流式细胞术检测食管鳞癌患者外周血循环肿瘤细胞

The detection of circulating tumor cells in peripheral blood of esophageal squamous cell carcinoma with flow cytometry

目的利用密度梯度离心法和流式细胞术联合检测食管鳞癌患者外周血循环肿瘤细胞(circulating tumor cells,CTCs),以建立一种高效简单易行的食管鳞癌CTCs分离和检测方法。方法选取中国人民解放军总医院第六医学中心胸外科2017年10月—2018年5月收治的20例食管鳞癌患者和5名健康志愿者,抽取外周血7.5 mL,用密度梯度离心法提取外周血中单个核细胞(peripheral blood mononuclear cell,PBMC),然后用流式细胞术的方法对提取的PBMC进行标记,用抗EpCAM、抗CD45以及抗Cytokeratin(CK)单克隆抗体对PBMC进行抗体染色标记,将其中CD45阴性,EpCAM阳性且CK阳性的细胞定义为CTCs。同时将健康志愿者外周血中加入不同数量的食管鳞癌细胞来检测该方法的敏感度。结果以CTCs≥3/7.5 mL基数定义为CTCs阳性;CTCs<3/7.5 mL基数定义为CTCs阴性。观察组中CTCs检出率达75%,对照组中未检出。用流式细胞术检测食管鳞癌外周血中CTCs的敏感度为10-6。结论初步建立了一种特异性好,敏感度较高,操作简单易行的食管鳞癌CTCs分离和检测方法。

Objective Density gradient centrifugation and flow cytometry were used to detect peripheral blood circulating tumor cells(CTCs)in patients with esophageal squamous cell carcinoma(ESCC),in order to establish a highly effective and simple method for the isolation and detection of CTCs.Methods Twenty ESCC patients were selected as observation group and five healthy subjects were selected as control group.Peripheral blood mononuclear cells(PBMC)were extracted from 7.5 mL EDTA anticoagulant bloods by density gradient centrifuge and stained with anti-CD45,anti-Ep-CAM and anti-Cytokeratin(CK)monoclonal antibody.The cells that labeled with CD45-EpCAM+CK+were consider as the CTCs.Meanwhile different numbers of ESCC cells were added to the peripheral blood of healthy volunteers to test the sensitivity.Results The base line was formulated as CTCs≥3/7.5 mL positive and CTCs<3/7.5 mL negative.In the observation group,CTCs positive rate was 75%,while no CTCs were detected in the control group.The sensibility of flow cytometry in detection CTCs of ESCC was 10-6.Conclusion A method for the isolation and detection of CTCs of ESCC with good specificity,high sensitivity and simple operation was established.