HBV血清学定量检测技术及应用

1、HBV血清学定量检测技术及应用1Company Confidential 2009 AbbottHBV血清学定量检测技术及应用 雅培诊断 钱学庆 博士Presentation TitleDate 2Company Confidential 200X AbbottTIMETreatment endpoints differ for antiviral and immune-based therapies乙型肝炎治疗终点乙型肝炎治疗终点Prof. Marcellin, France Communication/ presentation “Clinical strategies to CHB ma

2、tching the treatment to patients”Presentation TitleDate 3Company Confidential 200X Abbott免疫应答引起的抗体免疫应答引起的抗体Anti-HBc, Anti-HBc-IgMAnti-HBeAnti-HBs病毒标志物病毒标志物HBV DNAHBeAgHBsAgHepatitis B 诊断标志物诊断标志物Presentation TitleDate 4Company Confidential 200X Abbott两步法检测两步法检测Presentation TitleDate 5Company Confiden

3、tial 200X Abbott两步法可以消除HOOK效应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一步法和两步法试剂的比较分析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定量检测定量检测Presentation TitleDate 8Company Confidential 200X Abbott HBsAg的重要临床意义HBsAg是肝细胞受到是肝细胞受到HBV感染的标志感染的标志HBsAg清除是肝内清除是肝内HBV感染的免疫控制和抗病毒应答的标志感染的免

7、疫控制和抗病毒应答的标志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缓解清除能更好地代表疾病的持久缓解HBsAg清除可改善临床预后：降低肝硬化和肝癌的发生率；提高生存率清除可改善临床预后：降低肝硬化和肝癌的发生率；提高生存率最近的研究报告显示：肝细胞内最近的研究报告显示：肝细胞内 cccDNA 水平（水平（HBV感染细胞的标志）和血清感染细胞的标志）和血清HBsAg 水平存在显著相关性水平存在显著相关性目前缺少商业化目前缺少商业化 cccDNA检测试剂；是一种创伤性检测方法，应用受到限制检测试剂；是一种创伤性检测方法，应用受到限制 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 肝细胞内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 聚合酶的相关性聚合酶的相关性总计总计418 名名 慢性慢性HBV携带者，检测携带者，检测HBsAg 和和HBV-DNA 聚合

10、酶水平的结果聚合酶水平的结果.M. Deguchi et al. / Journal of Virological Methods 115 (2004) 217222Presentation TitleDate 11Company Confidential 200X Abbott 血清HBsAg可以反应被感染肝细胞的数量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 水平与 cccDNA水平正相关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的基线预测因素应答者(n=7)无应答者(n=19)P年龄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 肝内总肝内总 HBV DNA 1.46 (0.96, 2.69)2.76 (2.16, 3.56)0.001炎症坏死评分8 (3, 11)5 (1, 15)0.43纤维化评分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阳性持续应答预测值（PPV） 56%l阴性持续应答预测值（NPV） 92% Chan HLY, et al. Clin Gastroenterol Hepatol 2007HBsAg基线水平的预测效果好过基线水平的预测效果好过HBV DNAPresentation TitleDat

17、e 16Company Confidential 200X Abbott干扰素治疗干扰素治疗HBeAg阴性阴性CHB治疗后3年HBsAg清除率10 IU/mLn=171治疗后治疗后48周周HBsAg10 IU/mL是治疗结束后是治疗结束后3年年HBsAg清除率的预测因素清除率的预测因素而与而与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应用于干扰素治疗监测、预测的总结 监测时间点监测时间点 监测指标监测指标 结结 果果治疗12周 HBsAg水平 v.s.1500 IU/ml无论6个月和4年后，小于1500IU/ml的病人有显著提高的免疫应答水平治疗48周的e抗原阴性慢性乙肝病人比较治疗开始时和48周时的HBsAg下降水平 v.s. 100 IU/ml治疗三年后，下降大于100 IU/ml的病人有高的阴转率治疗48周的e抗原阴性慢性乙肝病人48周时的HBsAg水平 v.s. 10 IU/ml治疗三年后，如果HBsAg水平控制在10 IU/ml 之内，阴转率高HBsAg IU/ml 定量

20、检测是治疗路线管理、提高病人依从性的有力证据！Presentation TitleDate 20Company Confidential 200X Abbott 案例介绍患者，男，35岁，职员有乙肝家族史（母亲，姐姐）感染方式：母婴传播HBV DNA: 1.38E+06乙肝病毒抗原抗体测定： HBsAg250IU/L,HBsAb(-),HBeAg1336(S/Co)、HBeAb(-)肝功能： ALT 163 U/L慢性乙型肝炎诊断日期： 2006年04月04日既往抗病毒治疗： 无Presentation TitleDate 21Company Confidential 200X Abbott

21、抗病毒治疗治疗期限： 90周（06.508.3） 治疗剂量： 135g180g（自08.1起剂量由135g改为180g）联用药物 无不良反应： 轻度脱发 焦虑不安 体重下降：57.5kgPresentation TitleDate 22Company Confidential 200X Abbott治疗过程HBV DNA水平的变化ALT(U/L)28024020016012080400107106105104103102101HBV DNA(拷贝拷贝/ml)ALTHBV DNAHBV DNA转阴(第8周) 0 4 8 12 24 36 48 60 72 84 96 随访随访11月月时间(周)P

22、resentation TitleDate 23Company Confidential 200X Abbott治疗过程中HBeAg/HBeAb的变化HBeAg (S/CO) 100806040200HBeAgHBeAb120967248240HBeAg转阴(第57周)HBeAg血清转换(第57周) 0 4 8 12 24 36 48 60 72 84 96 随访随访11月月时间(周)0周周 HBeAg =1223 S/CO1/HBeAb (CO/S)Presentation TitleDate 24Company Confidential 200X Abbott 治疗过程中HBsAg/HBs

23、Ab的变化HBsAg (IU/ml) HBsAgHBsAb120967248240HBsAb(mIU/ml)HBsAg转阴(第90周)HBsAb血清转换(第90周)240180120600250 0 4 8 12 24 36 48 60 72 84 96 随访随访11月月时间(周)Presentation TitleDate 25Company Confidential 200X AbbottARCHITECT HBsAg 突变捕获ARCHITECT HBsAg: Detects HBsAg Mutations Mutationgly-145-argPresentation TitleDate

24、 26Company Confidential 200X AbbottARCHITECT HBsAg 量化定标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 标准曲线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 稀释流程及经验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：入选病例治疗前的HBsAg水平，最高为112,599 IU/mLHBsAg在香港研究中的浓度分布Presentation TitleDate 30Company Confidential 200X Abbott最高水平为100,000 IU/mL HBsAg在日本研究中的浓度分布Journal of Medical Virology,75:235-239(2005)Presentation TitleDate 31Company

27、Confidential 200X AbbottARCHITECT HBsAg 稀释后的线性 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 可以被定量检测吗可以被定量检测吗Presentat

28、ion TitleDate 33Company Confidential 200X AbbottHBeAg水平下降可以较好地预测水平下降可以较好地预测HBeAg的血清转换的血清转换HEPATOLOGY, Vol. 47, No. 2, 2008CHB病人中，有超过病人中，有超过30的的e抗原阴性病人抗原阴性病人Presentation TitleDate 34Company Confidential 200X AbbottHEPATOLOGY, Vol. 47, No. 2, 2008HBeAg水平下降可以较好地预测水平下降可以较好地预测HBeAg的血清转换的血清转换Presentation

29、TitleDate 35Company Confidential 200X AbbottPresentation TitleDate 36Company Confidential 200X Abbott预测预测HBeAg血清转换：血清转换：HBeAg优于优于HBV DNAHEPATOLOGY, Vol. 47, No. 2, 2008Presentation TitleDate 37Company Confidential 200X Abbott ARCHIETCT HBeAg 与 PEI U/ml的线性关系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