TSH互通性参考物质多系统赋值研究及其一致化

The international reference materials（TSH, WHO NIBSC 81/565) are freeze-dried powers, whose structure and antigen epitopes may be changed during production process. Nearly all the manufacturers claimed their calibrators were traced to these materials, but the antibodies they used were different. These antibodies have different affinity with the changed epitopes, which can be detected by “Commutability”. The diluents have different impact on the affinity among antigen epitopes and corresponding antibodies. The application of the antibody pairs which have appropriate combined capability with TSH molecules can pass the accurate value to targeted reference materials. Until now, the commutability of these freeze-dried materials was confined to “yes” or “no” among assays, the roles of "noncommutable" WHO materials have not elucidated. The reference materials whose values were assigned using “dilution method” included 470k, cystatin C, et al which were measured based on turbidimetry or nephelometry. The value of TSH reference materials which was measured based on chemiluminescence using“dilution method” have not reported. The aim of this study is to find the routine methods and commutable WHO materials diluted wich appropriate matrix, so as to provide correct value for targeted reference materials. Besides, the EQA materials which were commutable can identify the true difference and analytical specificity among methods, so as to prompt the manufactures which performed bad to improve their calibrators or reagents.

当前绝大多数TSH试剂生产商声称其校准品溯源到国际标准物质（WHO NIBSC 81/565）。生产商在溯源过程所使用的抗体和稀释液不尽相同，这可能是造成系统偏倚的重要原因之一。 课题组前期对TSH互通性的初步研究中，发现WHO NIBSC 81/565在不同基质中互通性不同。据此我们认为在WHO NIBSC 81/565制作过程中TSH分子结构及抗原表位可能发生了某种变化，使某些生产商不能正确的溯源，系统检测结果存在差异。我们通过选择适当的检测系统和稀释液，使其在多系统间具有互通性且在各系统上成线性。利用多系统将国际标准物质的值准确转移到目标参考物质（新鲜冰冻混合人血清）。进而将此参考物质应用于北京市临床检验中心开展正确度验证和一致化改进计划。此研究的完成将帮助提高TSH临床实验室检测结果的准确性和可比性，并为其他蛋白类检验项目的定值和一致化提供新思路。

量值溯源是使检测结果准确有效的重要手段和基本保证，对于TSH这类没有参考测量程序的蛋白类检验项目只有采用多系统将国际标准物质（WHO IRP 81/565）的值转移到TSH互换性目标参考物质（tRM），但国际标准物质本身的互换性及对多系统赋值的意义未知。我们首先采用11种基质将国际标准物质稀释为2-3个适当的浓度，再将之随机插入真实患者血清盘中，获得了国际标准物质在不同基质中的互换性信息。然后采用这些基质稀释的国际标准物质为11个浓度水平的tRM赋值，明确了只用采用使国际标准物质在不同系统间具有互换性的基质作为稀释液才能使tRM的赋值达到一致。采用生物学变异度和统计学方法剔除离群值后，经正态性检验多系统赋得的值符合正态性，最后取均值得到10种检测系统13种方法的11个TSH tRM赋值和扩展不确定度（包括均匀性和长期稳定性），从0.187±0.027μIU/mL到44.985±3.366 μIU/mL。本研究表明使国际标准物质具有互换性的重要意义在于能使tRM赋值达到一致，为TSH多系统赋值提供了新方法和思路；既往的研究表明采用互换性tRM（如酶学、CRP等）对检测仪器进行校准，实验室间检测结果的差异明显减小。但对于封闭系统，因为产品校准品的值是各厂商内部确定的，不能直接采用tRM，本研究然后采用称重法准确配置了6个厂家的专用校准品，采用22个低中高TSH浓度患者个体血清，使系统间的斜率从校准前的0.738-1.128变为校准后的0.954-1.053。以基于生物学变异度的中等允许总误差（23.74%）要求，校准前结果一致的比率为81.1%，校准后为94.7%，差异具有统计学意义(卡方值=11.52，P=0.0007)。室间质量评价（EQA）是监测实验室检测质量的重要工具，目前我国使用的TSH质评品存在互换性问题。本研究采用29个低中高TSH浓度患者冰冻血清在校正8个检测系统的系统偏差后得到系统之间真实的可比性关系，斜率从0.801到1.299。随后，我们校正了EQA物质互换性相关的偏倚，采用国家卫生健康委临床检验中心近3年的数据，得到了相似的比例关系，斜率从0.735到1.254。此项研究成果为EQA组织者采用非互换性EQA物质分析系统间可比性提供重要参考。