Eberlein Bernadette, Hann Rebekka, Eyerich Stefanie, Pennino Davide, Ring Johannes, Schmidt-Weber Carsten B, Buters Jeroen
Department of Dermatology and Allergy Biederstein, Technische Universität München, Germany, Munich, Germany.
Cytometry B Clin Cytom. 2015 May-Jun;88(3):183-9. doi: 10.1002/cyto.b.21203. Epub 2014 Nov 17.
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.
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.
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.
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.
通过流式细胞术鉴定嗜碱性粒细胞是使用IgE依赖性或IgE非依赖性刺激物测量嗜碱性粒细胞活化的前提条件。本研究的目的是在同步多色流式细胞术测量中比较不同的标志物组合。
本研究纳入了10名对草花粉过敏的患者和3名对照。使用抗CCR3、抗IgE、抗CRTH2、抗CD203c和抗CD3对嗜碱性细胞进行门控。细胞用单克隆抗FcεRI抗体、N-甲酰甲硫氨酰-亮氨酰-苯丙氨酸（fMLP）和变应原提取物早熟禾进行活化。使用活化标志物抗CD63。
抗CCR3+细胞、抗IgE+和抗IgE+/抗CD203c+细胞中嗜碱性粒细胞的相对数量最高，CRTH2+/CD203c+/CD3-细胞中最低。在所有实验中，CCR3+细胞与CCR3+/CD3-细胞和CRTH2+/CD203c+/CD3-细胞的一致性非常好。与所有其他标志物组合相比，CCR3+群体中CD3+细胞的污染以及IgE+群体中CCR3-细胞和CD203-细胞的污染最低。
由于在大多数情况下，抗CCR3之后是抗IgE和抗IgE/抗CD203c阳性群体鉴定出的嗜碱性粒细胞相对数量最高，这些标志物通常可推荐用于嗜碱性粒细胞的鉴定。如果需要非常高纯度的嗜碱性粒细胞群体，应选择抗IgE。
