唾液酸化胎球蛋白A lectin-ELISA方法的建立及其在原发性肝细胞癌中的诊断价值

Establishment of lectin-ELISA for sialylated fetuin-A and its diagnostic value in primary hepatocellular carcinoma

目的建立检测唾液酸化胎球蛋白A(Sia-fetuin A)的凝集素-酶联免疫吸附试验方法(lectin-ELISA),探讨其在原发性肝细胞癌(HCC)中的诊断价值。方法收集2017至2020年于上海东方肝胆外科医院就诊的HCC患者300例,疾病对照160例(包括肝硬化亚组36例、慢性乙型肝炎亚组124例),另同期收集健康体检者100名作为健康对照。根据接骨木凝集素(SNA)可与糖链末端α-2,6-半乳糖的唾液酸结合原理构建lectin-ELISA方法,检测各组血清Sia-fetuin A水平,以450 nm处吸光度值表示。采用logistic回归方法建立多指标联合检测模型,采用受试者工作特征曲线(ROC)评估单项指标及联合检测模型诊断HCC效能。结果建立了检测血清Sia-fetuin A的lectin-ELISA实验方法,其线性回归系数为0.9785,精密度评估及干扰实验均符合临床检测要求。采用该方法检测各组血清Sia-fetuin A水平,HCC组、疾病对照组和健康对照组分别为1.362±0.310、1.199±0.370、1.086±0.420,3组之间依次降低。Sia-fetuin A、AFP和二者联合检测模型鉴别诊断HCC的曲线下面积分别为0.790、0.809和0.860,诊断模型敏感度79.3%(238/300),特异度95.0%(247/260)。HCC组300例患者中138例(46%)患者血清AFP为阴性(<20μg/L),其血清Sia-fetuin A水平为1.364±0.305。将疾病对照组与健康对照组合并为非患癌组,其血清Sia-fetuin A水平为1.146±0.381。AFP阴性HCC患者血清Sia-fetuin A水平高于非患癌患者(t=6.134,P<0.001)。Sia-fetuin A与联合诊断模型诊断AFP阴性HCC的曲线下面积分别为0.776、0.919,联合诊断模型敏感度93.4%(129/138),特异度77.3%(201/260)。结论血清Sia-fetuin A及其联合检测模型可为AFP阴性HCC患者提供新的辅助诊断指标。

Objective To establish a lectin enzyme-linked immunosorbent assay(lectin-ELISA)for the dection of sialylated fetuin-A and to explore the clinical diagnostic value of sialylated fetuin-A in hepatocellular carcinoma(HCC).Methods From January 2017 to December 2020,300 HCC patients and 160 disease controls,including 36 liver cirrhosis subgroups and 124 chronic hepatitis B subgroups,were collected from Shanghai Eastern Hepatobiliary Surgery Hospital.At the same time,100 healthy subjects were collected as healthy controls.Lectin-ELISA method for detecting sialylated fetuin A was established based on the principle that Sambucus nigra lectin(SNA)can recognize the structure ofα-2,6-linked sialic acid residues.Differences between groups were compared using t-test or analysis of variance.Logistic regression method was used to establish the multi-index joint detection model,and receiver operating characteristic curve(ROC)was used to evaluate the efficacy of single index and joint detection model in the diagnosis of HCC.Results A lectin-ELISA method for the detection of serum Sia-fetuin A was established.The linear regression coefficient of the system was 0.9785,and the precision evaluation and interference experiments were in line with the clinical detection requirements.Using this method to detect serum Sia-fetuin A levels in each group,the levels of HCC group,disease control group and healthy control group were 1.362±0.310,1.199±0.370,1.086±0.420,respectively,and the three groups decreased in turn.The areas under the curve of Sia-fetuin A,α-fetoprotein,and their combined detection models for differential diagnosis of HCC were 0.790,0.809,and 0.860,respectively.The diagnostic model had a sensitivity of 79.3%(238/300)and a specificity of 95.0%(247/260).Among the 300 patients in the HCC group,138(46%)patients were negative for serum AFP(<20μg/L),and their serum Sia-fetuin A level was 1.364±0.305.Combining the disease control group and the healthy control group into the non-Cancer group,the serum Sia-fetuin A level was 1.146±0.381.The serum level of Sia-fetuin A in AFP-negative HCC patients was higher than that in non-HCC group(t=6.134,P<0.001).The areas under the curve of Sia-fetuin A and the combined diagnostic model for the diagnosis of AFP-negative HCC were 0.776 and 0.919,respectively.The combined diagnostic model had a sensitivity of 93.4%(129/138)and a specificity of 77.3%(201/260).Conclusion Serum Sia-fetuin A and combined determination model can provide a new auxiliary diagnostic index for AFP-negative HCC.