血清睾酮和雌二醇参考系统的建立研究

Serum testosterone and estradiol are one of the most important clinical endocrine measurements, and the prevention, diagnosis and treatment of many related diseases are dependent on the reliability of their test results. However, at present, the laboratory test results of testosterone and estradiol are lack of accuracy and comparability to achieve mutual recognition of test results from different time and different places, which would affect the diagnosis and treatment of disease. So it's urgent to carry out the standardization program to get the accurate and precise test results and achieve the mutual recognition among laboratories. The most effective way to resolve this problem is to establish the reference system and to realize the measurement standardization. Due to the extremely low level of testosterone and estradiol in vivo, these reference methods are very difficult to develop, which led to very low progress in this field. This program will develop the testosterone and estradiol reference system. The measurement procedure will performed based on isotope dilution liquid tandem mass spectrometry. We aim to develop a brand new sample preparation method - lipoprotein precipitation, combined with pre-column derivatization, to make sure a result with high sensitivity and high specifity. Some preliminary experiments have been performed and the desired results have been achieved. The development of the serum reference materials of testosterone and estradiol is an important solution to change the current international and domestic status of no reference material of testosterone and estradiol in serum. Thus, the reference materials and quality control materials for external quality assessment are also intended to be developed. And these serum materials are expected to be enrolled as the primary reference material in China. All these efforts are aimed at achieving the measurement standardization of serum testosterone and estradiol and improving the accuracy and comparability of the routine tests in clinical laboratories. And eventually, it will help to provide the reliable serum testosterone and estradiol results to physicians and patients, and help to improve the diagnosis, prevention and treatment of related diseases.

血清睾酮与雌二醇是临床重要的内分泌检测项目之一，多种相关疾病的预防、诊断和治疗都依赖于其检测结果的可靠性。然而目前这两个项目常规检测方法的准确性和可比性尚不理想，存在诸多问题。解决这些问题最有效的方法是开展量值溯源，建立参考系统。由于睾酮和雌二醇体内水平极低，参考系统建立的难度大，国内外相关研究一直进展缓慢。本项目将进行睾酮和雌二醇参考系统的相关研究，包括参考方法的建立和参考物质的研制。本项目采用先进的同位素稀释串联质谱法为拟建参考方法的基本分析原理；在以往相关激素测定研究的扎实工作基础上，首次提出全新的脂蛋白沉淀-提取样品前处理方法，并结合柱前衍生等新技术方法，使所建方法简便、可靠、具有高灵敏度和高特异性，以解决原有方法样本量大，操作极其复杂，费用高昂，难于维持运行等问题。本项目还针对目前国际上雌二醇项目及国内睾酮项目无冰冻血清基质参考物质的现状，研制雌二醇和睾酮标准物质，以解决上述项目无源可溯及有源难溯的问题。本项目的完成将为睾酮和雌二醇检测标准化提供必要的工作基础，并为常规方法检测结果的质量控制，实现结果互认提供有力的支持。

血清睾酮和雌二醇测定是临床上常用的甾体激素检测项目之一，多种相关疾病的诊断和治疗都依赖于其检测结果。但由于校准和方法特异性等原因，目前其临床常规测定方法在实际应用中还存在一些问题，造成结果准确性和可比性不理想。实现临床检验标准化，其核心方法就是开展量值溯源，建立参考系统。参考系统的基础是参考方法和参考物质。睾酮和雌二醇等甾体激素由于其浓度低，干扰多，国际上对其参考系统的研究进展缓慢。 以往国内在临床检验睾酮和雌二醇项目参考方法建立和标准物质研制方面均为空白，存在既无可靠参考方法也无标准物质、无源可溯的问题。.本项目以同位素稀释液相色谱串联质谱法为测量原理，建立了准确精密的血清睾酮和雌二醇候选参考测量程序。课题研究了测量过程的各个环节，包括标准溶液的配制，样品前处理的方案，衍生化方法、干扰研究和色谱和质谱条件等。所建方法使用羟丙基-β-环糊精提取的方法用于血清睾酮的前处理，使用脂蛋白沉淀-提取及丹磺酰氯衍生的方法用于雌二醇的样品前处理，极大地优化了分析过程，缩短了分析时间。血清睾酮测定方法的批内和总变异系数范围分别为0.92 %～1.9 %和0.96%～2.8 %，血清雌二醇为0.29%～1.58 %和0.72%～2.87%。两个方法参加相关项目的国际比对研究RELA，取得满意结果。.本项目成功研制了包括血清睾酮和雌二醇项目的冰冻人血清甾体激素国家一级标准物质。该套标准物质包括三个浓度水平，以新鲜冰冻血清为基础原料。考察了标准物质的稳定性、均一性和互通性，并用本项目建立的同位素稀释串联质谱法为标准物质确定靶值。经过国家标准物质委员会的评审，批准为国家一级标准物质。. 综上，本项目建立了可靠的血清睾酮和雌二醇候选参考测量程序，使用已建立的方法进行定值，研制了三个浓度水平的血清甾体激素国家一级标准物质。本项目的完成解决了我国临床检验领域睾酮和雌二醇项目多年来没有可靠准确定量方法以及血清标准物质的问题，为这两个项目的量值溯源体系的建立和标准化提供了重要工作基础。