馬 文,杜 娟,張先明,王 焰
(1.貴州醫(yī)科大學(xué)附院呼吸內(nèi)科,貴州貴陽(yáng) 550004; 2.貴州醫(yī)科大學(xué)附院中心試驗(yàn)室,貴州貴陽(yáng) 550004)
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脫落細(xì)胞DNA倍體分析及胸水CEA與血清CEA比值的診斷價(jià)值*?
馬文1,杜娟1,張先明1,王焰2
(1.貴州醫(yī)科大學(xué)附院呼吸內(nèi)科,貴州貴陽(yáng)550004; 2.貴州醫(yī)科大學(xué)附院中心試驗(yàn)室,貴州貴陽(yáng)550004)
[摘要]目的:探討胸水脫落細(xì)胞DNA倍體分析及胸水癌胚抗原(CEA)與血清CEA比值測(cè)定對(duì)惡性胸腔積液的診斷價(jià)值。方法: 41例惡性胸腔積液患者為試驗(yàn)組,84例良性胸腔積液患者為對(duì)照組,抽取2組患者胸水和血液,采用全自動(dòng)細(xì)胞圖像分析系統(tǒng)對(duì)胸水脫落細(xì)胞進(jìn)行DNA倍體分析,用蛋白芯片-化學(xué)發(fā)光法檢測(cè)血及胸水癌胚抗原水平,計(jì)算胸水CEA與血清CEA比值;比較2種方法單獨(dú)應(yīng)用或聯(lián)合應(yīng)用時(shí)診斷惡性胸腔積液的靈敏度、特異度及youden值。結(jié)果:試驗(yàn)組胸水脫落細(xì)胞DNA倍體分析陽(yáng)性率和胸水CEA與血清CEA比值陽(yáng)性率均高于對(duì)照組(P<0.05) ;胸水脫落細(xì)胞DNA倍體分析對(duì)惡性胸腔積液診斷靈敏度為85.37%、特異度為66.67%、youden指數(shù)0.52,胸水CEA與血清CEA比值對(duì)惡性胸腔積液診斷靈敏度為78.05%,特異度為77.38%,youden指數(shù)0.55,2種方法比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05) ; 2種方法聯(lián)合診斷惡性胸腔積液的靈敏度為96.79%,特異度為92.46%,youden指數(shù)0.59。結(jié)論:胸水脫落細(xì)胞DNA倍體分析聯(lián)合胸水CEA與血清CEA比值測(cè)定,對(duì)鑒別良、惡性胸腔積液具有更好的診斷價(jià)值。
[關(guān)鍵詞]胸水;脫落細(xì)胞; DNA;倍體分析;癌胚抗原
網(wǎng)絡(luò)出版時(shí)間: 2016-02-23網(wǎng)絡(luò)出版地址: http: / /www.cnki.net/kcms/detail/52.5012.R.20160223.1905.020.html
在胸水中查找腫瘤細(xì)胞是目前鑒別良、惡性胸腔積液的主要方法,該方法敏感度低,只有50%~60%[1];而利用血清學(xué)及免疫學(xué)等檢查鑒別良、惡性胸水的特異性又有限,所以研發(fā)新的診斷技術(shù)十分必要。有研究表明,DNA含量是監(jiān)測(cè)細(xì)胞增殖和腫瘤轉(zhuǎn)化的指標(biāo),DNA的非整倍體性質(zhì)是惡性腫瘤的特征之一,測(cè)定和分析細(xì)胞DNA含量的倍體對(duì)良、惡性腫瘤的診斷、鑒別診斷、療效判斷及預(yù)后測(cè)定等有重要價(jià)值[2-3]。癌胚抗原(CEA)是腫瘤細(xì)胞產(chǎn)生的物質(zhì),正常細(xì)胞和某些良性疾病只能分泌極少量[4-5]。本研究主要采用全自動(dòng)細(xì)胞圖像分析系統(tǒng)對(duì)胸水脫落細(xì)胞DNA含量進(jìn)行測(cè)定,探討其對(duì)惡性胸腔積液診斷的靈敏度、特異度及youden指數(shù);同時(shí)檢測(cè)胸水CEA與血CEA比值,評(píng)估2種方法獨(dú)立及聯(lián)合應(yīng)用對(duì)惡性胸腔積液的診斷價(jià)值。
1.1對(duì)象
選擇2011年6月~2013年6月在呼吸科、心內(nèi)科、感染科、腎病風(fēng)濕科住院的胸腔積液患者125例,男74例,女51例,年齡18~89歲,平均(61.78±7.03)歲。41例惡性胸腔積液為試驗(yàn)組,男24例,女17例,平均(62.42±7.35)歲,均經(jīng)胸水脫落細(xì)胞學(xué)、電子支氣管鏡肺活檢、淋巴結(jié)穿刺活檢、經(jīng)皮肺穿刺活檢或胸膜活檢、開胸肺活檢等病理檢查,并結(jié)合臨床常規(guī)檢查確診為惡性胸腔積液; 84例良性胸腔積液為對(duì)照組,男50例,女34例,平均(60.35±6.81)歲,根據(jù)病史、臨床表現(xiàn)、體征、血生化、胸水生化、腺苷脫氨酶(ADA)、抗酸染色等實(shí)驗(yàn)室檢查及肺功能、胸片、B超及對(duì)治療的反應(yīng)等,綜合確診為良性胸腔積液。
1.2主要儀器及試劑
全自動(dòng)DNA圖像分析儀、TDZ5-WS多管架自動(dòng)平衡離心機(jī)、Motic BA600 Mot顯微鏡系統(tǒng)、lu-07生物芯片閱讀器、電熱恒溫水箱5648、恒溫?fù)u床HDYC-2004B、Feulgen-thionin專用染液。
1.3方法
所有患者于治療前行標(biāo)準(zhǔn)胸腔穿刺術(shù),抽取胸水10 mL,均分為2管于2 h內(nèi)送檢,同時(shí)抽取外周血2 mL送檢。胸水脫落細(xì)胞DNA倍體分析采用全自動(dòng)細(xì)胞DNA分析系統(tǒng),測(cè)定細(xì)胞核積分光密度(IOD),按照公式DNA指數(shù)(DI) =被測(cè)細(xì)胞核DNA的IOD值/正常細(xì)胞核DNA的IOD值,計(jì)算胸水脫落細(xì)胞DI。CEA采用蛋白芯片-化學(xué)發(fā)光法進(jìn)行檢測(cè),將CEA的單克隆抗體固定在膜上制備成蛋白芯片,利用CEA的酶標(biāo)記抗體來(lái)測(cè)定血清和胸水中CEA的濃度,計(jì)算胸水CEA與血清CEA的比值。
1.4判斷標(biāo)準(zhǔn)
正常細(xì)胞的DI =1,DI>2.5提示細(xì)胞有癌變可能。根據(jù)文獻(xiàn)[6]標(biāo)準(zhǔn): (1)有3個(gè)或3個(gè)以上DI>2.5的細(xì)胞; (2)可識(shí)別的異倍體峰; (3)異常細(xì)胞增生>10%。滿足3條中的1條或以上者,判定為胸水脫落細(xì)胞DNA倍體(+),反之則為(-)。胸水CEA/血CEA>1為(+),<1為(-)。胸水脫落細(xì)胞DNA倍體分析和胸水CEA與血CEA比值2種方法聯(lián)合判斷的標(biāo)準(zhǔn)為只要有1種方法結(jié)果呈現(xiàn)(+)即診斷為(+) ; 2種方法獨(dú)立診斷的標(biāo)準(zhǔn)是必須兩項(xiàng)同時(shí)陽(yáng)性。
1.5統(tǒng)計(jì)學(xué)分析
采用SPSS(V13.0)統(tǒng)計(jì)軟件進(jìn)行數(shù)據(jù)分析,計(jì)量資料用均數(shù)±標(biāo)準(zhǔn)差(±s)表示,計(jì)算靈敏度、特異度和youden指數(shù),組間比較采用卡方檢驗(yàn)和u檢驗(yàn),P<0.05為差異有統(tǒng)計(jì)學(xué)意義。
2.1胸水脫落細(xì)胞DNA倍體或胸水CEA與血清CEA比值
胸水脫落細(xì)胞DNA倍體分析顯示,試驗(yàn)組41例患者陽(yáng)性35例,陰性6例,陽(yáng)性率85.37 %;對(duì)照組84例患者陽(yáng)性28例,陰性56例,陽(yáng)性率33.33%; 2組陽(yáng)性率比較有統(tǒng)計(jì)學(xué)意義(χ2= 14.47,P<0.05)。試驗(yàn)組41例患者胸水CEA與血清CEA比值陽(yáng)性32例,陰性9例,陽(yáng)性率78.05%;對(duì)照組84例患者胸水CEA與血清CEA比值陽(yáng)性19例,陰性65例,陽(yáng)性率22.62%; 2組陽(yáng)性率比較有統(tǒng)計(jì)學(xué)意義(χ2=19.32,P<0.05)。見(jiàn)表1和表2,表2結(jié)果提示,2種方法獨(dú)立應(yīng)用時(shí),只有兩項(xiàng)結(jié)果陽(yáng)性時(shí)才能診斷惡性胸水,試驗(yàn)組41例患者陽(yáng)性28例,可能漏診。
表1 胸水脫落細(xì)胞DNA倍體或胸水CEA/血清CEA檢測(cè)結(jié)果Tab.1 Results of DNA ploidy analysis of hydrothorax shedding cells and CEA ratio of hydrothorax/blood
表2 胸水脫落細(xì)胞DNA倍體和胸水CEA與血清CEA比值聯(lián)合檢測(cè)結(jié)果Tab.2 Results of DNA ploidy analysis of hydrothorax shedding cells combined with CEA ratio of hydrothorax/blood
2.2靈敏度、特異度及Youden指數(shù)
胸水脫落細(xì)胞DNA倍體分析或胸水CEA與血清CEA比值單獨(dú)應(yīng)用診斷惡性胸腔積液均有較高的靈敏度和特異度,且2種方法靈敏度、特異度及youden指數(shù)比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05),見(jiàn)表3。胸水脫落細(xì)胞DNA倍體分析和胸水CEA與血清CEA比值測(cè)定2種方法聯(lián)合診斷惡性胸水的特異度、靈敏度均高于2種方法單獨(dú)應(yīng)用時(shí),說(shuō)明2種方法聯(lián)合應(yīng)用更能提高對(duì)惡性胸水的診斷靈敏度和特異度,具有更高的診斷價(jià)值,可減少漏診,見(jiàn)表4。
表3 胸水脫落細(xì)胞DNA倍體分析和胸水CEA與血清CEA比值單獨(dú)診斷惡性胸水的靈敏度、特異度及Youden指數(shù)Tab.3 The sensitivity,specificity and youden's index of DNA ploidy analysis of hydrothorax shedding cells and CEA ratio of hydrothorax/blood in their separate diagnosis
表4 胸水脫落細(xì)胞DNA倍體分析和胸水CEA與血清CEA比值診斷惡性胸水的效果評(píng)價(jià)Tab.4 Effect evaluation of DNA ploidy analysis of hydrothorax shedding cells and CEA ratio of hydrothorax/blood in diagnosis of malignant pleural effusion
胸腔積液是臨床常見(jiàn)疾病,導(dǎo)致胸腔積液的原因很多,按病因可分為良性和惡性,兩者的鑒別非常重要。胸水脫落細(xì)胞檢查是診斷惡性胸腔積液的“金標(biāo)準(zhǔn)”,但因靈敏度較低,限制了該方法在臨床上的運(yùn)用。正常人體細(xì)胞具有較恒定的DNA含量,為整倍體細(xì)胞,而惡性腫瘤組織發(fā)生染色體突變,導(dǎo)致異倍體細(xì)胞的出現(xiàn),所以測(cè)定細(xì)胞DI,可以幫助發(fā)現(xiàn)異常的細(xì)胞,而且DI值越高,生存期越短,提示異倍體腫瘤的增值活性越高,預(yù)后越差[7]。CEA是目前研究最廣泛的腫瘤標(biāo)志物之一,在正常人血清中含量很低。CEA是一種大分子的糖蛋白,腫瘤發(fā)生胸膜轉(zhuǎn)移時(shí)合成的CEA釋放到胸水中,不易進(jìn)入血循環(huán),因此其在胸水中出現(xiàn)較早,含量也較血清水平高。本研究發(fā)現(xiàn),胸水脫落細(xì)胞DNA倍體分析和胸水CEA與血清CEA比值測(cè)定2種方法的陽(yáng)性率,試驗(yàn)組高于對(duì)照組(P<0.05),說(shuō)明這2種方法在臨床上均可用以診斷惡性胸腔積液。本研究對(duì)照組中有28例患者胸水脫落細(xì)胞DNA倍體分析為異倍體,而臨床診斷為良性疾病,出現(xiàn)假陽(yáng)性可能原因是: (1)反應(yīng)性間皮細(xì)胞增生,使G2M期細(xì)胞百分?jǐn)?shù)增高,造成假陽(yáng)性; (2)標(biāo)本制備不當(dāng)造成染色體機(jī)械缺損誤差。試驗(yàn)組中有6例患者未檢測(cè)到倍體細(xì)胞,出現(xiàn)假陰性的原因可能是: (1)腫瘤細(xì)胞的分化程度較高,二倍體腫瘤或近二倍體腫瘤的發(fā)生; (2)胸腔積液中惡性細(xì)胞含量少,有抽樣誤差可能性。本研究顯示胸水脫落細(xì)胞DNA倍體分析對(duì)惡性胸腔積液的診斷靈敏度為85.37%,特異度為66.67%,與文獻(xiàn)報(bào)道的相近[8]。文獻(xiàn)報(bào)道,以胸水與血清CEA比值>1為界診斷惡性胸腔積液的靈敏度約為40%~80%,特異度為70%~88%[9],本研究顯示胸水與血清CEA比值測(cè)定診斷惡性胸腔積液的靈敏度為78.05%,特異度為77.38%,與其基本一致,但2種方法比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)。臨床上為了提高篩檢效果,可采用多項(xiàng)篩檢試驗(yàn)檢查同一受試對(duì)象,以提高篩檢試驗(yàn)的靈敏度和特異度,這種方式稱為聯(lián)合檢驗(yàn),本研究顯示,聯(lián)合胸水脫落細(xì)胞DNA倍體分析和胸水與血清CEA比值測(cè)定2種方法診斷惡性胸腔積液的靈敏度為96.79%,特異度為68.29%。Youden指數(shù)為0.48,較2種方法獨(dú)立應(yīng)用時(shí)有較好的臨床價(jià)值。
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(2015-05-08收稿,2015-12-24修回)
中文編輯:戚璐;英文編輯:劉華
Diagnostic Value of DNA Ploidy Analysis of Hydrothorax Shedding Cells Combined with Ratio of Pleural Fluid CEA to Serum CEA in Diagnosis of Malignant Pleural Effusion
MA Wen1,DU Juan1,ZHANG Xianming1,WANG Yan2
(1.Departments of Respiratory,the Affiliated Hospital of Guizhoug Medical University,Guiyang 550004,Guizhou,China;2.Central Laboratory,Departments of Respiratory,the Affiliated Hospital of Guizhoug Medical University,Guiyang 550004,Guizhou,China)
[Abstract]Objective: To explore the diagnostic value of DNA ploidy analysis of hydrothorax shedding cells by automatic DNA image cytometry and the ratio of pleural fluid CEA to serum CEA in diagnosis of malignant pleural effusion.Methods: Forty-one cases of patients with malignant pleural effusions were enrolled in experimental group while 84 cases of patients with benign pleural effusion in control group.The serum and pleural fluid specimens were collected from two groups and DNA ploidy analysis of hydrothorax shedding cells was conducted by automatic DNA image cytometry.The levels of CEA were determined by protein chip-chemoluminescence and the ratio of pleural fluid CEA to serum CEA were calculated.Then,the sensitivity,specificity and youden's index were compared between two groups when single method or two methods combined were adopted.Results: The positive rates of DNA ploidy analysis of hydrothorax shedding cells and the ratio of pleural fluid CEA to serum CEA in experimental group were significantly higher than their counterparts in control group (P<0.05).The sensitivity of the DNA ploidy analysis of hydrothorax shedding cells in diagnosis of malignant pleural effusion was 85.37%,the specificity was 66.67%,and the youden's index was 0.52.The sensitivity of the ratio of pleural fluid CEA to serum CEA in diagnosis of malignant pleural effusion was 78.05%,the specificity was 77.38%,and the youden's index was 0.55.There was no statistical difference be-book=210,ebook=91tween the two methods (P>0.05).The sensitivity of the DNA ploidy analysis combined the CEA ratio of hydrothorax/blood was 66.63%,the specificity was 92.46%,and the youden's index was 0.59.Conclusion: The DNA ploidy analysis of hydrothorax shedding cells combined the CEA ratio of hydrothorax/blood may have a better diagnostic value in distinguish benign pleural effusion from malignant pleural effusion.
[Key words]pleural fluid; shedding cells; DNA; ploidy analysis; carcinoembryonic antigen
[中圖分類號(hào)]R734.2
[文獻(xiàn)標(biāo)識(shí)碼]A
[文章編號(hào)]1000-2707(2016) 02-0209-04