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). Several questions and influencing factors on the analytical feasibility and reliability of BATs still remain and generally the potentials and pitfall of these diagnostic tools are a point at issue (2,3). Activated basophils up-regulate several membrane proteins, such as the tetraspan CD63 and the ectoenzyme CD203c but also express many other molecules on the surface that can be used to characterize these cells allowing their separation from other leukocytes (4,5). A large number of gating strategies to phenotype basophils have been proposed in the past and most of them are currently in use: IgE, HLA-DR/CD123, CD45/ CCR3, CD3/CRTH2 (3). An approach for best basophil electronic capture is the use of a single selection marker, with a bright fluorochrome, as basophils gated with the brighter fluorochrome PE resulted in a better separation of the target basophilic cells compared to FITC (2). One candidate to isolate basophils in whole blood or in leukocyteenriched buffy coats might be CD203c, as this molecule is expressed only on basophilic cells (3,6); however this marker changes with activation, it is constitutively present on resting basophils with a wide interindividual variability in cell surface expression and it is influenced by preanalytical process and by autocrine interleukin 3 (2). Another well-known selection approach using the highly basophil specific marker anti-IgE has raised serious objections, because anti-IgE is a potential triggering agent, because other leucocytes express IgE-receptor (Fc‹RII) and because the density of IgE and Fc‹RI may vary widely among subjects and even among basophils in the same individual (3). So, recently published papers have suggested the use of eotaxin receptor CCR3, also known as CD193, as a single selection marker to separate basophils from other leukocytes in flow cytometry, though with some controversial issue (7,8). This marker has the indisputable advantage of being highly expressed in cells at the basal level with a comparable extent in different individuals but it changes its expression upon basophil stimulation (7). Phenotyping markers, which upor down-regulate membrane expression following cell stimulation, make it very difficult to perform a gating process without introducing cellular contamination into the gate and/or to attain a loss of target cells when activated. Choosing the best gating strategy often has to face all of these topics, to evaluate basophil function the best possible way. This work trying to address these controversial issues raises some comments and suggestions. A total of 32 volunteer randomized blood donors (mean age 44.61 6 4.57 SD, female/male ratio 5 1.28) enrolled and evaluated over a period of 2 years were considered in this study. All the subjects were non allergic and non atopic, they did not suffer from any immunological disorder and had never reported any previous history or genetic diathesis of chronic allergy. Moreover, none underwent either drug therapy or antihistamine therapy during the 48 hrs before withdrawal. All participants signed an informed consent. Basophils were collected and treated as leukocyteenriched buffy coats from venous K2-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). Cells were blocked in ice-cold buffer with 2.8 mmol/L Na3-EDTA, then stained with monoclonal antibodies (20 minutes at 14 C) and erythrolysed (4 min) with an ammoniumbuffered solution, centrifuged and pellets recovered and resuspended in a PBS-buffered saline solution (pH 7.4) for