytometry (PFC) is a proper tool to investigate basophil function, as it allows to distinguish the expression of several membrane markers upon activation in multiple experimental conditions.

Are Basophils and T helper 2 Cells Implicated in Mastocytosis?

“…In the literature the existence of "non responder" individuals in basophil activation tests has been reported when using CD63 to evaluate basophil activation [25]. However, it should be noted that cells "non-responding" to fMLP or anti-FcεRI (when evaluated using CD63) are fully responsive to the same agonists when evaluated with other activation-related markers, such as CD203c [26], which was not tested in the present study.…”

CCR3 as a single selection marker compared to CD123/HLADR to isolate basophils in flow cytometry: Some comments

“…Flow cytometry of activated basophils is extensively used to assess cell response to allergens or to soluble agonists and has immediately proved a successful approach for studying the function of basophils in vitro (1). The mechanism of expression of various membrane proteins is the basis of basophil activation tests (BATs).…”

“…All participants signed an informed consent. Basophils were collected and treated as leukocyteenriched buffy coats from venous K 2 -ethylendiaminetetraacetic acid (EDTA) anticoagulated peripheral blood from four screened healthy donors in each experiment performed, according to previously described methods (1). When indicated, activation was performed with 100 nmol/L of fMLP or 2 lg/ml of goat anti-human IgE, according to published methods (1).…”

The use of IL‐3 in basophil activation tests is the real pitfall

“…I wonder whether basophils could actually be primed by autocrine IL-3 in the 4 h following venous blood withdrawal, especially if receptor-ligand function is blocked by treating basophils in ice bath: a spontaneously induced priming by autocrine IL-3 should justify the conclusion with which Sturm EM et al suggest how to use BATs that evaluate CD203c upregulation. Hence, one issue is either to start experimental condition very early or by using ice bath for treating basophils, even during staining with fluorochrome-labeled antibodies (5). Factors other than IgE-mediated agonists, such as anaphylotoxins or bacterial products, are not able to induce autocrine IL-3 by nonstimulated basophils; hence, preanalytical errors due to environmental contamination are not relevant for basophil evaluation, whereas what seems to be more critical is the role of intracellular calcium (6).…”