循环肿瘤细胞富集和鉴定技术在乳腺癌患者中的临床验证

Clinical Validation of an Enrichment and Identificationmethod of Circulating Tumor Cells in Breast Cancer Patients

临床验证了一种有效的循环肿瘤细胞富集和鉴定方法,并探讨了该方法应用于乳腺癌循环肿瘤细胞(CTC)的临床检测。分别取10名健康志愿者外周抗凝静脉血7.5mL,加入经标记的培养乳腺癌细胞SKBR-3,抗白细胞抗体偶联的纳米磁珠阴性富集后在荧光显微镜下读取细胞并计数,通过细胞的回收率对富集方法予以评价;分别取20名健康志愿者、15名临床诊断为浸润性乳腺癌患者外周抗凝静脉血7.5mL,对所取血液进行编盲处理,经富集、免疫荧光加免疫细胞化学染色后在显微镜下读取并判定循环肿瘤细胞,通过两组间检测到的循环肿瘤细胞的差异对该研究方法的特异性和敏感性进行评价。研究结果表明,10名健康志愿者血液中加入培养细胞的平均回收率为75%;加入的培养细胞抗角化蛋白(CK18)染色阳性、抗Her2染色阳性、抗CD45染色阴性,符合乳腺癌细胞的特征;揭盲后结果显示,20名健康志愿者血液均未报告有循环肿瘤细胞,而在乳腺癌患者中,CTC数目小于3个(0—2)者占所检测患者的33.3%,大于3个者占66.7%。由此得出结论:该循环肿瘤细胞富集和鉴定方法对外周血CTC具有较好的回收率,且对于CTC的临床诊断具有很好的特异性和灵敏度。

An effective enrichment and identification method of Circulating Tumor Cells（CTC） was clinically validated and the specificity and sensitivity of the method in CTCs detection from breast cancer patients was evaluated as well in this paper.Fluorescent labeled cultured breast cancer cell line,SKBR-3 cells were added into 7.5mL peripheral blood obtained from ten of healthy volunteers respectively in this paper.After enrichment by using anti-leukocyte antigen antibody conjugated nanobeads,the slides were observed with fluorescent microscope and the fluorescent labeled cells were counted.The enrichment efficiency was evaluated by average cells recovery rate.The peripheral bloods from 20 of healthy volunteers and 15 of patients with clinical diagnosis of invasive breast cancer were extracted and blind coded.Then the CTCs were enriched and identified by immunofluorescent plus immunocytochemical stains.By comparing the difference of CTCs number between healthy volunteers and breast cancer patients,the specificity and sensitivity of the method were evaluated.The results show that the average spiked cells recovery rate was 75%.The spiked cells were anti-keratin（CK18） stain positive,anti-Her2 stain positive and anti-CD45 stain negative,which consist with the antigen expression characters of breast cancer cells.The unblinding result revealed that there were none of CTCs found in bloods from 20 of healthy volunteers,while among 15 of breast cancer patients,less than 3 CTCs（0—2） were detected in 33.3% patients and more than 3 CTCs were detected in 66.7% patients.The enrichment and identification method used could well recover spiked cells in peripheral blood and have significant specificity and sensitivity in CTCs clinical detection.