Kuwasaki T, Chihara J, Kayaba H, Kamata Y, Oyamada H, Saito N, Shioya T, Sasaki M, Kagaya M, Tsuda A
Division of Central Laboratory, Akita University Hospital, Akita, Japan.
Int Arch Allergy Immunol. 1998 Sep;117 Suppl 1:77-80. doi: 10.1159/000053578.
Using a simple technique detecting the eosinophil fraction in whole-blood flow cytometry, we measured intracellular antigen EG2 (a monoclonal antibody to eosinophil cationic protein) in 56 asthmatic patients (26 during an attack and 30 during an asymptomatic period) and 22 healthy subjects to determine whether EG2 reflects the pathological stages of allergy.
In brief, preparations of the sample included the following procedures: (1) hemolyzation of heparinized or EDTA-mixed whole blood; (2) fixation of white blood cells with 0.4% parabenzoquinone (PBQ) or paraformaldehyde (PFA); (3) permeabilizing the cell membrane with n-octyl-beta-D-glucopyranoside, and (4) staining of intracellular EG2 antigen with monoclonal EG2 antibody and FITC-labeled secondary antibody.
In PBQ-fixed samples, there was a clearer boundary of the eosinophil fraction with a higher yield and purity than in those fixed with PFA. The number of EG2-positive eosinophils was significantly greater in subjects during attacks than in asymptomatic patients. In addition, when compared with normal controls, asthmatic subjects had significantly greater numbers of EG2-positive eosinophils regardless of their current condition.
Eosinophil intracellular EG2 may indicate the pathological stage of asthma. This simple technique for analysing the properties of eosinophils using whole-blood flow cytometry would save time and labor in laboratories.
我们采用一种在全血流式细胞术中检测嗜酸性粒细胞比例的简单技术，对56例哮喘患者（26例发作期和30例无症状期）及22名健康受试者进行了细胞内抗原EG2（一种针对嗜酸性粒细胞阳离子蛋白的单克隆抗体）的检测，以确定EG2是否反映过敏的病理阶段。
简而言之，样本制备包括以下步骤：（1）对肝素化或乙二胺四乙酸（EDTA）混合的全血进行溶血；（2）用0.4%对苯醌（PBQ）或多聚甲醛（PFA）固定白细胞；（3）用正辛基-β-D-吡喃葡萄糖苷使细胞膜通透；（4）用单克隆EG2抗体和异硫氰酸荧光素（FITC）标记的二抗对细胞内EG2抗原进行染色。
在PBQ固定的样本中，嗜酸性粒细胞比例的边界比PFA固定的样本更清晰，产量和纯度更高。发作期受试者中EG2阳性嗜酸性粒细胞的数量明显多于无症状患者。此外，与正常对照组相比，无论哮喘患者当前处于何种状态，其EG2阳性嗜酸性粒细胞的数量均显著更多。
嗜酸性粒细胞内的EG2可能指示哮喘的病理阶段。这种使用全血流式细胞术分析嗜酸性粒细胞特性的简单技术将节省实验室的时间和人力。
