HBV血清学定量检测技术及应用.ppt

HBV血清学定量检测技术及应用,1,Company Confidential 2009 Abbott,HBV血清学定量检测技术及应用,雅培诊断 钱学庆 博士,Presentation Title Date,2,Company Confidential 200X Abbott,TIME,Loss of HBeAg,Loss of HBV DNA,Anti-HBe+,Loss of HBsAg,Anti-HBs+,Improved survival,Improved histology,Treatment endpoints differ for antiviral and immune-based therapies,乙型肝炎治疗终点,Prof. Marcellin, France Communication/ presentation “Clinical strategies to CHB matching the treatment to patients”,Presentation Title Date,3,Company Confidential 200X Abbott,免疫应答引起的抗体 Anti-HBc, Anti-HBc-IgM Anti-HBe Anti-HBs,病毒标志物 HBV DNA HBeAg HBsAg,Hepatitis B 诊断标志物,Presentation Title Date,4,Company Confidential 200X Abbott,两步法检测,Presentation Title Date,5,Company Confidential 200X Abbott,两步法可以消除HOOK效应,Step 1,+,WASH,WASH,Step 2,Sample 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 Title Date,6,Company Confidential 200X Abbott,一步法和两步法试剂的比较分析,Presentation Title Date,7,Company Confidential 200X Abbott,ARCHITECT HBsAg定量检测,Presentation Title Date,8,Company Confidential 200X Abbott,HBsAg的重要临床意义,HBsAg是肝细胞受到HBV感染的标志 HBsAg清除是肝内HBV感染的免疫控制和抗病毒应答的标志 HBsAg清除最接近治愈 相比HBV DNA， HBsAg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 最近的研究报告显示：肝细胞内 cccDNA 水平（HBV感染细胞的标志）和血清HBsAg 水平存在显著相关性 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 Colombatto P. et al, Antiviral Therapy 2006； Werle-Lapostolle et al, Gastroenterology 2004；Volz et al, Gastroenterology 2007,Presentation Title Date,9,Company 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.,Infectious HBV virion,Viral polymerase converts pregenomic RNA to partially ds DNA,Partially dsDNA,Subviral particles,Hepatocyte,mRNA,Cytoplasm,Nucleus,Precore/core,HBeAg,ER,HBcAg,HBsAg,cccDNA,Minus strand DNA,Encapsulated pregenomic mRNA,Presentation Title Date,10,Company Confidential 200X Abbott,HBsAg 和 HBV-DNA 聚合酶的相关性,总计418 名 慢性HBV携带者，检测HBsAg 和HBV-DNA 聚合酶水平的结果.,M. Deguchi et al. / Journal of Virological Methods 115 (2004) 217222,Presentation Title Date,11,Company Confidential 200X Abbott,血清HBsAg可以反应被感染肝细胞的数量,cccDNA acts as transcription template Serum HBsAg levels reflect cccDNA (A) and intrahepatic HBV DNA (B) Non-invasive marker of infected cells,A,B,Chan et al. Clin Gastro Hepatol 2007,HBsAg at baseline,HBsAg at baseline,Log (cccDNA) at baseline,Log (intrahepatic HBV DNA) at baseline,A,Presentation Title Date,12,Company Confidential 200X Abbott,血清 HBsAg 水平与 cccDNA水平正相关,p0.01,-3,-2,-1,0,1,-3,-2,-1,0,ADV+PEG,Change in Serum HBsAg,(log,10,ng/mL),Change in cccDNA,(log,10,copies/cell),Petersen EASL 2005,Presentation Title Date,13,Company Confidential 200X Abbott,Serum Hepatitis B Surface Antigen Quantitation Can Reflect Hepatitis B Virus in the Liver and Predict Treatment Response HENRY LIKYUEN CHAN, VINCENT WAISUN WONG, ADA MEILING TSE, CHIHANG TSE, ANGEL MEILING CHIM,HOIYUN CHAN, GRACE LAIHUNG WONG, and JOSEPH JAOYIU SUNG Department of Medicine and Therapeutics, and Institute of Digestive Disease, The Chinese University of Hong Kong, Hong Kong SAR, China,CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2007;5:14621468,Presentation Title Date,14,Company Confidential 200X Abbott,SVR的基线预测因素,CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2007;5:14621468,Presentation Title Date,15,Company Confidential 200X Abbott,AUC for HBsAg = 0.80 (95% 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),P0.0001,Baseline HBsAg level 10,000 IU/ml Sensitivity 86% Specificity 56% 阳性持续应答预测值（PPV） 56% 阴性持续应答预测值（NPV） 92%,Chan HLY, et al. Clin Gastroenterol Hepatol 2007,HBsAg基线水平的预测效果好过HBV DNA,Presentation Title Date,16,Company Confidential 200X Abbott,干扰素治疗HBeAg阴性CHB,治疗后3年HBsAg清除率,HBsAg IU/ml定量检测的临床应用 -用来预测经干扰素治疗患者的疗效,Presentation Title Date,17,Company Confidential 200X Abbott,表面抗原定量的临床应用：48周评价干扰素治疗预后,Marcellin P, et al. Hepatology,2007;46:673A,干扰素治疗48周： 在26例HBsAg下降水平超过100 IU/ml的患者中,有11(42.3%)例在治疗结束结束后三年发生HBsAg转阴。 在172例HBsAg下降水平小于100 IU/ml的患者中,只有5(2.9%)例在治疗结束结束后三年发生HBsAg转阴。 治疗一定时间HBsAg下降水平，可以作为评价愈后的指标。,Presentation Title Date,18,Company Confidential 200X Abbott,HBsAg IU/ml 预测干扰素应答的长期效应,4-yr and 6-mo response rates higher with Wk 12 HBsAg level 1500 IU/mL vs 1500 IU/mL in long-term cohort,Marcellin P, et al. AASLD 2008. Abstract 919.,6 mos posttreatment,4 yrs posttreatment,HBsAg 1500 IU/mL,HBV DNA 10,000 copies/mL,HBV DNA 400 copies/mL,HBsAg Clearance,Patients With Response (%),59,39,39,31,7,23,60,40,20,0,6 mos posttreatment,4 yrs posttreatment,HBsAg 1500 IU/mL,HBV DNA 10,000 copies/mL,HBV DNA 400 copies/mL,HBsAg Clearance,Patients With Response (%),34,12,9,8,2,4,60,40,20,0,100,80,100,80,Presentation Title Date,19,Company Confidential 200X Abbott,HBsAg IU/ml应用于干扰素治疗监测、预测的总结,HBsAg IU/ml 定量检测是治疗路线管理、提高病人依从性的有力证据！,Presentation Title Date,20,Company 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 Title Date,21,Company Confidential 200X Abbott,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：57.5kg,Presentation Title Date,22,Company Confidential 200X Abbott,治疗过程HBV DNA水平的变化,ALT(U/L),280 240 200 160 120 80 40 0,107106 105 104 103 102 10 1,HBV DNA (拷贝/ml),ALT,HBV DNA,HBV DNA转阴 (第8周),0 4 8 12 24 36 48 60 72 84 96 随访11月,时间(周),Presentation Title Date,23,Company Confidential 200X Abbott,治疗过程中HBeAg/HBeAb的变化,HBeAg (S/CO),100 80 60 40 20 0,HBeAg,HBeAb,120 96 72 48 24 0,HBeAg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11月,时间(周),0周 HBeAg =1223 S/CO,1/HBeAb (CO/S),Presentation Title Date,24,Company Confidential 200X Abbott,治疗过程中HBsAg/HBsAb的变化,HBsAg (IU/ml),HBsAg,HBsAb,120 96 72 48 24 0,HBsAb(mIU/ml),HBsAg转阴 (第90周),HBsAb 血清转换(第90周),240 180 120 60 0,250,0 4 8 12 24 36 48 60 72 84 96 随访11月,时间(周),Presentation Title Date,25,Company Confidential 200X Abbott,ARCHITECT HBsAg 突变捕获,ARCHITECT HBsAg: Detects HBsAg Mutations,Presentation Title Date,26,Company Confidential 200X Abbott,ARCHITECT HBsAg 量化定标,Calibrated to WHO standard preparation,Assay range : 0.05 250 IU/ml Off-line dilution : 0.05-125,000 IU/ml,Presentation Title Date,27,Company Confidential 200X Abbott,ARCHITECT HBsAg 标准曲线,100,1,000,10,000,100,000,1,000,000,10,000,000,0.01,0.10,1.00,10.00,100.00,1,000.00,HBs,抗原濃度,(IU/mL),Lot1,Lot2,Lot3,ARCHITECT HBsAg QT (RLU),HBsAg Concentration (IU/mL),Cutoff: 0,05 IU/mL,Result or = 0.05 IU/ml, reactive,Presentation Title Date,28,Company Confidential 200X Abbott,HBsAg 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 48 weeks of treatment,Presentation Title Date,29,Company Confidential 200X Abbott,香港研究：入选病例治疗前的HBsAg水平，最高为112,599 IU/mL,HBsAg在香港研究中的浓度分布,Presentation Title Date,30,Company 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 Title Date,31,Company Confidential 200X Abbott,ARCHITECT HBsAg 稀释后的线性,0.001,0.01,0.1,1,10,100,1000,10000,100000,0.0000001,0.000001,0.00001,0.0001,0.001,0.01,0.1,1,希釈倍率,HBsAg QT (IU/mL),Sample 1,Sample 2,Sample 3,Cutoff,ARCHITECT HBsAg QT (IU/mL),Dilution Factor,Presentation Title Date,32,Company Confidential 200X Abbott,HBe 可以被定量检测吗,Presentation Title Date,33,Company Confidential 200X Abbott,HBeAg水平下降可以较好地预测HBeAg的血清转换,HEPATOLOGY, Vol. 47, No. 2, 2008,CHB病人中，有超过30的e抗原阴性病人,Presentation Title Date,34,Company Confidential 200X Abbott,HEPATOLOGY, Vol. 47, No. 2, 2008,HBeAg水平下降可以较好地预测HBeAg的血清转换,Presentation Title Date,35,Company Confidential 200X Abbott,HBeAg 在24周的血清转换阴性预测价值,Negative predictive value (NPV) of HBV DNA at week 24 as a predictor of HBeAg seroconversion.,Presentation Title Date,36,Company Confidential 200X Abbott,预测HBeAg血清转换：HBeAg优于HBV DNA,HEPATOLOGY, Vol. 47, No. 2, 2008,Presentation Title Date,37,Company Confidential 200X Abbott,ARCHIETCT HBeAg 与 PEI U/ml的线性关系,Using Relative Quant on HBeAg Monitoring,Using PEI Standards to Plot a Curve for S/CO Index,PEI (Paul Ehrlich Institute) is an agency of Germany Federal Ministry of Health,Presentation Title Date,38,Compa