中华检验医学杂志
中華檢驗醫學雜誌
중화검험의학잡지
CHINESE JOURNAL OF LABORATORY MEDICINE
2013年
8期
727-732
,共6页
嵇金陵%何晓东%索美芳%张培%黄蓉%孙余婕%沈佐君
嵇金陵%何曉東%索美芳%張培%黃蓉%孫餘婕%瀋佐君
혜금릉%하효동%색미방%장배%황용%손여첩%침좌군
肺肿瘤%肿瘤循环细胞%免疫磁化分离%抗原,肿瘤%细胞黏附分子
肺腫瘤%腫瘤循環細胞%免疫磁化分離%抗原,腫瘤%細胞黏附分子
폐종류%종류순배세포%면역자화분리%항원,종류%세포점부분자
Lung neoplasms%Neoplasm circulating cells%Immunomagnetic separation%Antigens,neoplasm%Cell adhesion molecules
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2 ml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 掺入肿瘤细胞回收试验的回收率在68％～82％之间,掺人细胞间回归方程为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R2=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.
目的 建立一種新型免疫磁珠方法,檢測肺癌患者外週血中循環腫瘤細胞(CTCs).方法 實驗探索性研究.選擇經手術切除組織病理確診為非小細胞肺癌的42例初治患者,其中Ⅰ期7例、Ⅱ期9例、Ⅲ期16例、Ⅳ期10例,健康誌願者20名.將不同數量(50、100、200、500、1000箇)的肺癌細胞株(A549)混入2 ml健康成人血標本中以模擬腫瘤患者的血樣,對該血樣進行紅細胞裂解和離心,沉澱所得細胞與經過免疫磁珠標記的上皮細胞黏附分子(EpCAM)的抗體反應,然後經一套自製的檢測裝置對細胞進行富集和分離,與磁標記的EpCAM抗體反應後的上皮細胞因為磁力的作用被集中在一張玻片上,最後用經典的HE染色鑒定循環腫瘤細胞併計數.計算腫瘤細胞的迴收率驗證該方法的敏感性,同時檢測20名健康誌願者血標本驗證該方法的特異性.用上述方法檢測42例肺癌患者的CTCs,將檢測到的結果根據患者的臨床特徵進行分組,以卡方檢驗分析CTCs的檢齣率與患者的年齡、性彆、臨床分期、腫瘤大小等臨床特徵的相關性,同時以直線相關法研究摻入細胞數與迴收細胞數的相關性.結果 摻入腫瘤細胞迴收試驗的迴收率在68％～82％之間,摻人細胞間迴歸方程為y =0.6419X+8.8875,迴收的細胞與添加細胞間數量有顯著的相關性(相關繫數R2=0.9916,P ＜0.05).42例肺癌患者中有18例(42.9％) CTCs暘性,其中,Ⅰ期CTCs檢齣率為0,Ⅱ期CTCS檢齣率為11.1％,Ⅲ期CTCs檢齣率為62.5％,Ⅳ期CTCs檢齣率為70％.CTCs檢測的暘性率與患者的年齡、性彆、腫瘤大小均無明顯關繫(P＞0.05),而與臨床分期(TNM)有密切關繫(P＜0.05).特異性檢測結果為20名健康誌願者外週血標本均未檢測到CTCs.結論 新型免疫磁珠富集技術可以有效分離和鑒定肺癌患者外週血中的循環腫瘤細胞,有較高的敏感性和特異性；隨著肺癌患者分期增高和腫瘤增大,肺癌細胞髮生遠處轉移逐漸增多,CTCs檢齣的水平亦增高,對于早期髮現肺癌隱形微轉移和重新確定肺癌分期具有重要意義,但其臨床價值仍需進一步多中心、大樣本病例的臨床研究加以證實.
목적 건립일충신형면역자주방법,검측폐암환자외주혈중순배종류세포(CTCs).방법 실험탐색성연구.선택경수술절제조직병리학진위비소세포폐암적42례초치환자,기중Ⅰ기7례、Ⅱ기9례、Ⅲ기16례、Ⅳ기10례,건강지원자20명.장불동수량(50、100、200、500、1000개)적폐암세포주(A549)혼입2 ml건강성인혈표본중이모의종류환자적혈양,대해혈양진행홍세포렬해화리심,침정소득세포여경과면역자주표기적상피세포점부분자(EpCAM)적항체반응,연후경일투자제적검측장치대세포진행부집화분리,여자표기적EpCAM항체반응후적상피세포인위자력적작용피집중재일장파편상,최후용경전적HE염색감정순배종류세포병계수.계산종류세포적회수솔험증해방법적민감성,동시검측20명건강지원자혈표본험증해방법적특이성.용상술방법검측42례폐암환자적CTCs,장검측도적결과근거환자적림상특정진행분조,이잡방검험분석CTCs적검출솔여환자적년령、성별、림상분기、종류대소등림상특정적상관성,동시이직선상관법연구참입세포수여회수세포수적상관성.결과 참입종류세포회수시험적회수솔재68％～82％지간,참인세포간회귀방정위y =0.6419X+8.8875,회수적세포여첨가세포간수량유현저적상관성(상관계수R2=0.9916,P ＜0.05).42례폐암환자중유18례(42.9％) CTCs양성,기중,Ⅰ기CTCs검출솔위0,Ⅱ기CTCS검출솔위11.1％,Ⅲ기CTCs검출솔위62.5％,Ⅳ기CTCs검출솔위70％.CTCs검측적양성솔여환자적년령、성별、종류대소균무명현관계(P＞0.05),이여림상분기(TNM)유밀절관계(P＜0.05).특이성검측결과위20명건강지원자외주혈표본균미검측도CTCs.결론 신형면역자주부집기술가이유효분리화감정폐암환자외주혈중적순배종류세포,유교고적민감성화특이성；수착폐암환자분기증고화종류증대,폐암세포발생원처전이축점증다,CTCs검출적수평역증고,대우조기발현폐암은형미전이화중신학정폐암분기구유중요의의,단기림상개치잉수진일보다중심、대양본병례적림상연구가이증실.
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 Ⅲ 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 cells 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 cells was 62.5％ at stage Ⅲ 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 multicenter and large sample clinical research are needed to confirm its clinical value.
