中华结核和呼吸杂志
中華結覈和呼吸雜誌
중화결핵화호흡잡지
Chinese Journal of Tuberculosis and Respiratory Diseases
2012年
9期
673-678
,共6页
杨俊俊%徐兴祥%闵凌峰%LIN Ping%钱桂生
楊俊俊%徐興祥%閔凌峰%LIN Ping%錢桂生
양준준%서흥상%민릉봉%LIN Ping%전계생
纳米粒子%胸腔积液,恶性%原位杂交,荧光
納米粒子%胸腔積液,噁性%原位雜交,熒光
납미입자%흉강적액,악성%원위잡교,형광
Nanoparticles%Pleural effusion,malignant%In situ hybridization,fluorescence
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20 ml心力衰竭患者的胸腔积液中[约含(1～10)×106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 分别加入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(x2=4.00,P=0.039;x2=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出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.
目的 建立免疫磁珠陰性法富集噁性胸腔積液腫瘤細胞的方法,探討該方法富集胸腔積液中腫瘤細胞的敏感性、特異性及其應用價值.方法 將5、10、20、50、100箇4,6-二瞇-2苯吲哚( DAPI)染色的肺腺癌細胞繫A549細胞分彆加入20 ml心力衰竭患者的胸腔積液中[約含(1～10)×106箇細胞],通過免疫磁珠陰性法富集癌細胞,熒光顯微鏡檢測體外加入癌細胞的迴收率.收集2010年7月至2011年7月江囌省囌北人民醫院呼吸科53例患者的胸腔積液,其中包括經活檢病理證實的36例腫瘤患者的胸腔積液標本,男25例,女11例,年齡40 ～78歲,平均(63±9)歲；17例非腫瘤患者的胸腔積液標本,男8例,女9例,年齡25 ～81歲,平均(53±18)歲.應用免疫磁珠陰性法及密度梯度離心法富集53份胸腔積液標本併塗片,然後分彆應用瑞姬染色、免疫熒光染色及熒光原位雜交( FISH)法進行腫瘤細胞鑒定.結果 分彆加入5、10、20、50、100箇經DAPI染色的肺腺癌A549細胞的迴收率分彆為75％、78％、82％、85％和88％,平均迴收率為81.6％.應用免疫磁珠陰性法聯閤瑞姬染色檢測噁性胸腔積液中腫瘤細胞的暘性率為81％ (29/36),免疫磁珠陰性法聯閤免疫熒光染色檢齣錶達細胞角蛋白18(CK18)和DAPI 暘性、白細胞共同抗原(CD45)陰性細胞的暘性率均為100％,免疫磁珠陰性法聯閤熒光原位雜交(FISH)檢齣腫瘤細胞的暘性率為86％(31/36);密度梯度離心法聯閤瑞姬染色檢齣腫瘤細胞的暘性率為61％ (22/36).免疫磁珠陰性法聯閤瑞姬染色或FISH與密度梯度離心法聯閤瑞姬染色比較,差異均有統計學意義(x2=4.00,P=0.039;x2=5.818,P=0.012).良性胸腔積液患者應用免疫磁珠陰性法聯閤瑞姬染色或FISH、密度梯度離心法聯閤瑞姬染色均未檢齣腫瘤細胞,而應用免疫磁珠陰性法聯閤免疫熒光染色檢齣CK18及DAPI暘性、CD45陰性細胞的暘性率為100％.結論 應用免疫磁珠陰性法富集胸腔積液腫瘤細胞的方法可行,且該方法聯閤瑞姬染色或FISH對噁性胸腔積液的診斷具有較高的敏感性和特異性,對于良噁性胸腔積液的鑒彆具有重要意義；而聯閤CK18、DAPI、CD45標記的免疫熒光染色不能有效鑒彆胸腔積液中的腫瘤細胞和間皮細胞,還需尋找更特異的腫瘤細胞抗體標誌物.
목적 건립면역자주음성법부집악성흉강적액종류세포적방법,탐토해방법부집흉강적액중종류세포적민감성、특이성급기응용개치.방법 장5、10、20、50、100개4,6-이미-2분신타( DAPI)염색적폐선암세포계A549세포분별가입20 ml심력쇠갈환자적흉강적액중[약함(1～10)×106개세포],통과면역자주음성법부집암세포,형광현미경검측체외가입암세포적회수솔.수집2010년7월지2011년7월강소성소북인민의원호흡과53례환자적흉강적액,기중포괄경활검병리증실적36례종류환자적흉강적액표본,남25례,녀11례,년령40 ～78세,평균(63±9)세；17례비종류환자적흉강적액표본,남8례,녀9례,년령25 ～81세,평균(53±18)세.응용면역자주음성법급밀도제도리심법부집53빈흉강적액표본병도편,연후분별응용서희염색、면역형광염색급형광원위잡교( FISH)법진행종류세포감정.결과 분별가입5、10、20、50、100개경DAPI염색적폐선암A549세포적회수솔분별위75％、78％、82％、85％화88％,평균회수솔위81.6％.응용면역자주음성법연합서희염색검측악성흉강적액중종류세포적양성솔위81％ (29/36),면역자주음성법연합면역형광염색검출표체세포각단백18(CK18)화DAPI 양성、백세포공동항원(CD45)음성세포적양성솔균위100％,면역자주음성법연합형광원위잡교(FISH)검출종류세포적양성솔위86％(31/36);밀도제도리심법연합서희염색검출종류세포적양성솔위61％ (22/36).면역자주음성법연합서희염색혹FISH여밀도제도리심법연합서희염색비교,차이균유통계학의의(x2=4.00,P=0.039;x2=5.818,P=0.012).량성흉강적액환자응용면역자주음성법연합서희염색혹FISH、밀도제도리심법연합서희염색균미검출종류세포,이응용면역자주음성법연합면역형광염색검출CK18급DAPI양성、CD45음성세포적양성솔위100％.결론 응용면역자주음성법부집흉강적액종류세포적방법가행,차해방법연합서희염색혹FISH대악성흉강적액적진단구유교고적민감성화특이성,대우량악성흉강적액적감별구유중요의의；이연합CK18、DAPI、CD45표기적면역형광염색불능유효감별흉강적액중적종류세포화간피세포,환수심조경특이적종류세포항체표지물.
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 adenocarcinoma) cells were labeled with DAPI and added into 20 ml pleural effusions [ containing ( 1 - 10 ) × 106cells ]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 identification with 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 A549 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 (x2 =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 cells was 86％ (31/36),much higher than that using density gradient centrifugation combined with cytology analysis ( x2 =5.818,P =0.012 ). In 17cases 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 cells using immunofluorence staining with CK18,DAPI and CD45 label. More specific markers were needed to recognize tumor cells from pleural effusions.
