评价高通量酶联免疫吸附试验(enzyme-linked immunosorbent assay, ELISA)定量检测血清甲胎蛋白(alpha fetoprotein, AFP)的方法学性能及临床应用价值。

收集2013年3月至12月，青岛市中心医院住院的肝癌、肝炎、肝硬化患者新鲜血清标本170份，医院体检中心健康查体者新鲜血清标本200份。参考美国临床和实验室标准化协会EP5-A2、EP15-A2文件及相关文献，评价高通量ELISA测定血清AFP的线性范围、精密度、正确度、参考区间及其对原发性肝癌的诊断性能，并与电化学发光法比对，评价其检测准确度和临床应用价值。

高通量ELISA测定AFP的线性范围为0.0~200.0 μg/L。低、中、高浓度的批内变异系数(coefficient of variation, CV )分别为4.9%、3.1%、3.7%，批间CV分别为3.8%、1.5%、2.1%；总CV分别为5.0%、6.0%、5.0%。与电化学发光法比对，相对平均偏倚为7.9%。回收率为91%~109%，平均97.76%。高通量ELISA测定本实验室血清AFP的参考区间为 0.00~7.20 μg/L，绘制ROC曲线，确定原发性肝癌的诊断临界值为195.7 μg/L，敏感度39.5%，特异度93.3%。

本研究所用的高通量ELISA定量检测血清AFP的标准操作规程，检测血清AFP结果稳定，可用于诊断原发性肝癌。

To analyze the methodology of serum alpha fetoprotein (AFP) assay by high-throughput enzyme-linked immunosorbent assay (ELISA) and appraise its clinical value.

A total of 170 serum samples of patients (hepatic carcinoma, hepatitis and hepatic cirrhosis patients) and 200 serum samples of healthy people were recruited from Qingdao Central Hospital from March to December 2013 in this study. The linear range, precision, accuracy, reference interval and diagnostic performance for primary liver cancer were estimated in accordance with EP5-A2 files and EP15-A2 files from the American association of Clinical Laboratory Standards Institute and related literatures. Compared with the electrochemical luminescence assay was performed to evaluated the detection accuracy and clinical value of high-throughput ELISA.

The linear range of high-throughput ELISA for AFP was 0.0-200.0μg/L. The intra-batch coefficient of variation (CV ) of different concentrations (low, medium and high) were 4.9%, 3.1%, 3.7% respectively, the inter-batch CVs of different concentrations were 3.8%, 1.5%, 2.1% respectively, while total CVs of different concentrations were 5.0%, 6.0%, 5.0% respectively. Compared with the results of the electrochemical luminescence, the relative bias was 7.9%. The recovery rate was 91%-109%, 97.76% on average. The reference interval of serum AFP detected by high-throughput ELISA in our laboratory was 0.00~7.20 μg/L. The diagnostic threshold of primary liver cancer was 195.7μg/L, with sensitivity of 39.5% and specificity of 93.3%.

This study established standard operating procedures for the quantitative detection of serum AFP by a high-throughput ELISA method. High-throughput ELISA assay for quantitation of serum AFP is an accurate, stable performance in diagnosis of primary liver cancer.