利用新型免疫磁珠富集原理检测肺癌患者外周血循环肿瘤细胞

A novel method for detection of circulating tumor cells in peripheral blood of patients with lung cancer using immunomagnetic beads device

目的建立一种新型免疫磁珠方法，检测肺癌患者外周血中循环肿瘤细胞（CTCs）。方法实验探索性研究。选择经手术切除组织病理确诊为非小细胞肺癌的42例初治患者，其中I期7例、Ⅱ期9例、Ⅲ期16例、Ⅳ期10例，健康志愿者20名。将不同数量（50、100、200、500、1000个）的肺癌细胞株（A549）混入2ml健康成人血标本中以模拟肿瘤患者的血样，对该血样进行红细胞裂解和离心，沉淀所得细胞与经过免疫磁珠标记的上皮细胞黏附分子（EpCAM）的抗体反应，然后经一套自制的检测装置对细胞进行富集和分离，与磁标记的EpCAM抗体反应后的上皮细胞因为磁力的作用被集中在一张玻片上，最后用经典的HE染色鉴定循环肿瘤细胞并计数。计算肿瘤细胞的回收率验证该方法的敏感性，同时检测20名健康志愿者血标本验证该方法的特异性。用上述方法检测42例肺癌患者的CTCs，将检测到的结果根据患者的临床特征进行分组，以卡方检验分析CTCs的检出率与患者的年龄、性别、临床分期、肿瘤大小等临床特征的相关性，同时以直线相关法研究掺入细胞数与回收细胞数的相关性。结果掺人肿瘤细胞回收试验的回收率在68％～82％之间，掺入细胞间回归方程为Y=0．6419X＋8．8875，回收的细胞与添加细胞间数量有显著的相关性（相关系数尺。=0．9916，P〈0．05）。42例肺癌患者中有18例（42．9％）CTCs阳性，其中，I期CTCs检出率为0，II期CTCs检出率为11．1％，Ⅲ期CTCs检出率为62．5％，IV期CTCs检出率为70％。CTCs检测的阳性率与患者的年龄、性别、肿瘤大小均无明显关系（P〉0．05），而与临床分期（TNM）有密切关系（P〈0．05）。特异性检测结果为20名健康志愿者外周血标本均未检测到CTCs。结论新型免疫磁珠富集技术可以有效分离和鉴定肺癌患者外周血中的循环肿瘤细胞，有较高的敏感性和特异性；随着肺癌患者分期增高和肿瘤增大，肺癌细胞发生远处转移逐渐增多，CTCs检出的水平亦增高，对于早期发现肺癌隐形微转移和重新确定肺癌分期具有重要意义，但其临床价值仍需进一步多中心、大样本病例的临床研究加以证实。

Objective To establish a novel method for detecting circulating tumor cells （CTCs） in phripheral blood of lung cancer patients with high sensitivity and specificity. Methods Experimental study. 42 cases of initial treatment patient who underwent resection and diagnosed to be non-small cell lung cancer by biopsy were studied, including 7 patients at stage I , 9 patients at stage II , 16 patients at stage IU and 10 patients at stage IV. As a control group, 20 cases of healthy volunteers were selected. A series of experiments was conducted to determine the efficiency of tumor cells isolation, in which varied concentration （50,100,200,500,1000 cells） of A549 cells spiked into 2 ml peripheral blood drawn from healthy donors. The blood was removed of unwanted erythrocytes by lysis buffer, and made the rest of nucleated ceils incubate with anti-EpCAM magnetic beads, then separated and enriched by a specific detector. All epithelia cells were retained on a slide because of a magnetic force and identified by H＆E staining protocol. On the basis of cell recovery rate we calculated the sensitivity of tumor cells isolation. 20 blood samples taken from healthy individuals were also detected to validate the specificity of this method. Samples of 42 patients with lung cancer were assayed for CTCs detection by above method. The correction of CTCs quantity with the patients＇ clinical features, for example, ages, gender, clinical stage, tumor size was analyzed in lung cancer patients by chi-square statistics. The correction of recovery cells with the spiked cells were assayed by linear correlation. Results The recovery rate was ranging from 68% to 82% by spiking varying numbers of A549 lung cancer cells into 2ml blood samples of healthy volunteers. Regression analysis of number of recovered vs. spiked A549 cells yielded a regression equation of Y = 0. 6419X ＋ 8. 8875. The number of CTCs detected has signification correlate with the cells spiked （ R2 = 0. 9916, P 〈 0. 05 ）, Eighteen of the 42 patients （43%） were found have CTCs in peripheral blood. The detection rate of lung cancer cells was 0 at stage I , the detection rate of lung cancer cells was 11.1% at stage II, the detection rate of lung cancer ceils was 62. 5% at stage m and the detection rate of lung cancer cells was 70% at stage IV. The positive rate of CTCs has no signification correlate with ages and gender of patients and tumor size （ P 〉 0. 05 ）, has signification with the clinical stage （ P 〈 0. 05 ）. None of the peripheral blood samples of the 20 healthy subjects analyzed was found to have CTCs. Conclusions This novel immunomagnetic separation technology is a sensitive and specific method, which provides a new tool allowing for feasible and specific detection of CTCs in lung cancer patients. The level of CTCs increases with the clinical stage and tumor size increased, which has important value to discover the early stage micrometastasis and redefine the clinical stage. But further muhicenter and large sample clinical research are needed to confirm its clinical value.