胸腔积液肺癌脱落细胞的流式检测及其初步临床应用

A preliminary study on the detection of lung cancer cells in pleural effusion by flow cytometry for non-small cell lung cancer

目的探讨特异性荧光抗体标记的流式细胞术检测胸腔积液肺癌脱落细胞的临床应用价值。方法采用特异性识别非小细胞肺癌（NSCLC）的单克隆抗体NJ001进行荧光标记，建立流式细胞术检测胸腔积液肺癌脱落细胞的方法。采用病例对照研究，收集2014年2至8月南京医科大学第一附属医院经临床确诊的32例NSCLC（包括29例肺腺癌和3例肺鳞癌）和26例肺部良性疾病患者的胸腔积液，流式细胞术进行肺癌脱落细胞水平检测，采用秩和检验分析组间差异，并绘制ROC曲线进行分析；对32例NSCLC的流式法和巴氏染色法检测结果进行，检验统计比较。对22例NSCLC胸腔积液脱落细胞涂片采用直接荧光法检测，观察脱落细胞上NJ001特异性抗原的表达，并与流式法检测结果进行，检验统计比较。结果NSCLC组流式检测的阳性计数水平为100（2—988）个／10万，显著高于肺部良性疾病组的5（0～18）个／10万（Z=-5．647，P〈0．001）；ROC曲线下面积为0．934；最佳诊断临界值为18个／10万，对应的敏感度为87．5％（28／32），特异度为100．0％（26／26）。其中29例肺腺癌胸腔积液流式法检测的阳性率为93．1％（27／29），显著高于巴氏染色法检测的阳性率58．6％（17／29），差异有统计学意义（x2=7．627，P=0．006），12例巴氏染色法检测阴性的肺腺癌胸腔积液，流式法检测的阳性率为11／12；3例肺鳞癌胸腔积液流式法检测的阳性率为1／3，对应的巴氏染色法检测均为阴性。22例NSCLC胸腔积液脱落细胞涂片直接荧光法检测的阳性率为59．1％（13／22）：对应的流式法的阳性率为90．9％（20／22），显著高于直接荧光法的阳性率59．1％（13／22）（X2=4．364，P=0．037）。结论特异性荧光抗体标记的流式细胞术检测胸腔积液肺癌尤其是肺腺癌脱落细胞具有极高的敏感度和特异度，具有重要的临床应用价值。

Objective To investigate the clinical application value of flow cytometry （FCM） established by specific fluorescent monoclonal antibody in detecting lung cancer cells in pleural effusion with non-small cell lung cancer （ NSCLC ） . Methods The FCM assay was established by using fluorescent monoclonal antibody NJ001 which could specifically recognize NSCLC. A case-control study was conducted to evaluate the level of lung cancer cells in pleural effusion among 32 cases of NSCLC （ including 29 lung adenocarcinoma cases and 3 lung squamous cell carcinoma cases） and 26 cases of benign disease with definite diagnosis collected from the First Affiliated Hospital of Nanjing Medical University from February 2014 to August 2014. The data of FCM assay between NSCLC and benign disease were evaluated by the rank sum test. The data were analyzed by ROC curve to assess the diagnosis value. Those FCM results among 52 cases of NSCLC were compared with Papanicolaou staining by the chi-square test. Meanwhile, NJO01 specific antigen on exfoliate cells was detected by direct immunofluorescence assay and comparing with FCM assay by the chi-square test among 22 cases of NSCLC. Results The number of positive events by FCM assay was 5 （0 -18 ）/100 000 in benign pleural effusion and 100 （2 -988 ）/100 000 in pleural effusion of NSCLC, which was significantly higher compared with the benign disease （Z = - 5. 647 ,P 〈 0. 001 ）. The area under the ROC curve was 0. 934 and the optimal diagnostic critical value was 18/100 000. This method performed a high sensitivity[ 87. 5% （28/32） ] and specificity[ 100. 0% （26/26） ]. The positive rate in 29 lung adenocarcinoma cases was significantly higher by FCM assay than Papanicolaou staining [ 93.1% （27/ 29） vs 58.6% （ 17/29 ）, X2 = 7. 627, P = 0. 006 ] , and in 12 negative Papanicolaou staining lung adenocarcinoma cases the positive rate of FCM assay was 11/12; In 3 lung squamous cell carcinoma cases the positive rate of FCM assay was 1/3 while all of Papanicolaou staining presented negative results. The FCM assay positive rate of 22 NSCLC cases was significantly higher than the direct immunofluorescence assay [90. 9% （20/22） vs 59. 1% （13/22）, X2 =4. 364, P =0. 0371. Conclusion The FCM assay established by specific fluorescent monoclonal antibody applied for lung cancer cells especially for lung adenocarcinoma detection in pleural effusion had a high sensitivity and specificity, which was confirmed to be a valuable technological application for NSCLC clinical diagnosis.