免疫磁珠负性富集结合免疫荧光抗体技术检测卵巢癌患者外周血CTCs的方法建立和应用

Establishment and Application of Immunomagnetic Bead Negative Enrichment and Immunofluorescence Antibody Technique for Detection of CTCs in Peripheral Blood of Patients with Ovarian Cancer

目的建立基于细胞学水平的循环肿瘤细胞负性富集技术来检测卵巢上皮性癌(卵巢癌)患者外周血循环肿瘤细胞。方法体外培养卵巢癌SKOV-3细胞,按一定比例掺入到2 ml健康人外周静脉血中,应用免疫磁珠为基础的负性富集结合免疫荧光抗体技术对循环肿瘤细胞进行定量检测。评价该方法富集靶细胞的回收率,并用上述方法检测32例卵巢癌患者和10例对照者外周血中的循环肿瘤细胞。结果①在健康志愿者2 ml外周血掺入不同数目SKOV-3细胞的条件下,肿瘤细胞的回收率在64%~80%之间,掺入细胞间回归方程为Y=0.782X-1.408,相关系数为0.998。②10例对照者外周血中未检测到CK8/18+,18例卵巢癌患者的外周血中检测到CK8/18+,阳性率为56.3%(18/32,X^2=7.681,P<0.01)。③CTCs阳性与远处转移密切相关(X^2=5.776,P<0.05)。结论免疫磁珠负性富集结合免疫荧光抗体技术检测卵巢癌患者外周血循环肿瘤细胞的方法可能具有一定的临床应用价值。

Objective To establish a cell level-based negative enrichmen technique to detect circulating tumor cells （CTCs） in the peripheral blood of patients with epithelial ovarian cancer. Methods The colon cancer SKOV-3 cells were mixed with 2 ml whole blood from healthy donors at different ratio. Quantification of CTCs was performed using immunomagnetic bead based negative enrichment combined with immunofluorescence antibody method. The method was evaluated the recovery rate of target cells,Samples of 32 patients with ovarian cancer and 10 controls were assayed for CTCs detection by above method. Results ①The recovery rate was ranging from 64% - 80% by spiking varying numbers of SKOV-3 into 2 ml blood samples of healthy volunteers. Regression analysis of number of recovered SKOV-3 cells yielded a regression equation of Y= 0. 782X-1. 408 and a coefficient of determination of R2 =0. 998. ②Did not detect CK8/18＋ circulating tumor cells in 10 controls,and CK8/18＋ circulating tumor cells in 18 cases of 32 Patients with ovarian cancer. The positive rate of CK 8/18 ＋ circulating tumor cells was significantly differences between the two groups （X^2 = 7. 681 ,P〈0.01）. ③The presence of- CTCs was significantly correlated with distant metastasis （X^2 = 5. 776,P〈0.05）. Conclusion The method of immunomagnetic bead based negative enrichment combined with immunofluorescence antibody technique for CTCs detection in peripheral blood of patients with ovarian cancer has a clinical value of application and extension.