免疫磁珠阳性筛选联合流式细胞术对胰腺癌外周血循环肿瘤细胞的诊断价值

The Diagnosis Value about the Peripheral Blood Circulating Tumor Cells of Pancreatic cancer by Positive-Screened Immune Magnetic Bead Combine with Flow Cytometry

目的通过免疫磁珠阳性筛选联合流式细胞术检测胰腺癌患者外周血循环肿瘤细胞(CTCs),探讨其敏感性及诊断价值。方法将我院自2013年06月至2014年12月期间收治67例胰腺癌患者作为观察组,同时选取健康体检者19例作为对照组。采用ACD采血管收集抗凝血7.5mL,人淋巴细胞分离液分离出单个核细胞,红细胞裂解处理后加入EPCAM(CD326)抗体标记磁珠,通过MAC分离柱收集CD326抗体阳性细胞,最后加入CD45、Cytokeratin、CD326抗体标记上流式仪检测以获得CTCs个数。将CTCs个数与患者年龄、肿瘤大小、位置、分期、是否远端转移及肿瘤标志物CA19-9进行相关性分析。同时通过对健康体检者血液分别加入5、50、100、500个胰腺癌肿瘤细胞以检测该方法的敏感性。结果观察组中CTCs阳性率达80.6%。CTCs数量与肿瘤分期、是否发生远端转移、血清肿瘤标志物CA19-9显著相关(P<0.01),与性别、肿瘤位置及大小无关。健康体检者检出CTCs数量均为0个。CTCs的回收率分别为66.7%、86.7%、87.3%及83.3%。结论免疫磁珠阳性筛选联合流式细胞术可有效诊断胰腺癌CTCs,并对评估肿瘤风险有一定的临床价值。

Objective To explore the sensitivity and diagnostic value about the detect method of peripheral blood circulating tumor cells（CTCs）by immune magnetic beads combined with flow cytometry in pancreatic cancer patients.Methods From June 2013 to December 2014,67 patients with pancreatic cancer were selected as observation group,and19 healthy patients were selected as control group.After 7.5mL EDTA anticoagulant blood was drawn and stored with ACD tube,the mononucleocytes were separated from the blood over Ficoll-Paque.the RBCs were removed using a lysis buffer.Labeled cells with CD326 antibodies microbeads,then collected CD326 antibody positive cells through MACS separator.Labeled cells with CD45,Cytokeratin,CD326 antibody and detected the number of CTCs with flow cytometry.We used CTCs number,patient age,tumor size,location,stage,whether the distal metastasis,tumor marker CA19-9for correlation analysis.At the same time,5,50,100,500 pancreatic cancer cell line cells was added respectively into the control group blood to detect the sensitivity.Results In the observation group,CTCs positive rate was 80.6%.The analyses showed that there was statistically significant difference（P0.01）in the Number of CTCs between the distal metastasis,tumor stage and serum tumor marker CA19-9,but hasn＇t difference between with the sex,tumor location,and tumor size（P0.05）.Control group were detected in CTCs quantity to zero.The recovery rate of CTCs are66.7%,86.7%,87.3% and 83.3% respectively.Conclusion Positive-screened immune magnetic bead combine with flow cytometry can detect CTCs effectively and has certain clinical value to evaluate the risk of tumor.