摘要
目的建立免疫磁珠阴性法富集恶性胸腔积液肿瘤细胞的方法，探讨该方法富集胸腔积液中肿瘤细胞的敏感性、特异性及其应用价值。方法将5、10、20、50、100个4，6-二眯-2苯吲哚（DAPI）染色的肺腺癌细胞系A549细胞分别加入20ml心力衰竭患者的胸腔积液中[约含（1～10）×10^6个细胞]，通过免疫磁珠阴性法富集癌细胞，荧光显微镜检测体外加入癌细胞的回收率。收集2010年7月至2011年7月江苏省苏北人民医院呼吸科53例患者的胸腔积液，其中包括经活检病理证实的36例肿瘤患者的胸腔积液标本，男25例，女11例，年龄40～78岁，平均（63±9）岁；17例非肿瘤患者的胸腔积液标本，男8例，女9例，年龄25～81岁，平均（53±18）岁。应用免疫磁珠阴性法及密度梯度离心法富集53份胸腔积液标本并涂片，然后分别应用瑞姬染色、免疫荧光染色及荧光原位杂交（FISH）法进行肿瘤细胞鉴定。结果分别加人5、10、20、50、100个经DAPI染色的肺腺癌A549细胞的回收率分别为75％、78％、82％、85％和88％，平均回收率为81．6％。应用免疫磁珠阴性法联合瑞姬染色检测恶性胸腔积液中肿瘤细胞的阳性率为81％（29／36），免疫磁珠阴性法联合免疫荧光染色检出表达细胞角蛋白18（CK18）和DAPI阳性、白细胞共同抗原（CD45）阴性细胞的阳性率均为100％，免疫磁珠阴性法联合荧光原位杂交（FISH）检出肿瘤细胞的阳性率为86％（31／36）；密度梯度离心法联合瑞姬染色检出肿瘤细胞的阳性率为61％（22／36）。免疫磁珠阴性法联合瑞姬染色或FISH与密度梯度离心法联合瑞姬染色比较，差异均有统计学意义（X^2=4．00，P=0．039；X^2=5．818，P=0．012）。良性胸腔积液患者应用免疫磁珠阴性法联合瑞姬染色或FISH、密度梯度离心法联合瑞姬染色均未检出肿瘤细胞，而应用免疫磁珠阴性法联合免疫荧光染色检卅CK18及DAPI阳性、CD45，阴性细胞的阳性率为100％。结论应用免疫磁珠阴性法富集胸腔积液肿瘤细胞的方法可行，且该方法联合瑞姬染色或FISH对恶性胸腔积液的诊断具有较高的敏感性和特异性，对于良恶性胸腔积液的鉴别具有重要意义；而联合CK18、DAPI、CD45，标记的免疫荧光染色不能有效鉴别胸腔积液中的肿瘤细胞和间皮细胞，还需寻找更特异的肿瘤细胞抗体标志物。
Objective To establish a method （negative enrichment by immunomagnetic beads） for detection of tumor cells in pleural effusions and to evaluate the sensitivity and specificity of the method for clinical application. Methods Five, 10, 20, 50 and 100 A549 （lung adenoeareinoma） cells were labeled with DAPI and added into 20 ml pleural effusions [ containing （ 1 - 10 ） ×10^6cells ] from heart failure patients, followed by immunomagnetic negative enrichment method. Recovered cancer cells were enumerated using a fluorescent microscope. Tumor cells were enriched from pleural effusion samples by means of density gradient centrifugation and negative enrichment by immunomagnetic beads method, followed by identificationwith cytology analysis （Wright＇ s Giemsa＇ s staining）, immunofluorence staining （IF） and fluorescence in situ hybridization （FISH） using centromere DNA probes of chromosome 7 and 8. Cytology, IF and FISH evaluations were performed in 53 pleural effusion samples, including 36 cases of malignant disease （25 male and 11 female patients aging 40 to 78 years, mean age （63 ± 9） and 17 cases of benign disease （8 male and 9 female patients aging 25 to 81 years, mean age （53 ± 18）. Results After DAPI staining and mixing with pleural effusions from heart failure patients, the cell recovery rates of Asg9 cells evaluated under fluorescence microscope were 75%, 78%, 82%, 85%, 88%, and the average recovery rate was 81.6%. Using negative enrichment method and density gradient centrifugation combined with cytology analysis, the positive rates of tumor cells in 36 malignant pleural effusion samples were 81% （29/36） and 61% （22/36）, respectively （X^2 =4. 00, P =0. 039）. Using negative enrichment method combined with IF, the positive rate of CK18（ ＋）, DAPI（ ＋ ） , CD45 （ - ） cells was 100%. Moreover, using negative enrichment method combined with FISH analysis, the positive rate of tumor ceils was 86% （31/36）, much higher than that using density gradient centrifugation combined with cytology analysis （X^2 = 5. 818, P =0. 012 ）. In 17 cases of benign pleural effusions, using negative enrichment method combined with IF, the positive rate was 100%. But other methods didn＇ t find cancer cells from benign pleural effusions. Conclusions It was applicable to enrich tumor cells from pleural effusions using negative enrichment method by immunomagnetic beads. This method combined with cytology analysis or FISH significantly enhanced the sensitivity and specificity of tumor cell detection in pleural effusions. But it was difficult to distinguish cancer cells from mesothelial ceils using immunofluorence staining with CK18, DAPI and CD45 label. More specific markers were needed to recognize tumor cells from pleural effusions.
出处
《中华结核和呼吸杂志》
CAS
CSCD
北大核心
2012年第9期673-678,共6页
Chinese Journal of Tuberculosis and Respiratory Diseases
基金
江苏省“六大人才高峰”项目（2010WS007）
关键词
纳米粒子
胸腔积液
恶性
原位杂交
荧光
Nanoparticles
Pleural effusion, malignant
In situ hybridization, fluorescence
