不同稀释法检测HBsAg定量的比较及临床意义
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摘要
背景与目的慢性乙型肝炎(CHB)是威胁人类健康的重大疾病。乙型肝炎表面抗原(HBsAg)是乙型肝炎病毒(HBV)的外膜蛋白,由病毒S基因编码,在病毒感染肝细胞过程中起重要作用。近年来的研究显示血清HBsAg水平不仅在一定程度上反映HBV复制水平,更重要的是其与肝细胞内共价闭合环状DNA(cccDNA)这一乙肝病毒复制的模板有一定的相关性。随着抗病毒药物的研发和治疗方法的改进,HbsAgd的定量检测引起了越来越多研究者和临床医生的关注,尤其是早期HBsAg水平下降对持续病毒学应答(SVR)的预测价值备受重视。准确地检测HBsAg定量水平,对于判断HBV感染和清除,指导抗病毒治疗有着重要意义。目前仅美国雅培公司生产的HBsAg定量试剂盒(ARCHITECT(?) HBsAg)获美国食品药品监督管理局(FDA)正式批准。由于Architect HBsAg定量检测的线性范围在0～250IU/mL,对超过线性范围上限的HBsAg精确定量检测研究很少,主要原因是专用配套稀释液价格昂贵,限制了其临床应用。本研究用生理盐水作为稀释液对超出线性范围的标本进行稀释后检测,与专用配套稀释液进行比较,分析不同稀释法检测HBsAg定量结果的一致性,并对HBsAg定量与临床观察指标的相关性进行初步研究。
材料和方法从山东大学第二医院门诊及住院病人中挑选96例慢性HBV感染者,所有患者Architect HBsAg定量检测均超过250IU/mL,并排除甲肝、丙肝、戊肝病毒感染,诊断符合2005年《慢性乙型肝炎防治指南》的诊断标准。对所有标本分别用配套原液标准稀释(A法)、生理盐水标准稀释(B法)、生理盐水
一步法稀释(C法)后进行检测,稀释后A法和B法分别有4例和5例无法得到具体测定值(>125000IU/mL)。将3种稀释方法均能得到具体测定值的91例患者纳入统计学分析,比较B法、C法与A法检测HBsAg定量结果的一致性。以A法检测的HBsAg定量结果为标准,选取未接受过抗病毒治疗的44例患者,分析HBsAg定量与疾病状态、HBeAg状态、HBeAg水平、HBV DNA水平、性别、年龄及ALT水平的相关性。
结果
1.A法、B法、C法测定值的对数值范围分别为(2.72～4.92) log10IU/mL、(2.52～4.97) log10IU/mL、(2.54～4.84) log10IU/mL,对数值的中位数分别是3.83 log10IU/mL、3.81 log10IU/mL、3.54 log10IU/mL。B法检测结果与A法检测结果的差异无统计学意义(P=0.712),C法检测结果与A法检测结果的差异有统计学意义(P=0.000)。B法、C法与A法检测结果的相关系数分别为0.969、0.919(P=0.000)。采用Bland-Altman方法分析,在95%的一致性界限,绝大多数差值都位于该区间内。在不同的HBsAg水平分层中,B法、C法的检测结果亦与A法有较好的相关性。
2.慢性乙型肝炎组的HBsAg定量高于肝硬化组(P=0.001), HBeAg阳性组的HBsAg定量高于HBeAg阴性组(P=0.001),不同性别之间HBsAg定量的差别无统计学意义(P=0.448)。在HBeAg阳性组,HBsAg定量与HBeAg水平呈正相关(r=0.675,P=0.000),在HBV DNA阳性组,HBsAg定量与HBV DNA水平呈正相关(r=0.613,P=0.000)。HBsAg定量与年龄(r=-0.291, P=0.055)、ALT水平(r=0.162,P=0.306)不相关。
结论B法的检测结果与A法的检测结果有较好的一致性。HBsAg定量在一定程度上能够反映HBV的复制水平。
Background and Objective Chronic hepatitis b (CHB) is a serious disease to threaten the health of human. Hepatitis b surface antigen (HBsAg), the embrane protein of hepatitis b virus (HBV) which coded by S genetic of virus plays an important role in the process of infecting liver cells. Recent research shows that serum HBsAg level is not only reflect HBV replication level to a certain extent, more important it has certain correlation with covalently closed circular DNA (cccDNA), which is the replication template of HBV in liver cells. With the research and development of anti-viral drugs and improving of treatment methods, the detection of HBsAg quantitative attracts more and more attention of researchers and clinicians, especially the early serum HBsAg drop has predictive value to the ongoing sustained virological response (SVR). Detection of HBsAg quantitative accurately has important significance in judging if infect or clear HBV and guiding antiviral treatment. Resently, HBsAg quantitative kit (ARCHITECT(?) HBsAg) produced by Abbott is only approved by the U. S. Food and Drug Administration (FDA). The linear range detected by Architect HBsAg quantitative is 0-250IU/mL, there are few studies on accurate detection about quantitative of HBsAg which are over than the maximum linear range, the main reason is exclusive ancillary dilutions expensive thus limiting its clinical application. In the study, normal saline was used to dilute samples for them over than the linear range, then tested and compared with that diluted by special supporting dilution to analyze the consistency of different quantitative results, and also studied preliminarily the association between HBsAg quantitative and clinical indicators.
Materials and Methods:96 cases were selected from the outpatient and inpatient with chronic HBV infection, Architect HBsAg quantitative of patients were all over than 250IU/mL, and ruled out hepatitis A, hepatitis C,. hepatitis E virus infection, Diagnosis accord to the diagnostic criteria of guideline on prevention and treatment of chronic hepatitis B in China (2005). All the specimens were respectively diluted in matching standard dilution (Method A), physiological saline (Method B) according to the instructions, physiological saline but one-step dilution (Method C), and then tested. There were respectively 4 cases and 5 cases who can not get concrete determination value diluted according to Method A and B (> 125000U/mL).91 cases that all had concrete determination value diluted according to three dilution method were chosen into statistical analysis to compare the consistency of HBsAg quantitative. HBsAg quantitative diluted by Method A as the standard,44 cases who had not received any antiviral treatment were selected. The correlation were respectively analyzed between HBsAg quantitative and disease status, HBeAg status, HBeAg level, HBV DNA level, gender, age, ALT. Results l.The logarithmic range of determination value were respectively (2.72～4.92) log10IU/mL, (2.52～4.97) log10IU/mL, (2.54～4.84) log10IU/mL in Method A, Method B and Method C, medians were respectively 3.83 log10 IU/mL,3.81 log10 IU/mL and 3.54 log10IU/mL. Difference of testing results between Method B and Method A was not statistically significant (P= 0.712), while difference of testing results between Method C and A was statistically significant (P=0.000). There were good correlation between Method B and Method A, between Method C and Method A(r=0.919 0.969, P=0.000). Adopt Bland-Altman method analysis, in 95% of consistency boundary, most differences were located within the region. In different MBsAg horizontal, Method B and C had good correlation with Method A.
2. HBsAg quantitative of Chronic hepatitis b group was higher than that of cirrhosis group (P=0.001), HBsAg quantitative of HBeAg-positive group was higher than that of HBeAg-negative group (P=0.001), differentce of HBsAg quantitative between genders had no statistically significant (P=0.448). In HBeAg-positive group, Levels between HBsAg quantitative and HBeAg were positively correlated (r=0.675, P=0.000), in HBV DNA positive group, levels of HBsAg quantitative and HBV DNA were positively correlated (r=0.613, P=0.000). HBsAg quantitative had no correlation to age (r=-0.291,P=0.055) and ALT (r=0.162, P=0.306). CONCLUSIONS The results tested by Method B and Method A showed good agreement. HBsAg quantitative can reflect the level of HBV replication to a certain extent.
材料和方法从山东大学第二医院门诊及住院病人中挑选96例慢性HBV感染者,所有患者Architect HBsAg定量检测均超过250IU/mL,并排除甲肝、丙肝、戊肝病毒感染,诊断符合2005年《慢性乙型肝炎防治指南》的诊断标准。对所有标本分别用配套原液标准稀释(A法)、生理盐水标准稀释(B法)、生理盐水
一步法稀释(C法)后进行检测,稀释后A法和B法分别有4例和5例无法得到具体测定值(>125000IU/mL)。将3种稀释方法均能得到具体测定值的91例患者纳入统计学分析,比较B法、C法与A法检测HBsAg定量结果的一致性。以A法检测的HBsAg定量结果为标准,选取未接受过抗病毒治疗的44例患者,分析HBsAg定量与疾病状态、HBeAg状态、HBeAg水平、HBV DNA水平、性别、年龄及ALT水平的相关性。
结果
1.A法、B法、C法测定值的对数值范围分别为(2.72～4.92) log10IU/mL、(2.52～4.97) log10IU/mL、(2.54～4.84) log10IU/mL,对数值的中位数分别是3.83 log10IU/mL、3.81 log10IU/mL、3.54 log10IU/mL。B法检测结果与A法检测结果的差异无统计学意义(P=0.712),C法检测结果与A法检测结果的差异有统计学意义(P=0.000)。B法、C法与A法检测结果的相关系数分别为0.969、0.919(P=0.000)。采用Bland-Altman方法分析,在95%的一致性界限,绝大多数差值都位于该区间内。在不同的HBsAg水平分层中,B法、C法的检测结果亦与A法有较好的相关性。
2.慢性乙型肝炎组的HBsAg定量高于肝硬化组(P=0.001), HBeAg阳性组的HBsAg定量高于HBeAg阴性组(P=0.001),不同性别之间HBsAg定量的差别无统计学意义(P=0.448)。在HBeAg阳性组,HBsAg定量与HBeAg水平呈正相关(r=0.675,P=0.000),在HBV DNA阳性组,HBsAg定量与HBV DNA水平呈正相关(r=0.613,P=0.000)。HBsAg定量与年龄(r=-0.291, P=0.055)、ALT水平(r=0.162,P=0.306)不相关。
结论B法的检测结果与A法的检测结果有较好的一致性。HBsAg定量在一定程度上能够反映HBV的复制水平。
Background and Objective Chronic hepatitis b (CHB) is a serious disease to threaten the health of human. Hepatitis b surface antigen (HBsAg), the embrane protein of hepatitis b virus (HBV) which coded by S genetic of virus plays an important role in the process of infecting liver cells. Recent research shows that serum HBsAg level is not only reflect HBV replication level to a certain extent, more important it has certain correlation with covalently closed circular DNA (cccDNA), which is the replication template of HBV in liver cells. With the research and development of anti-viral drugs and improving of treatment methods, the detection of HBsAg quantitative attracts more and more attention of researchers and clinicians, especially the early serum HBsAg drop has predictive value to the ongoing sustained virological response (SVR). Detection of HBsAg quantitative accurately has important significance in judging if infect or clear HBV and guiding antiviral treatment. Resently, HBsAg quantitative kit (ARCHITECT(?) HBsAg) produced by Abbott is only approved by the U. S. Food and Drug Administration (FDA). The linear range detected by Architect HBsAg quantitative is 0-250IU/mL, there are few studies on accurate detection about quantitative of HBsAg which are over than the maximum linear range, the main reason is exclusive ancillary dilutions expensive thus limiting its clinical application. In the study, normal saline was used to dilute samples for them over than the linear range, then tested and compared with that diluted by special supporting dilution to analyze the consistency of different quantitative results, and also studied preliminarily the association between HBsAg quantitative and clinical indicators.
Materials and Methods:96 cases were selected from the outpatient and inpatient with chronic HBV infection, Architect HBsAg quantitative of patients were all over than 250IU/mL, and ruled out hepatitis A, hepatitis C,. hepatitis E virus infection, Diagnosis accord to the diagnostic criteria of guideline on prevention and treatment of chronic hepatitis B in China (2005). All the specimens were respectively diluted in matching standard dilution (Method A), physiological saline (Method B) according to the instructions, physiological saline but one-step dilution (Method C), and then tested. There were respectively 4 cases and 5 cases who can not get concrete determination value diluted according to Method A and B (> 125000U/mL).91 cases that all had concrete determination value diluted according to three dilution method were chosen into statistical analysis to compare the consistency of HBsAg quantitative. HBsAg quantitative diluted by Method A as the standard,44 cases who had not received any antiviral treatment were selected. The correlation were respectively analyzed between HBsAg quantitative and disease status, HBeAg status, HBeAg level, HBV DNA level, gender, age, ALT. Results l.The logarithmic range of determination value were respectively (2.72～4.92) log10IU/mL, (2.52～4.97) log10IU/mL, (2.54～4.84) log10IU/mL in Method A, Method B and Method C, medians were respectively 3.83 log10 IU/mL,3.81 log10 IU/mL and 3.54 log10IU/mL. Difference of testing results between Method B and Method A was not statistically significant (P= 0.712), while difference of testing results between Method C and A was statistically significant (P=0.000). There were good correlation between Method B and Method A, between Method C and Method A(r=0.919 0.969, P=0.000). Adopt Bland-Altman method analysis, in 95% of consistency boundary, most differences were located within the region. In different MBsAg horizontal, Method B and C had good correlation with Method A.
2. HBsAg quantitative of Chronic hepatitis b group was higher than that of cirrhosis group (P=0.001), HBsAg quantitative of HBeAg-positive group was higher than that of HBeAg-negative group (P=0.001), differentce of HBsAg quantitative between genders had no statistically significant (P=0.448). In HBeAg-positive group, Levels between HBsAg quantitative and HBeAg were positively correlated (r=0.675, P=0.000), in HBV DNA positive group, levels of HBsAg quantitative and HBV DNA were positively correlated (r=0.613, P=0.000). HBsAg quantitative had no correlation to age (r=-0.291,P=0.055) and ALT (r=0.162, P=0.306). CONCLUSIONS The results tested by Method B and Method A showed good agreement. HBsAg quantitative can reflect the level of HBV replication to a certain extent.
引文
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