基于质谱法原理的血清糖化白蛋白参考测量体系的建立及其在糖尿病诊断中的应用价值研究

As a threat to the global human health, diabetes mellitus is one of the most important non-communicable diseases. Blood glucose monitoring plays an important role in diabetes management. Glycated albumin is more effective to reflect the change of blood glucose than HbA1c, the accurate and reliable results can provide important basis for clinical evaluation of curative effect and treatment. Because the standardization of domestic GA is not enough, there are quite differences among various methods, thus it is urgent to establish the reference measurement method and ensure the traceability of test results. Based on the previous research projects, we will attempt to establish improved reference measurement methods for GA by isotope dilution liquid chromatography/tandem mass spectrometry. We will firstly choose MRM detection mode, use 2H and 13C respectively as internal standards of lysine and DOF-lys, which can ensure the proposed methods are accurate and reliable. At the same time, we will also develop serum standard reference materials of GA, which can solve the problem of metrological traceability. Then glycated albumin standard material will be applied to EQA and accuracy verification plan to realize the consistency and mutual recognition of accurate detection results. On this basis, the establishment of reliable cut-off value in patients with diabetes and abnormal glucose tolerance will be useful to improve the diagnosis rate of DM. The completion of this project will greatly improve the clinical laboratory quality of GA in our country, and finally develop its application value in the diagnosis of diabetes mellitus.

糖尿病是当前威胁全球人类健康最重要的非传染性疾病之一，血糖监测对于糖尿病管理至关重要。糖化白蛋白（GA）较HbA1c能更及时地反映血糖的变化，其准确、可靠的实验室检测结果，能为临床评估疗效、调整治疗提供重要依据。由于目前国内GA标准化程度不够，不同试剂方法的结果差异较大，建立参考测量方法、保证检验结果的溯源性已成为迫切需要。本项目在以往研究工作基础上，首次采用三重四级杆质谱MRM模式检测，建立基于同位素稀释质谱法原理的GA参考方法，选用氘及13C标记的赖氨酸及糖化赖氨酸为内标，保证拟建方法的准确、可靠。同时首次研制国内血清基质的GA标准物质，有效解决量值溯源传递问题，并应用于室间质量评价和正确度验证，最终实现该项目在准确科学基础上的互认。在此基础上，建立糖尿病及糖耐量异常人群的真实可信的诊断切点，提高糖尿病的诊断率。该项目完成将大大改进我国GA临床检验质量,发挥其在糖尿病诊断中的应用价值。

血糖监测对于糖尿病管理至关重要，糖化白蛋白（GA）较HbA1c能更及时地反映血糖的变化，能为临床评估疗效、调整治疗提供重要依据。本项目在以往研究工作基础上，首次采用三重四级杆质谱MRM模式检测，建立了基于同位素稀释质谱法原理的GA参考方法，同时首次研制国内血清基质的GA标准物质，有效解决量值溯源传递问题，并应用于室间质量评价和正确度验证，最终实现该项目在准确科学基础上的互认。.在实验前处理方面，分别采用了三种不同方法提纯白蛋白，分别是阴离子交换色谱法，SDS-PAGE电泳法，亲和层析联合SEC法，其中亲和层析联合SEC法经HPLC测纯度可达98%，提纯的白蛋白纯品经LC-MS测量计算得到GA值，并对标准物质定值。因目前国内外尚无商品化的糖化赖氨酸、13C标记糖化赖氨酸纯品，为保证实验准确性，研制了糖化赖氨酸和13C标记糖化赖氨酸纯品，经HPLC纯度分析均可达到99%。对建立的糖化白蛋白参考方法进行方法学考察，以本研究方法测定三种不同混合浓度的血清三天，精密度范围在0.99%-2.51%；JCCRM611-1标准物质评价其准确度，范围在2.27%-3.2%；参考方法和常规系统测定糖化白蛋白线性相关系数R2>0.975。采用单因素方差分析法评价候选标准物质的均匀性，三个水平的F值为1.07、1.67、1.79均小于F0.05(14,30)；其稳定性在-80℃,-20℃，冷藏（2-8℃），室温（20-25℃），37℃分别至少稳定180天、22天、7天、2天、8小时。2017年和2018年糖化白蛋白三个水平的正确度验证通过率分别为46.6%，53.3%，53.3%；75%，70%，60%。.本项目通过建立可靠的参考方法，建立糖尿病及糖耐量异常人群的真实可信的诊断切点，提高糖尿病的诊断率。该项目完成将大大改进我国GA临床检验质量,发挥其在糖尿病诊断中的应用价值。