Optimizing of the basophil activation test: Comparison of different basophil identification markers

Background Flowcytometric identification of basophils is a prerequisite for measuring activation of basophils with IgE-dependent or IgE-independent stimuli. Aim of this study was to compare different marker combinations in a simultaneous multicolor flowcytometric measurement. Methods Ten patients with a grass pollen allergy and three controls were included in the study. Basophilic cells were gated by using anti-CCR3, anti-IgE, anti-CRTH2, anti-CD203c, and anti-CD3. Cells were activated by a monoclonal anti-FcεRI antibody, N-formyl-methionyl-leucyl-phenylalanine (fMLP), and the allergen extract Phleum pratense. The activation marker anti-CD63 was used. Results The highest relative number of basophils was found with anti-CCR3⁺ cells, anti-IgE⁺ and anti-IgE⁺/anti-CD203c⁺ cells, the lowest with CRTH2⁺/CD203c⁺/CD3^- cells. A very good and good concordance of CCR3⁺ cells was seen with CCR3⁺/CD3^- cells and CRTH2⁺/CD203c⁺/CD3^- cells in all experiments. The contamination of the CCR3⁺ population with CD3⁺ cells and the contamination of the IgE⁺-population with CCR3^- cells and CD203^- cells were the lowest compared to all other marker combinations. Conclusions As the highest relative number of basophils was identified by anti-CCR3 followed by the anti-IgE and anti-IgE/antiCD203c positive population in most cases, these markers can generally be recommended for identification of basophils. If a basophil population with very high purity is needed, anti-IgE should be chosen.