血清T4、T3和rT3测定参考系统的建立及标准化研究

Except for diabetes, thyroid disease has now become the second largest endocrine disorders in the worldwide. In China, the prevalence of thyroid disease is increasing year by year, which is nearly one-third in the entire population. It's known to all, thyroxine (T4) and 3,3',5-triiodothyronine (T3) and 3,3',5'-triiodothyronine (rT3) are the diagnosis indicators of thyroid disease, however, the laboratory test results of T4, T3 and rT3 are lack of accuracy and comparability to achieve mutual recognition of test results from different time and different places. The results from External Quality Assessment (EQA) plan in 2011 showed that the variation coefficient of T4 and T3 among laboratories were 6.1%-63.3% and 6.0%-40.8%, respectively. The results were not accurate and precise, which would affect the diagnosis and treatment of disease. So it's urgent to carry out the standardization plan to acquire the accurate and precise test results and achieve the mutual recognition among laboratories. Some preliminary experiments have been carried out to measure T4, T3 and rT3 in serum and the desired results have been achieved. Based on the data from the study, we want to establish the accurate method of serum T4 and T3, using the primary measurement principle of isotope dilution mass spectrometry, which is expected to become a candidate reference measurement procedures of serum T4 and T3. A rapid and easy LC/MS/MS method to determine the serum T4, T3 and rT3 at the same time is also planned, which is expected to become the designated comparison method for routine testing. The established methods will be used in the following aspects: Development of the serum reference materials of T4, T3 and rT3 to change the current international and domestic status of no reference material of thyroid hormone in serum medium and to be used in the calibration and accuracy quality control process. And the serum materials are expected to be enrolled as the primary reference material in China. The serum panel and quality control materials for external quality assessment (EQA) are intended to be developed. All these efforts are aimed at achieving the measurement standardization of serum T4, T3 and rT3; improving the accuracy and comparability of the routine tests in clinical laboratories; and at last providing the reliable results to physicians and patients to improve the diagnosis, prevention and treatment of thyroid diseases and related diseases.

甲状腺疾病在中国的发病率逐年上升，患病人群接近整个人群的1/3。然而用于疾病诊断的实验室测定结果却缺乏准确性和可比性，我国室间质量评价计划显示T4、T3不同方法测定值的变异分别为6.1%~63.3%和6.0%~40.8%，测定结果不准确、不精密势必会影响疾病的诊断和治疗。要实现检验结果准确测定和互认，必须建立参考系统并开展标准化计划。课题组已对血清T4、T3、rT3的测定方法进行了初步研究并取得预期结果，本课题欲进一步进行优化，建立血清T4、T3的参考测量方法。同时建立快速、简便地同时测定T4、T3和rT3的方法，有望成为常规检测的指定比对方法。并将研制T4、T3国家一级标准物质，解决目前国际上无血清基质T4、T3参考物质的现状；并开展血清T4、T3、rT3测定的标准化计划。此研究的完成将大大提高临床实验室测定结果的准确性和可比性，为甲状腺疾病及相关疾病的诊断和防治提供可靠的依据。

在我国甲状腺疾病呈逐年上升的趋势，已成为除糖尿病外内分泌领域的第二大疾病。血清甲状腺素的常规测定方法主要是免疫分析方法，这些方法快速、灵敏，能满足临床大批量样品的测定需求，但均存在交叉反应等非特异的问题，因此测定结果缺乏准确性和可比性。测定结果不准确、不精密势必会影响疾病的诊断和治疗。要保证检测结果的准确性和可比性，需要建立完善的临床检验量值溯源体系，其中包括参考方法的建立和不同级别参考物质的制备，这是实现实验室间结果互认的基础。目前国际上公认的T4和T3参考方法均为同位素稀释质谱法，参考物质是IRMM468（T4）和IRMM469（T3），均为纯度标准物质(16)。尚无用于准确性验证的血清基质的参考物质。我国在此方面为空白。.本研究采用同位素稀释质谱技术，对校准溶液的准确配制、样品制备、色谱质谱条件进行了详细的研究，建立了ID-LC/MS/MS测定血清T4和T3的候选参考测量程序，并建立了LC/MS/MS快速测定血清T4、T3、rT3的方法。建立T4候选参考测量程序多次参加IFCC组织的参考实验室比对，结果同国际参考实验室一致。将建立的T4和T3候选参考测量程序同6个常规免疫方法同时测定了120份以上临床标本，评价常规方法的校准偏差和准确性，并评价了全国室间质评物质等制备物的互通性。通过稳定性、均匀性、互通性评价和定值测量过程，研制了4种血清T4和T3候选标准物质。4种候选标准物质的研究结果显示均匀性良好，在-70℃保存至少可稳定1年。研制的血清T4标准物质均匀、稳定，定值可靠，已被批准为国家一级标准物质（GBW 09127、GBW 09128、GBW 09129和GBW 09130）。.本研究建立了血清T4和T3候选参测量程序，应用此程序评价了常规方法的准确性、制备物的互通性、研制了4个浓度水平的血清T4和T3候选标准物质，血清T4候选标准物质被批准为国家一级标准物质。这些工作的开展填补了我国在甲状腺激素准确测定方面的空白，血清基质标准物质的研制更是达到国际领先水平，可用于常规试剂的校准，将降低我国临床检验事业对进口检测技术及仪器试剂的依赖，促进民族科技与产业的发展。有望在我国甲状腺激素测定标准化方面发挥重要作用。