Stability of FibroTouch in determining liver stiffness measurement and controlled attenuation parameter

Zhao XiaoBo; Yu Jing; Zhang ZhenHua; Zou GuiZhou

doi:10.3969/j.issn.1001-5256.2020.01.023

Volume 36 Issue 1

Jan. 2020

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Article Navigation > Journal of Clinical Hepatology > 2020 > 36(1): 102-106

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Stability of FibroTouch in determining liver stiffness measurement and controlled attenuation parameter

DOI: 10.3969/j.issn.1001-5256.2020.01.023

Department of Hepatology,The Second Affiliated Hospital of Anhui Medical University

Research funding:

Received Date: 2019-07-19
Published Date: 2020-01-20

Abstract

Abstract

Objective To investigate the stability of FibroTouch(FT) in the determination of liver stiffness measurement(LSM) and controlled attenuation parameter(CAP). Methods A total of 309 patients with chronic hepatitis B virus(HBV) infection,nonalcoholic fatty liver disease(NAFLD),liver function abnormalities,or other diseases who underwent FT in the outpatient service of Department of Hepatology,The Second Affiliated Hospital of Anhui Medical University,were enrolled. FT was performed at three different measurement points for the same subject,and intraclass correlation coefficient(ICC) and the Bland-Altman method were used to evaluate the stability of measured values. The Kendall's W test and pie charts were used to evaluate the stability of diagnostic results. Results LSM and CAP values of the three different measurement points for the same subject had an ICC of > 0. 75 in different disease groups(all P < 0. 001),among which the chronic HBV infection group had the highest ICC of LSM of 0. 905,the other disease group had the highest ICC of CAP of 0. 805,the NAFLD group had the lowest ICC of LSM of 0. 785 and the lowest ICC of CAP of 0. 780. The Bland-Altman plots were generated for the three measurement points,and the dots within the limits of agreement accounted for about 95%. The diagnostic results of LSM and CAP staging of the three measurement points had a Kendall's W coefficient of 0. 825 and 0. 858,respectively(all P < 0. 001). The proportion of patients with consistency between the diagnostic results for F≥2 stage(LSM > 7. 3 k Pa) and mild(or more severe) fatty liver stage(CAP >240 dB/m) based on mean value of measurement points and all three measurement points was > 60%,while the proportion of patients with consistency between the diagnostic results based on mean value of measurement points and only one measurement point was < 10%. Conclusion The values and diagnostic results of FT measurement have good stability; however,in order to improve its stability in diagnosing F ≥2 stage and mild(or more severe) fatty liver,it is suggested to use the mean value of multiple measurement points for diagnosis.
- liver cirrhosis,
- fatty liver,
- FibroTouch

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慢性HBV感染的自然史划分为4个期，即免疫耐受期、免疫清除期、免疫控制期和再活动期^[1]。目前，对于处于免疫清除期以及再活动期的慢性乙型肝炎(CHB)患者，各大指南均推荐抗病毒治疗，对于免疫耐受期则不推荐抗病毒治疗，建议长期随访^[1-4]。然而，有研究^[5-11]表明，10%~49%免疫耐受期CHB (Immune-tolerant CHB，IT-CHB) 患者经肝组织病理学检查证实存在明显的肝脏炎症和/或纤维化，若不积极治疗，发展至肝硬化及肝癌的风险增加。IT-CHB患者是否抗病毒治疗尚存在争议^[12-18]，而评估肝组织学显著肝脏炎症及纤维化对于抗病毒治疗具有重要意义，肝活检仍然是金标准，但其有创性及不易重复等缺点限制了临床应用。本研究通过分析IT-CHB患者显著肝损伤(≥G2/S2)的高危因素，构建无创的个体化列线图预测模型，旨在为指导IT-CHB抗病毒治疗提供参考依据。

1. 资料和方法

1.1 研究对象

回顾性选取2002年8月—2017年12月在解放军总医院第五医学中心住院的IT-CHB患者。免疫耐受期的诊断标准符合2018年版美国肝病学会CHB指南^[2]中的定义。纳入标准：(1)年龄＞18岁；(2)HBsAg阳性及HBeAg阳性＞1年；(3)ALT水平持续正常(男性35 U/L，女性25 U/L)＞1年；(4)HBV DNA＞1×10⁶ IU/ml；(5)接受肝活检。排除标准：(1)合并其他病毒感染；(2)其他类型肝脏疾病；(3)失代偿期肝硬化；(4)肝癌或其他恶性肿瘤病史；(5)严重的心脏、肾脏或者其他脏器的原发疾病或精神系统疾病。

1.2 肝组织学检查

采用16G活检针进行超声引导下经皮肝活检，要求肝组织长度≥15 mm，至少包括11个汇管区^[19]。由2名经验丰富的病理医师进行双盲法阅片，肝组织炎症分级和纤维化分期标准参照《慢性乙型肝炎防治指南(2015年版)》^[20]。显著肝损伤(≥G2/S2)定义为肝组织学存在明显的肝脏炎症(≥G2)或纤维化(≥S2)。

1.3 血清学检测

采用贝克曼库尔特AU5421全自动生化仪检测血清ALT、AST、TBil、PLT等。乙型肝炎血清学标志物采用罗氏E170电化学发光法检测。计算APRI指数和FIB-4指数，APRI = (AST/正常值上限×100)/PLT，FIB-4=(年龄×AST)/(PLT×ALT^1/2)^[21]。

1.4 伦理学审查

本研究通过解放军总医院第五医学中心伦理委员会审批，批号：2020056D。

1.5 统计学方法

采用SPSS 22.0进行统计分析。正态分布的计量数据以x±s表示，2组间比较采用独立样本t检验；非正态分布数据以M(P₂₅~P₇₅)表示，2组间比较采用Mann- Whitney U检验; 多组比较采用Kruskal-Wallis H检验；计数资料2组间比较采用χ²检验。相关性分析采用Spearman秩相关。通过多因素logistic回归模型进入法筛选显著肝损伤的相关因素，采用R语言(3.6.1)的RMS(Regression Modeling Strategies)程序包构建列线图模型，通过Bootstrap重抽样法对模型进行内部验证，用一致性指数(C-指数)、ROC曲线、校准曲线来评价列线图的区分度及校准度。P＜0.05为差异有统计学意义。

2. 结果

2.1 一般资料

共纳入382例IT-CHB患者，其中82例(21.5%)存在显著肝损伤。肝组织炎症活动度分级: G0 29例(7.6%)、G1 301例(78.8%)、G2 50例(13.1%)、G3 2例(0.5%)；肝组织纤维化分期: S0 57例(14.9%)、S1 251例(65.7%)、S2 39例(10.2%)、S3 23例(6.0%)、S4 12例(3.1%)。按照是否存在显著肝损伤(≥G2/S2)分为2组，2组年龄、HBV DNA载量、ALT、AST、PLT比较差异均有统计学意义(P值均＜0.001)(表 1)。

表 1 患者基线的一般资料

指标	总体(n=382)	非显著肝损伤组(n=300)	显著肝损伤组(n=82)	统计值	P值
男性[例(%)]	261(68.3)	201(67.0)	60(73.2)	χ²=1.133	0.287
年龄(岁)	33.3±10.2	31.5±9.1	39.9±11.2	t=-7.071	＜0.001
年龄段[例(%)]				χ²=56.472	＜0.001
＜30岁	161(42.1)	147(49.0)	14(17.1)
30~39岁	130(34.0)	106(35.3)	24(29.3)
40~49岁	64(16.8)	35(11.7)	29(35.4)
≥50岁	27(7.1)	12(4.0)	15(18.3)
乙型肝炎家族史[例(%)]	221(57.9)	173(57.7)	48(58.5)	χ²=0.020	0.888
BMI(kg/m²)	23.2±3.53	23.0±3.4	23.7±3.9	t=-1.021	0.308
HBV DNA(log₁₀IU/ml)	8.4(7.8~8.8)	8.4(8.0~8.8)	7.9(6.9~8.5)	Z=-4.924	＜0.001
ALT(U/L)	23.0(18.0~28.0)	23.0(18.0~28.0)	25.5(21.0~32.0)	Z=-3.693	＜0.001
AST(U/L)	23.0(19.0~27.0)	21.0(19.0~26.0)	28.0(23.0~34.0)	Z=-6.945	＜0.001
TBil(μmol/L)	11.1(8.3~15.3)	10.9(8.3~15.3)	11.5(8.6~15.4)	Z=-0.585	0.559
PLT(×10⁹/L)	202(164~234)	208(176~239)	161(137~209)	Z=-5.723	＜0.001

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2.2 年龄与肝组织损伤病理学的关系

为了评估年龄对IT-CHB显著肝损伤的影响，将患者分为4个年龄段，即＜30岁、30~39岁、40~49岁和≥50岁。随年龄的增加，肝组织炎症及纤维化程度逐渐升高，趋势性检验结果表明差异均具有统计学意义(P值均＜0.001)。Spearman等级相关分析显示，两者呈正相关(r值分别为0.222、0.275，P值均＜0.001)(图 1)。Logistic单因素分析结果显示，较年龄＜30岁组，30~39岁组、40~49岁组、年龄≥50岁组出现显著肝损伤的可能性分别为2.4倍(95%CI: 1.175~4.811)、8.7倍(95%CI: 4.165~18.175)、13.1倍(95%CI: 5.146~33.477)(P值均＜0.05)。

图 1 不同年龄段与肝组织病理炎症及纤维化的关系

下载: 全尺寸图片幻灯片

2.3 HBV DNA与肝组织损伤病理学的关系

随着肝脏坏死性炎症的加剧，HBV DNA水平呈下降趋势(H=34.161，P＜0.001)，组间两两比较结果显示：G0组、G1组与≥G2组之间差异有统计学意义(H值分别为80.688、96.903，P值均＜0.05)，而GO组与G1组无差异(图 2)。伴随肝纤维化的进展，HBV DNA同样表现出下降的趋势(H=26.627，P＜0.001)，组间两两比较显示，S0与S4、S1与S4、S1与S3之间差异均有统计学意义(H值分别为112.287、125.953、74.354，P值均＜0.05)(图 2)。

图 2 HBV DNA载量与不同阶段肝组织病理炎症及纤维化的关系

下载: 全尺寸图片幻灯片

2.4 显著肝损伤的单因素及多因素分析

为进一步构建无创预测模型，基于无创参数中单因素分析P＜0.05的变量作为自变量，以显著肝损伤作为因变量进行logistic回归分析。结果显示，年龄、HBV DNA水平、AST以及PLT是显著肝损伤的独立影响因素(P值均＜0.01)(表 2)。

表 2 显著肝损伤的logistic回归分析

因素	单因素分析		多因素分析
因素	OR(95%CI)	P值	OR(95%CI)	P值
年龄	1.084 (1.057~1.113)	＜0.001	1.074(1.043~1.107)	＜0.001
HBV DNA	0.437 (0.324~0.589)	＜0.001	0.442(0.314~0.624)	＜0.001
ALT	1.076(1.036~1.119)	＜0.001	1.009(0.959~1.060)	0.736
AST	1.132 (1.089~1.177)	＜0.001	1.096(1.051~1.142)	＜0.001
PLT	0.985 (0.98~0.991)	＜0.001	0.992(0.986~0.998)	0.006

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2.5 列线图的制作与检验

基于logistic回归分析结果，将独立影响因素引入R软件建立预测显著肝损伤的个体化列线图预测模型，并绘制校准曲线和ROC曲线。结果显示，列线图模型预测IT-CHB发生显著肝损伤的C-指数的ROC曲线下面积(AUC)为0.845(95%CI: 0.795~0.895), 明显优于单独使用APRI(AUC=0.781, 95%CI: 0.723~0.840)以及FIB-4(AUC=0.802, 95%CI: 0.746~0.859)，差异有统计学意义。校正曲线贴近于理想曲线(对角线)，斜率为1.017，Hosmer-Lemeshow拟合优度检验χ²=8.224，P=0.412，提示模型预测值与实际观测值之间的差异无统计学意义，预测模型有良好的校准度。ROC曲线分析显示，列线图的AUC高于APRI、FIB-4，预测IT-CHB患者显著肝损伤的最佳界值为141.4，其敏感度、特异度分别为74.4%、84.7%，差异有统计学意义(P＜0.05)(表 3, 图 3)。

表 3 列线图、APRI、FIB-4诊断显著肝损伤的效能比较

诊断参数	AUC	95%CI	界值	敏感度(%)	特异度(%)	阳性预测值(%)	阴性预测值(%)	Youden指数
列线图	0.845	0.795 ~ 0.895	141.4	74.4	84.7	57.0	92.4	0.59
APRI	0.781	0.723 ~ 0.840	0.338	70.7	77.7	46.4	90.7	0.48
FIB-4	0.802	0.746 ~ 0.859	0.882	78.1	72.3	43.5	92.3	0.50

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图 3 预测IT-CHB患者显著肝损伤的列线图

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3. 讨论

全球慢性HBV感染者约2.92亿人，其中约5940万处于免疫耐受期，我国的IT-CHB患者约有1584万例^[22]。目前国内外指南对于免疫耐受期的定义尚存在争议^{[1-4, 23-25]}，ALT正常上限的标准亦不同, 按照美国肝病学会的标准意味我国IT-CHB患者并非全部处于免疫耐受阶段。单纯用病毒学、ALT水平评估免疫耐受可能存在临床误判，“真正”的免疫耐受需在肝活检基础上进一步确诊，目前多项研究^[5-9]表明，10%~49% IT-CHB患者存在明显的肝细胞炎症坏死和肝纤维化病理学改变, 此类患者是否应抗病毒治疗逐渐成为热点问题。

本研究发现IT-CHB患者中21.5%(82/382)存在显著肝损伤，19.4%(74/382)呈显著肝纤维化，其中12例患者(3.1%)处于S4期，提示并不是全部IT-CHB患者均不需要治疗，如何筛选出需要治疗的患者尤为重要。本研究筛选出4个显著肝损伤的高危因素，包括年龄、HBV DNA水平、AST以及PLT，其中AST、PLT作为APRI、FIB-4的参数之一，已被充分证实与肝纤维化程度有关^{[21, 26]}。既往研究^[27-28]表明，年龄是CHB患者疾病进展的独立危险因素，尤其年龄＞30岁时，HBV相关性肝纤维化、肝硬化、肝癌患者的比例显著增加。Xing等^[6]发现年龄是影响肝组织炎症及纤维化的独立预测因子，这一点与本研究结果一致，将IT-CHB患者的年龄分为4个年龄亚组，结果表明，随年龄的增加，肝组织炎症及纤维化程度逐渐升高。关于HBV DNA，我国台湾的大样本研究^[29]发现高HBV DNA水平CHB患者进展至肝硬化的风险增加，但其中81.6%(2923/3582) 为HBeAg阴性患者，不属于IT-CHB患者，因此该研究无法准确反映高HBV DNA水平与IT-CHB患者肝纤维化的关系。而本研究发现，IT-CHB患者随着肝脏炎症及纤维化程度的加重，HBV DNA呈下降趋势；并且轻度肝损伤(＜G2/S2)的IT- CHB患者中位HBV DNA水平更高(8.4 log₁₀ IU/ml)，因此单纯HBV DNA水平并不能准确反映出IT-CHB患者纤维化程度。基于上述分析，本研究建立了无创的列线图模型用于预测IT-CHB患者的显著肝损伤，该模型具有无创的优势，并将多因素分析结果可视化、量化、个体化，具有可重复性，可作为肝活检的有效替代方式。根据该列线图模型，假设某40岁的IT-CHB患者，HBV DNA水平为6.56 log₁₀ IU/ml，AST 35 U/L，PLT 166×10⁹/L，则该患者总得分为188.6分，发生显著肝损伤的概率高达85%，需积极抗病毒治疗。

综上所述，免疫耐受期具有显著肝损伤的患者比例并不少见，基于年龄、HBV DNA、AST、PLT 4个因素构建的列线图模型具有良好的预测准确性，可用于个体化预测IT-CHB患者的显著肝损伤，减少肝活检，为抗病毒的精准治疗提供参考。

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Stability of FibroTouch in determining liver stiffness measurement and controlled attenuation parameter

DOI: 10.3969/j.issn.1001-5256.2020.01.023