目的:研制同型半胱氨酸冰冻人血清国家标准品,用于血清中同型半胱氨酸检测的正确度评价及标准化研究。方法:收集含同型半胱氨酸的外观清澈、无溶血黄疸和乳糜的人血清,经多重滤过及除菌后分装处理,制备成正常(水平Ⅰ)和异常(水平Ⅱ)两水平的标准品,放置于-70 ℃或以下保存。标准品的均匀性采用单因素方差分析法评估;稳定性采用线性回归分析法评估。选择4家实验室采用同位素稀释质谱参考方法定值,同时评估标准品的不确定度及互通性。结果:共制备了2900套同型半胱氨酸标准品。均匀性评估中,水平Ⅰ和水平Ⅱ的F值分别为1.3749和1.1269,均小于F0.05(1.5117);水平Ⅰ在(20~25) ℃、(2~8) ℃和-20 ℃均至少能稳定30 d;水平Ⅱ在(20~25)、(2~8)及-20 ℃分别能稳定3、10和30 d。水平Ⅰ、水平Ⅱ定值结果分别为(10.81±0.30) μmol·L-1 (k=2)、(32.11±0.60) μmol·L-1 (k=2)。水平Ⅰ、水平Ⅱ测定值均在临床血清样本测定值所建立的回归直线95%置信区间范围内,表明互通性良好。结论:该标准品定值准确,均匀稳定且互通性良好,可作为国家标准品使用。该同型半胱氨酸血清基质国家标准品的建立,对促进同型半胱氨酸量值溯源及同型半胱氨酸检测试剂盒的质量评价具有重要意义。
Objective: To develop the national standard material of homocysteine in frozen human serum, to evaluate the accuracy and to promote standardization of homocysteine detection in serum. Methods: Human serum containing homocysteine with clear appearance and without hemolytic, jaundice or chyle were collected. After multiple filtration and sterilization, the samples were prepared into normal (level Ⅰ) and abnormal (level Ⅱ) standards and stored at-70 ℃ or below. The homogeneity for standards was evaluated by one-way ANOVA, and the stability was evaluated by linear regression analysis. The values for standards were assigned with reference method of isotope dilution mass spectrometry by four laboratories. The uncertainty and interoperability of standard were also evaluated. Results: 2900 sets of homocysteine standard were prepared. The F values of homogeneity for level Ⅰ and level Ⅱ were 1.3749 and 1.1269, respectively. All of them were less than F0.05 (1.5117). Level Ⅰ is stable for at least 30 d at (20 -25) ℃, (2-8) ℃ and-20 ℃. Level Ⅱ is stable for 3 d, 10 d and 30 d at (20-25) ℃, (2- 8) ℃ and -20 ℃, respectively. The assigned value of level Ⅰ and level Ⅱ were: (10.81±0.30) μmol·L-1 (k=2) and (32.11±0.60) μmol·L-1 (k=2) respectively. The measured values of level Ⅰ and Ⅱ were all within the 95% confidence interval of the regression line established by the measured values of clinical serum samples, which showed good commutability. Conclusion: The standard of homocysteine is accurate, uniform and stable, and can be used as national standard. The establishment of homocysteine serum standard is of great significance to promote the traceability of homocysteine and the quality evaluation of homocysteine kit.
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