HBV血清學(xué)定量檢測技術(shù)及應(yīng)用

1、HBV血清學(xué)定量檢測技術(shù)及應(yīng)用1Company Confidential 2009 AbbottHBV血清學(xué)定量檢測技術(shù)及應(yīng)用 雅培診斷 錢學(xué)慶 博士Presentation TitleDate 2Company Confidential 200X AbbottTIMETreatment endpoints differ for antiviral and immune-based therapies乙型肝炎治療終點乙型肝炎治療終點Prof. Marcellin, France Communication/ presentation “Clinical strategies to CHB ma

2、tching the treatment to patients”Presentation TitleDate 3Company Confidential 200X Abbott免疫應(yīng)答引起的抗體免疫應(yīng)答引起的抗體Anti-HBc, Anti-HBc-IgMAnti-HBeAnti-HBs病毒標(biāo)志物病毒標(biāo)志物HBV DNAHBeAgHBsAgHepatitis B 診斷標(biāo)志物診斷標(biāo)志物Presentation TitleDate 4Company Confidential 200X Abbott兩步法檢測兩步法檢測Presentation TitleDate 5Company Confiden

3、tial 200X Abbott兩步法可以消除HOOK效應(yīng)Step 1+WASHWASHStep 2Sample antigen fills all binding sites on the solid phase, but extra antigen is washed away.Conjugate label binds normally to antigens, sample will record high value on detection, leading to flagging of the result needing dilution.Presentation TitleD

4、ate 6Company Confidential 200X Abbott一步法和兩步法試劑的比較分析1-step assay2-step assayStrenghtsEasy to automatePotentially better precision vs 2-StepPotentially faster assayInexpensive to develop (assay & instrument optimization)Suitable to almost all assaysExcellent clinical performance: No High-Dose-Hook

5、 effect No interference by Thyroid Hormone Autoantibodies Less interferences by non-specific bindingWeaknessesMore risk for clinically significant interferences, ie. thyroid, microbiology, cancer marker assays.Risk of High-Dose-Hook effectMore complex to automatePotentially slower assaysPotentially

6、poorer precisionMore expensive to develop (assay & instrumentation optimization)Presentation TitleDate 7Company Confidential 200X AbbottARCHITECT HBsAg定量檢測定量檢測Presentation TitleDate 8Company Confidential 200X Abbott HBsAg的重要臨床意義HBsAg是肝細(xì)胞受到是肝細(xì)胞受到HBV感染的標(biāo)志感染的標(biāo)志HBsAg清除是肝內(nèi)清除是肝內(nèi)HBV感染的免疫控制和抗病毒應(yīng)答的標(biāo)志感染的免

7、疫控制和抗病毒應(yīng)答的標(biāo)志HBsAg清除最接近治愈清除最接近治愈相比相比HBV DNA， HBsAg清除能更好地代表疾病的持久緩解清除能更好地代表疾病的持久緩解HBsAg清除可改善臨床預(yù)后：降低肝硬化和肝癌的發(fā)生率；提高生存率清除可改善臨床預(yù)后：降低肝硬化和肝癌的發(fā)生率；提高生存率最近的研究報告顯示：肝細(xì)胞內(nèi)最近的研究報告顯示：肝細(xì)胞內(nèi) cccDNA 水平（水平（HBV感染細(xì)胞的標(biāo)志）和血清感染細(xì)胞的標(biāo)志）和血清HBsAg 水平存在顯著相關(guān)性水平存在顯著相關(guān)性目前缺少商業(yè)化目前缺少商業(yè)化 cccDNA檢測試劑；是一種創(chuàng)傷性檢測方法，應(yīng)用受到限制檢測試劑；是一種創(chuàng)傷性檢測方法，應(yīng)用受到限制 Col

8、ombatto P. et al, Antiviral Therapy 2006； Werle-Lapostolle et al, Gastroenterology 2004；Volz et al, Gastroenterology 2007Presentation TitleDate 9Company Confidential 200X Abbott 肝細(xì)胞內(nèi)HBV的生活史Adapted from Lai CL, et al. J Med Virol. 2000;61:367-373.InfectiousHBV virionViral polymeraseconverts pregenomi

9、c RNAto partially ds DNAPartiallydsDNASubviralparticlesHepatocytemRNACytoplasmNucleusPrecore/coreHBeAgERHBcAgHBsAgcccDNAMinus strand DNAEncapsulated pregenomic mRNAPresentation TitleDate 10Company Confidential 200X AbbottHBsAg 和和 HBV-DNA 聚合酶的相關(guān)性聚合酶的相關(guān)性總計總計418 名名 慢性慢性HBV攜帶者，檢測攜帶者，檢測HBsAg 和和HBV-DNA 聚合

10、酶水平的結(jié)果聚合酶水平的結(jié)果.M. Deguchi et al. / Journal of Virological Methods 115 (2004) 217222Presentation TitleDate 11Company Confidential 200X Abbott 血清HBsAg可以反應(yīng)被感染肝細(xì)胞的數(shù)量cccDNA acts as transcription templateSerum HBsAg levels reflect cccDNA (A) and intrahepatic HBV DNA (B)Non-invasive marker of infected cell

11、sABChan et al. Clin Gastro Hepatol 2007HBsAg at baselineHBsAg at baselineLog (cccDNA) at baselineLog (intrahepatic HBV DNA) at baselineAPresentation TitleDate 12Company Confidential 200X Abbott 血清 HBsAg 水平與 cccDNA水平正相關(guān)p0.01-3-2-101-3-2-10ADV+PEGChange in Serum HBsAg(log10 ng/mL)Change in cccDNA(log1

12、0 copies/cell)Petersen EASL 2005Presentation TitleDate 13Company Confidential 200X AbbottHENRY LIKYUEN CHAN, VINCENT WAISUN WONG, ADA MEILING TSE, CHIHANG TSE, ANGEL MEILING CHIM,HOIYUN CHAN, GRACE LAIHUNG WONG, and JOSEPH JAOYIU SUNGDepartment of Medicine and Therapeutics, and Institute of Digestiv

13、e Disease, The Chinese University of Hong Kong, Hong Kong SAR, ChinaCLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2007;5:14621468CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2007;5:14621468Presentation TitleDate 14Company Confidential 200X Abbott SVR的基線預(yù)測因素應(yīng)答者(n=7)無應(yīng)答者(n=19)P年齡35163070.40男性 (n, %)6 (86%)13 (

14、68%)0.63Log 血清 HBV DNA9.10 (7.54, 9.60)8.54 (5.76, 9.79)0.40血清血清 HBsAg（IU/ml)3375 (192, 15018)31264 (1164, 112599)0.022ALT 水平151 (60, 386)163 (91, 709)0.53Log cccDNA-0.55 (-1.17, 0.56)0.24 (-0.21, 1.18)0.004Log 肝內(nèi)總肝內(nèi)總 HBV DNA 1.46 (0.96, 2.69)2.76 (2.16, 3.56)0.001炎癥壞死評分8 (3, 11)5 (1, 15)0.43纖維化評分1

15、(0, 3)2 (0, 5)0.061CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2007;5:14621468CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2007;5:14621468Presentation TitleDate 15Company Confidential 200X Abbott1.00.80.60.40.20.01 - Specificity1 - Specificity1.00.80.60.40.20.0SensitivitySensitivityAUC for HBsAg = 0.80 (95

16、% CI 0.62, 0.98; p=0.022)AUC for log serum HBV DNA = 0.39 (95% CI 0.14, 0.64; p=0.40) P0.0001Baseline HBsAg level 10,000 IU/ml Sensitivity 86%lSpecificity 56%l陽性持續(xù)應(yīng)答預(yù)測值（PPV） 56%l陰性持續(xù)應(yīng)答預(yù)測值（NPV） 92% Chan HLY, et al. Clin Gastroenterol Hepatol 2007HBsAg基線水平的預(yù)測效果好過基線水平的預(yù)測效果好過HBV DNAPresentation TitleDat

17、e 16Company Confidential 200X Abbott干擾素治療干擾素治療HBeAg陰性陰性CHB治療后3年HBsAg清除率10 IU/mLn=171治療后治療后48周周HBsAg10 IU/mL是治療結(jié)束后是治療結(jié)束后3年年HBsAg清除率的預(yù)測因素清除率的預(yù)測因素而與而與HBV DNA 1500 IU/mL in long-term cohortMarcellin P, et al. AASLD 2008. Abstract 919.6 mos posttreatment4 yrs posttreatmentHBsAg 1500 IU/mLHBV DNA 10,000 c

18、opies/mLHBV DNA 400 copies/mLHBsAg ClearancePatients With Response (%)5939393172360402006 mos posttreatment4 yrs posttreatmentHBsAg 1500 IU/mLHBV DNA 10,000 copies/mLHBV DNA 400 copies/mLHBsAg ClearancePatients With Response (%)3412982460402001008010080Presentation TitleDate 19Company Confidential 2

19、00X AbbottHBsAg IU/ml應(yīng)用于干擾素治療監(jiān)測、預(yù)測的總結(jié) 監(jiān)測時間點監(jiān)測時間點 監(jiān)測指標(biāo)監(jiān)測指標(biāo) 結(jié)結(jié) 果果治療12周 HBsAg水平 v.s.1500 IU/ml無論6個月和4年后，小于1500IU/ml的病人有顯著提高的免疫應(yīng)答水平治療48周的e抗原陰性慢性乙肝病人比較治療開始時和48周時的HBsAg下降水平 v.s. 100 IU/ml治療三年后，下降大于100 IU/ml的病人有高的陰轉(zhuǎn)率治療48周的e抗原陰性慢性乙肝病人48周時的HBsAg水平 v.s. 10 IU/ml治療三年后，如果HBsAg水平控制在10 IU/ml 之內(nèi)，陰轉(zhuǎn)率高HBsAg IU/ml 定量

20、檢測是治療路線管理、提高病人依從性的有力證據(jù)！Presentation TitleDate 20Company Confidential 200X Abbott 案例介紹患者，男，35歲，職員有乙肝家族史（母親，姐姐）感染方式：母嬰傳播HBV DNA: 1.38E+06乙肝病毒抗原抗體測定： HBsAg250IU/L,HBsAb(-),HBeAg1336(S/Co)、HBeAb(-)肝功能： ALT 163 U/L慢性乙型肝炎診斷日期： 2006年04月04日既往抗病毒治療： 無Presentation TitleDate 21Company Confidential 200X Abbott

21、抗病毒治療治療期限： 90周（06.508.3） 治療劑量： 135g180g（自08.1起劑量由135g改為180g）聯(lián)用藥物 無不良反應(yīng)： 輕度脫發(fā) 焦慮不安 體重下降：57.5kgPresentation TitleDate 22Company Confidential 200X Abbott治療過程HBV DNA水平的變化ALT(U/L)28024020016012080400107106105104103102101HBV DNA(拷貝拷貝/ml)ALTHBV DNAHBV DNA轉(zhuǎn)陰(第8周) 0 4 8 12 24 36 48 60 72 84 96 隨訪隨訪11月月時間(周)P

22、resentation TitleDate 23Company Confidential 200X Abbott治療過程中HBeAg/HBeAb的變化HBeAg (S/CO) 100806040200HBeAgHBeAb120967248240HBeAg轉(zhuǎn)陰(第57周)HBeAg血清轉(zhuǎn)換(第57周) 0 4 8 12 24 36 48 60 72 84 96 隨訪隨訪11月月時間(周)0周周 HBeAg =1223 S/CO1/HBeAb (CO/S)Presentation TitleDate 24Company Confidential 200X Abbott 治療過程中HBsAg/HBs

23、Ab的變化HBsAg (IU/ml) HBsAgHBsAb120967248240HBsAb(mIU/ml)HBsAg轉(zhuǎn)陰(第90周)HBsAb血清轉(zhuǎn)換(第90周)240180120600250 0 4 8 12 24 36 48 60 72 84 96 隨訪隨訪11月月時間(周)Presentation TitleDate 25Company Confidential 200X AbbottARCHITECT HBsAg 突變捕獲ARCHITECT HBsAg: Detects HBsAg Mutations Mutationgly-145-argPresentation TitleDate

24、 26Company Confidential 200X AbbottARCHITECT HBsAg 量化定標(biāo)Calibrated to WHO standard preparationAssay range : 0.05 250 IU/mlOff-line dilution : 0.05-125,000 IU/mlPresentation TitleDate 27Company Confidential 200X AbbottARCHITECT HBsAg 標(biāo)準(zhǔn)曲線1001,00010,000100,0001,000,00010,000,0000.010.101.0010.00100.001

25、,000.00HBs抗原濃度 (IU/mL)Lot1Lot2Lot3ARCHITECT HBsAg QT (RLU)HBsAg Concentration (IU/mL)Cutoff: 0,05 IU/mLResult or = 0.05 IU/ml, reactivePresentation TitleDate 28Company Confidential 200X AbbottHBsAg 稀釋流程及經(jīng)驗From in house experience, a 1:150 dilution is recommended for use in patients before and up to

26、48 weeks of treatmentPresentation TitleDate 29Company Confidential 200X Abbott香港研究：入選病例治療前的HBsAg水平，最高為112,599 IU/mLHBsAg在香港研究中的濃度分布Presentation TitleDate 30Company Confidential 200X Abbott最高水平為100,000 IU/mL HBsAg在日本研究中的濃度分布Journal of Medical Virology,75:235-239(2005)Presentation TitleDate 31Company

27、Confidential 200X AbbottARCHITECT HBsAg 稀釋后的線性 0.0010.010.11101001000100001000000.00000010.0000010.000010.00010.0010.010.11希釈倍率HBsAg QT (IU/mL)Sample 1Sample 2Sample 3CutoffARCHITECT HBsAg QT (IU/mL)Dilution FactorPresentation TitleDate 32Company Confidential 200X AbbottHBe 可以被定量檢測嗎可以被定量檢測嗎Presentat

28、ion TitleDate 33Company Confidential 200X AbbottHBeAg水平下降可以較好地預(yù)測水平下降可以較好地預(yù)測HBeAg的血清轉(zhuǎn)換的血清轉(zhuǎn)換HEPATOLOGY, Vol. 47, No. 2, 2008CHB病人中，有超過病人中，有超過30的的e抗原陰性病人抗原陰性病人Presentation TitleDate 34Company Confidential 200X AbbottHEPATOLOGY, Vol. 47, No. 2, 2008HBeAg水平下降可以較好地預(yù)測水平下降可以較好地預(yù)測HBeAg的血清轉(zhuǎn)換的血清轉(zhuǎn)換Presentation

29、TitleDate 35Company Confidential 200X AbbottPresentation TitleDate 36Company Confidential 200X Abbott預(yù)測預(yù)測HBeAg血清轉(zhuǎn)換：血清轉(zhuǎn)換：HBeAg優(yōu)于優(yōu)于HBV DNAHEPATOLOGY, Vol. 47, No. 2, 2008Presentation TitleDate 37Company Confidential 200X Abbott ARCHIETCT HBeAg 與 PEI U/ml的線性關(guān)系Using Relative Quant on HBeAg MonitoringUsi

30、ng PEI Standards to Plot a Curve for S/CO Index PEI (Paul Ehrlich Institute) is an agency of Germany Federal Ministry of HealthPresentation TitleDate 38Company Confidential 200X Abbott HBeAg滴度的人群分布滴度的人群分布 In 40% the HBeAg titre 1000 PE IU/ml051015202530350 -10001000+2000+3000+4000+5000+6000+7000+8000+9000+10000+HBeAg titre (PE IU/ml)No of patientsNumber o