劉巧,吳俊
(1.北京積水潭醫(yī)院檢驗(yàn)科,北京 100035;2.華中科技大學(xué)同濟(jì)醫(yī)學(xué)院附屬梨園醫(yī)院檢驗(yàn)科,武漢 430077)
·臨床實(shí)驗(yàn)研究·
血漿游離DNA在老年骨折患者深靜脈血栓形成中的輔助診斷價(jià)值
劉巧1,2,吳俊1
(1.北京積水潭醫(yī)院檢驗(yàn)科,北京 100035;2.華中科技大學(xué)同濟(jì)醫(yī)學(xué)院附屬梨園醫(yī)院檢驗(yàn)科,武漢 430077)
目的探討血漿游離DNA(cfDNA)水平對(duì)老年骨折患者深靜脈血栓形成(DVT)的輔助診斷價(jià)值。方法根據(jù)加壓雙向超聲將106例股骨骨折患者分為骨折DVT組50例和骨折非DVT組56例。用熒光染料法定量測(cè)定血漿cfDNA濃度,同時(shí)檢測(cè)患者外周血WBC、PLT、C反應(yīng)蛋白(CRP)、D-二聚體(DD)水平。結(jié)果骨折DVT組血漿cfDNA水平為337.38(272.89,436.42)ng/mL,高于骨折非DVT組232.56(208.54,280.89)ng/mL,P<0.01;PLT、DD均高于骨折非DVT組(P均<0.01),而WBC、CRP低于骨折非DVT組(P均<0.01。血漿cfDNA與PLT、DD均呈正相關(guān),與WBC呈負(fù)相關(guān),相關(guān)系數(shù)(r)分別為0.273、0.365、-0.255,P均<0.01。cfDNA、DD診斷DVT的ROC曲線下面積分別為0.825、0.733,臨界值分別為287.86 ng/mL和3.4 mg/L FEU(fibrinogen equivalent units)時(shí),敏感性分別為72%、96%,特異性分別為85.5%、43.6%。結(jié)論DVT患者cfDNA水平升高,其臨床診斷效能優(yōu)于DD,可作為輔助診斷DVT的生物學(xué)指標(biāo)之一。
深靜脈血栓形成;游離DNA;骨折
游離DNA(cell-free DNA,cfDNA)是一種網(wǎng)狀的細(xì)胞外DNA,廣泛存在于包括血液在內(nèi)的多種體液中,在腫瘤[1]、創(chuàng)傷[2]等病理情況下會(huì)升高。目前,國(guó)內(nèi)外大多采用實(shí)時(shí)熒光定量PCR法檢測(cè)血液cfDNA[3],但外周血中cfDNA水平極低限制了其臨床應(yīng)用。Sytox green(S7020)熒光染料親和力高,能特異性結(jié)合DNA,可極大提高檢測(cè)的敏感性和特異性。骨科手術(shù),如股骨、頸骨骨折手術(shù)后深靜脈血栓形成(deep venous thrombosis,DVT)發(fā)病率高,但早期診斷一直沒有較好的指標(biāo),只能用影像學(xué)如超聲、計(jì)算機(jī)斷層掃描(CT)或造影予以明確診斷。cfDNA可誘導(dǎo)血小板聚集,促進(jìn)凝血活化,抑制纖維蛋白溶解并直接干擾凝塊的穩(wěn)定性[4]。關(guān)于cfDNA與DVT關(guān)系的報(bào)道較少,有學(xué)者檢測(cè)47例加壓超聲確診DVT患者的cfDNA,發(fā)現(xiàn)其水平明顯高于超聲陰性患者和健康人對(duì)照組[5]。McIlroy等[6]用實(shí)時(shí)熒光定量PCR在創(chuàng)傷和創(chuàng)傷后手術(shù)患者的血標(biāo)本中檢測(cè)出異常表達(dá)的cfDNA。本研究用基于熒光染液Sytox green(S7020)特異性結(jié)合DNA的技術(shù)檢測(cè)DVT患者血漿cfDNA水平,并探討其作為DVT輔助診斷生物標(biāo)志物的價(jià)值。
1.1研究對(duì)象 2017年4月至8月北京積水潭醫(yī)院因股骨骨折入院的患者,納入標(biāo)準(zhǔn):(1)年齡大于65歲;(2)股骨骨折。排除標(biāo)準(zhǔn):(1)感染急性期,(2)合并腫瘤活動(dòng)期、肝硬化、腎衰竭及免疫系統(tǒng)疾病且正在治療中。共納入研究對(duì)象106例,按加壓雙向超聲標(biāo)準(zhǔn)[5],分為骨折DVT組和骨折非DVT組,其中,骨折DVT組50例,男15例,女35例,年齡65~89歲,平均77歲;骨折非DVT組56例,男14例,女52例,年齡65~95歲,平均78歲,兩組間年齡、性別比差異均無(wú)統(tǒng)計(jì)學(xué)意義(P均>0.05)。
1.2標(biāo)本采集與處理 研究對(duì)象入院時(shí)采集肘靜脈血3管,分別為無(wú)抗凝劑、EDTA-K2抗凝劑,0.109 mol/L枸櫞酸鈉(1∶9)抗凝劑管。將無(wú)抗凝劑管和枸櫞酸鈉抗凝劑管于室溫下以1 500×g離心10 min,分別取上層血清和血漿;將分離的血漿于室溫下以1 500×g離心10 min以獲取乏血小板血漿,取上層血漿,-80 ℃保存。
1.3主要儀器和試劑 XN-20全自動(dòng)血液分析儀、 CS-5100全自動(dòng)血凝分析儀及其配套WBC、PLT、D-二聚體(D-dimer,DD)試劑(日本希森美康公司);Immage 800特定蛋白分析儀及其配套C反應(yīng)蛋白(C-reactive protein,CRP)試劑(美國(guó)貝克曼庫(kù)爾特公司);TECAN infinite M200 pro多功能酶標(biāo)儀(瑞士帝肯公司);熒光染液Sytox Green(S7020) 購(gòu)自美國(guó)Thermo fisher公司;DNA標(biāo)準(zhǔn)品及樣品稀釋液購(gòu)自美國(guó)Sigma公司。
1.4相關(guān)指標(biāo)檢測(cè) 用XN-20全自動(dòng)血液分析儀及其配套試劑檢測(cè)EDTA-K2抗凝血WBC和PLT;用免疫比濁法檢測(cè)血漿DD;用免疫比濁法檢測(cè)血清CRP。實(shí)驗(yàn)室內(nèi)質(zhì)量控制均在控。
1.5血漿cfDNA檢測(cè) 參照文獻(xiàn)檢測(cè)血漿cfDNA[7]。吸取20 μL待測(cè)血漿于1.5 μL離心管中,加入180 μL 1×buffer(貨號(hào)F7171,Sigma公司),混勻,取50 μL 稀釋后樣品,再加入50 μL 熒光染液Sytox Green(S7020)(終濃度2 μmol/L),室溫下避光放置5 min,用TECAN infinite M200 pro多功能酶標(biāo)儀(激發(fā)光485 nm,發(fā)射光538 nm,震蕩5 s)檢測(cè),其熒光強(qiáng)度與樣品中DNA濃度成正比。每個(gè)樣品設(shè)雙孔(測(cè)試孔=稀釋后血漿+染液,本底孔=稀釋后血漿+稀釋液)。用1 mg/mL DNA標(biāo)準(zhǔn)品(貨號(hào)D4810,Sigma公司)分別配成終濃度為0、10、20、30、40、50、80、100 ng/mL的溶液,繪制標(biāo)準(zhǔn)曲線,以定量檢測(cè)血漿中DNA濃度。分別收集20個(gè)健康人、20個(gè)血栓患者,分別制作混合乏血小板血漿,并檢測(cè)批間CV<7%,批內(nèi)CV<5%。
2.1骨折DVT組和骨折非DVT組的基本資料 骨折DVT組的PLT、DD均高于骨折非DVT組(P均<0.01),WBC、CRP均低于骨折非DVT組(P均<0.01),見表1。
表1 骨折DVT組和骨折非DVT組的基本資料
2.2骨折DVT組和骨折非DVT組的血漿cfDNA水平 骨折DVT組血漿cfDNA水平為337.38(272.89,436.42)ng/mL,高于骨折非DVT組232.56(208.54,280.89)ng/mL(U=483.0,P<0.01)。
2.3血漿cfDNA與其他指標(biāo)的相關(guān)性 血漿cfDNA與PLT、DD呈正相關(guān),與WBC呈負(fù)相關(guān),相關(guān)系數(shù)(r)分別為0.273、0.365、-0.255,P均<0.01,與年齡、性別、CRP均無(wú)相關(guān)性(P均>0.05)。
2.4血漿cfDNA和DD診斷骨折DVT的效能 根據(jù)骨折DVT組和骨折非DVT組血漿cfDNA和DD水平制作ROC曲線,見圖1,ROC曲線下面積(AUCROC)分別為0.825、0.733。約登指數(shù)(約登指數(shù)=敏感性+特異性-1)取最大值時(shí),cfDNA和DD最佳診斷界值分別為287.86 ng/mL、3.4 mg/L FEU,敏感性分別為72.0%和96.0%、特異性分別為85.5%和43.6%,陽(yáng)性預(yù)測(cè)值分別為81.8%和60.0%,陰性預(yù)測(cè)值分別為77.4%和92.3%,準(zhǔn)確性分別為79.2%和67.9%。
圖1 血漿cfDNA和DD診斷骨折DVT的ROC曲線
DVT易發(fā)部位為下肢深靜脈,常見于骨科大手術(shù)后。血栓形成后,除少數(shù)能自行消融或局限于發(fā)生部位外,大部分會(huì)擴(kuò)散至整個(gè)肢體的深靜脈主干,若不能及時(shí)診斷和處理,多數(shù)會(huì)演變?yōu)檠ㄐ纬珊筮z癥,長(zhǎng)時(shí)間影響患者的生活質(zhì)量;還有一些患者可能并發(fā)肺栓塞,造成非常嚴(yán)重的后果。因此,早期采取有效措施,預(yù)防術(shù)后DVT十分重要。DVT發(fā)生后DD有不同程度升高,血漿cfDNA是DD的良好補(bǔ)充。目前,血栓形成的診斷主要依賴超聲,但超聲不能動(dòng)態(tài)觀測(cè)血栓形成速率或提示血栓是否處于活動(dòng)期。造影雖是診斷DVT的金標(biāo)準(zhǔn),但有創(chuàng)和放射性限制了其使用頻率和范圍。
研究顯示,多種疾病血漿cfDNA水平升高,因此,骨折后DVT的發(fā)生發(fā)展過程中可能也存在cfDNA水平的改變。目前,血液cfDNA的定量檢測(cè)方法包括放射免疫法[8]、免疫熒光法、ELISA[9]、qPCR[10]等?,F(xiàn)有技術(shù)提取的DNA水平和純度達(dá)不到理想結(jié)果且費(fèi)時(shí),限制了cfDNA的臨床應(yīng)用。Sytox染液無(wú)需洗滌,避免了洗滌造成樣品損耗。與Hoechst 系列[11](結(jié)合Minor Groove)和DAPI不一樣,Sytox Green極少有堿基選擇性。結(jié)合了核酸的Sytox Green熒光增強(qiáng)1 000倍,量子產(chǎn)率高。Sytox Green是一種極好的DNA染液,幾乎不受血漿RNA干擾,以上特性使得Sytox Green染液成為一個(gè)簡(jiǎn)單、靈敏、一步定量DNA的指標(biāo)。
本研究結(jié)果表明,骨折DVT組血漿cfDNA濃度升高,與骨折非DVT組比較差異有統(tǒng)計(jì)學(xué)意義(P<0.05);與Fuchs等[12]和Diaz等[5]報(bào)道基本相符。Fuchs等[12]用Sytox Green熒光染液檢測(cè)cfDNA,其血栓組cfDNA水平均低于本研究,但結(jié)論相似:DVT患者血漿cfDNA升高。本研究結(jié)果顯示,cfDNA水平與PLT、DD呈正相關(guān),與WBC呈負(fù)相關(guān)。cfDNA水平升高,可能提示發(fā)生血栓風(fēng)險(xiǎn)。cfDNA的AUCROC0.825,診斷效能優(yōu)于DD。cfDNA特異性高于DD,敏感性低于DD,在輔助診斷DVT時(shí),是DD的良好補(bǔ)充。
本研究顯示cfDNA與DD呈正相關(guān),也有研究報(bào)道血漿中cfDNA與血小板消耗和凝血相關(guān),如血小板減少癥[13]、FⅫ因子介導(dǎo)的內(nèi)源性凝血與DIC預(yù)后不良[14]、DD[15]。動(dòng)物實(shí)驗(yàn)表明,血栓時(shí)中性粒細(xì)胞外誘捕網(wǎng)(neutrophil extracellular traps, NETs)形成與cfDNA升高具有一致性[16-17]。cfDNA提供血小板黏附支架,促使纖維蛋白沉積[18],有助于血栓形成和穩(wěn)定[19]。骨折可能激活了免疫細(xì)胞,釋放核內(nèi)物質(zhì)[20],這種核內(nèi)物質(zhì)是DNA與胞漿蛋白如組蛋白等形成的復(fù)合物[21],組蛋白誘導(dǎo)血小板聚集和增強(qiáng)凝血酶生成[22]。鑒于上述分析,骨折可能引起血栓里有核細(xì)胞的釋放,也可能是骨折的應(yīng)激反應(yīng)釋放細(xì)胞因子[23],激活免疫細(xì)胞釋放核內(nèi)物質(zhì)。cfDNA也可能具有相似的出凝血效果,引發(fā)進(jìn)一步高凝狀態(tài),一旦下一步手術(shù),“二次打擊”下可能有更多患者發(fā)生DVT事件。
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Auxiliarydiagnosticvalueofplasmacell-freeDNAindeepvenousthrombosisformationinelderlypatientswithfractures
LIUQiao1,2,WUJun1
(1.DepartmentofClinicalLaboratory,BeijingJishuitanHospital,Beijing100035, 2.DepartmentofLaboratoryMedicine,LiyuanHospitalofTongjiMedicalCollegeofHuazhongUniversityofScienceandTechnology,Wuhan430076,Hubei,China)
ObjectiveTo investigate the role of plasma cell-free DNA (cfDNA) for diagnosis of deep venous thrombosis formation (DVT) in elder patients with fractures.MethodsAccording to the results of compression duplex ultrasound, 106 elderly patients with femoral fracture were divided into fracture with deep venous thrombosis (DVT) group (n=50, DVT group) and fracture without DVT group (n=50, non-DVT group). The concentrations of cfDNA in plasma were measured by fluorescence method. White blood cell count (WBC), platelet count (PLT), C-reactive protein (CRP) and D-dimer (DD) were simultaneously determined.ResultsThe concentration of plasma cfDNA in DVT group was 337.38 (272.89, 436.42) ng/mL, which was significantly higher than that in the non-DVT group [232.56 (208.54, 280.89) ng/mL,P<0.01]. The levels of PLT and DD in fracture DVT group were significantly higher than those in non-DVT group (allP<0.01). The levels of WBC and CRP were lower than those in non-DVT group (allP<0.01). Plasma cfDNA level was positively correlated with PLT and DD and negatively correlated with WBC, correlation coefficient(r) is 0.273,0.365 and -0.255, respectively, allP<0.01. ROC curves showed that the area under the curve cfDNA and DD were 0.825 and 0.733, and when cut-off value were 287.86 ng/mL and 3.4 mg/L FEU(fibrinogen equivalent units), sensitivity were 72% and 96% while specificities were 85.5% and 43.6%.ConclusionThe level of cfDNA in DVT patients is higher, and its clinical diagnostic efficiency is better than that of DD. It can be used as one of the biological indicators to assist the diagnosis of DVT.
deep venous thrombosis; cell-free DNA; bone fracture
10.13602/j.cnki.jcls.2017.12.09
北京市衛(wèi)生系統(tǒng)第五批高層次人才培養(yǎng)計(jì)劃(2015-3-034);人社部留學(xué)人員科技活動(dòng)項(xiàng)目([2015]192);首都特色臨床醫(yī)療研究(Z141107006614012);教育部留學(xué)歸國(guó)人員科研啟動(dòng)基金([2013]693)。
劉巧,1989年生,女,碩士研究生,主要從事血栓與止血研究。
吳俊,主任醫(yī)師,博士,碩士研究生導(dǎo)師,E-mail:wujunpostbox@sina.com。
R446.11;R619
A
2017-09-06)
王海燕)