【作者】 张顺利；

【导师】 陈文祥；

【作者基本信息】 北京协和医学院 ， 临床检验诊断学， 2014， 博士

【摘要】 目的研究常用蛋白类检验项目主要检验方法检测结果的可比性,为我国相关检验项目标准化和实验室检测的有效临床应用提供科学依据；研究常用蛋白类检验项目可能参考物质的互通性,为我国相关检验项目室间质评计划、标准化计划等遴选可能的参考物质。方法将在两医院检验科收集的病人剩余血血清盘(含50或75个样本)和相应的可能参考物质分成5-9份,将可能参考物质随机插入血清盘中,统一标签后分发。根据不同的项目,每个样本检测2次或3次。涉及可能的参考物质包括：(1)新鲜冰冻混合人血清(HSP),由人剩余血清汇集而成。(2)国际标准物质,制作方法按照说明书。(3)室间质评物,按照室间质评计划的使用方法制备。(4)纯化物质,购自清华长三角有限公司,提取自人的组织液。(5)动物血清,来自屠宰场和模型动物实验室。(6)人胸水,由检验科直接收集。本研究包括的检验项目共22项,分别是：IgA、IgG、IgM、C3、C4、PA、β2M、Cystatin C、RF、ASO、TRF、CRP/HsCRP、 cTnI、CK-MB、MYO、TSH、PSA、AFP、CEA、CA125、CA153和CA199;涉及的检验方法共27种,分别是：进口A1-A4、进口B1-B3、C1、C2、D1-D3、E、F、 G, H、I、J、国产A1、A2、B、C、D、E、F、G和H。对某一具体项目,剔除不完整结果和离群值后进行统计分析：(1)相关性与可比性分析,根据两两比较的最小二乘法得到平均值剩余标准差(Sy.x)。将某方法与其他方法的2倍Sy.x与血清盘中位值的平均百分比(R)与生物学变异度导出的最低允许总误差(mTEa)相比较作为判断依据,同时参照散点图,按项目将方法间相关性分为好、中、差。若多数方法R小于mTEa,则认为方法间的相关性好,剔除少数R>mTEa的方法后进行相关性分析；若多数方法R略大于mTEa,则认为方法间的相关性一般,则剔除R值异常大的方法；若多数方法R远大于mTEa,则认为方法间的相关性差,仅对R值较小的方法进行相关性分析；对以上进入相关性分析的方法,考察其可比性。以各方法2倍均值的CV与mTEa相比较,如果前者小于后者,则认为方法间具有可比性。(2)互通性：采用C53的方案,对浓度范围窄的项目(IgA、IgG、C3、C4、PA和TRF),直接进行可能参考物质互通性评价；对浓度范围宽的项目(IgM、p2M、Cystatin C、RF、ASO、CRP/HsCRP、cTnl、CK-MB、 MYO、TSH、PSA、AFP、CEA、CA125、CA153和CA199), log转化后进行互通性评价。结果1.相关性和可比性依照相关性同时参照散点图,可将所有项目分成3类。第1类,多数方法间相关性好(R小于mTEa),包括11项,即：IgA、IgG、IgM、C3、C4、PA、β2M、 Cystatin C、TRF、CRP/HsCRP和MYO,其中IgA、IgM、PA、CRP/HsCRP等4项结果方法之间具有可比性,其余7项某一(些)方法存在系统误差,结果不可比。第2类,多数方法的相关性一般(R略大于mTEa),包括9项,即：ASO、cTnI、 CK-MB、TSH、PSA、AFP、CEA、CA125和CA153,其中CK-MB和PSA结果方法之间具有可比性,其余7项方法之间结果不可比。第3类,多数方法间相关性差(R远大于mTEa),包括2项,即：RF和CA199。RF涉及的进口B2、进口D3和国产D之间相关性好,方法间结果不可比；而CA199涉及的进口B4、进口D2、进口E、国产A1之间相关性好,结果可比。2.参考物质互通性在评估的22个项目中,新鲜冰冻混合人血清(HSP)方法间几乎全有互通性；欧洲标准物质ERM470k、471和474包含的IgA,IgG, IgM, C4, PA, TRF, CRP和Cystatin C所有方法间全有互通性,但在C3仅部分组合有互通性；SRM2921(cTnI)仅部分组合有互通性；采用生理盐水和牛血清白蛋白稀释的国际标物NIBSC81/565(TSH)全有互通性,但是采用人血清和猪血清稀释时,互通性不足；采用生理盐水,牛血清白蛋白稀释的国际标物NIBSC96/670(PSA)互通性不足。EQA物质包含的IgM, PA, β2M, TRF, CRP, MYO, TSH, AFP, CEA和CA153全具有互通性,其余项目仅部分互通或全不互通。人血清稀释长三角物质在PA, β2M, CA125有互通性,在PSA部分有互通性。猪血清(包括心肌梗死猪和屠宰猪)包含的IgG, IgM, cTnI, CK-MB和MYO方法间不具有互通性,而IgA, C3, C4和PA所有检测试剂对其均无反应性。大鼠血清包含的cTnI, MYO仅少部分组合有互通性,对CK-MB无反应性。采用猪血清稀释的人高值血清包含的β2M和RF有互通性,而PA, Cystatin C, CRP/HsCRP, TSH, PSA和CA125部分组合具有互通性。人血清或猪血清稀释的人CA125高值胸水具有互通性。结论蛋白类检验项目不同检验方法结果可比性依检验项目而异,部分检验项目主要检验方法结果无明显差异,部分项目检验方法存在明显校准偏倚,另有部分项目检验方法存在特异性问题；不同类型参考物质表现不同互通性,新鲜冰冻混合人血清对各检验项目和各检验方法表现良好互通性。各种制备物和动物血清在不同程度上缺乏互通性。针对不同的情况选择适宜参考物质,建立适宜检验质量监测和改进机制,将是蛋白类检验项目标准化下一步工作任务。更多还原

【Abstract】 ObjectiveTo investigate thecomparabilityof measurement results for serum proteins and to provide the scientific evidence for effective use of the measurement results and for standardization of serum protein measurements; To evaluate the commutability of possiblereference materials and to select candidatereference materials for EQA scheme and standardization programs.MethodSerum panels, which consisted of50or70individual patient specimens, were collected at the clinical laboratoriesof two Beijing Hospitals. Possible reference materials were randomly interspersed among thepatient specimens, and all of which were aliquoted into5-9vials andthen unified labeled. Each specimen was analyzed in duplicate or triplicate based on the measurand. The possible reference materials included:(1)Fresh frozen serum pools(HSP), pooled byleftover sera of patient samples.(2) International standard materials, prepared following manufacturer’s instructions.(3)EQA materials, prepared following our EQA instructions.(4) Purified materials, purchased from YangtzeDeltaRegionlnstituteofTsinghuaUniversity.(5)Animal sera, collected from an abattoir or model animals.(6) Pleural effusion, collected at department of clinical laboratory of xx hospital. A total of22measurands(ie, IgA, IgG, IgM, C3,1C4, PA, β2M, Cystatin C, RF, ASO, TRF, CRP/HsCRP, cTnI, CK-MB, MYO, TSH, PSA, AFP, CEA, CA125, CA153and CA199) and27analytical reagents (ie, imported A1-A4, B1-B3, Cl, C2, D1-D3, E, F, G, H, I and J, and domestic reagents A1, A2,B,C,D,E,F,G andH) were evaluated.For eachmeasurand, Correlations and comparabilities between/among methods were analyzed after outliers and incomplete data were excluded. Correlations were evaluated based on scatter plots and the differences between the ratio [R,2times of residual standard deviation(Sy.x) to median of the serum panel]and minimum allowable total error (mTEa) derived from biological variations. Measurands were divided into3groups and correlations were analyzed accordingly. If R<mTEa formost the methods, correlations were regarded to be "good"and methods withR> mTEa were deleted from correlation analysis. If R was slightly higher than mTEa, correlations were designated to be "average" in which methods with extremely high R values were excluded. Correlations were"poor" if most of the methods had R values significantly higher than mTEa, and in this case, only methods with relatively low R were analyzed. Furthermore, comparability analysis was done for all the above methods thatwere included in the correlation analysis.Method comparabilities were evaluated based on the difference between2times of CV derived fromthe mean of assays and mTEa. If2CV was higher than mTEa, the results among assays were regarded to be consistent. In addition, commutabilities of possible reference materials were evaluated according to C53-A protocol. Deming regression was applied directly for measurands with narrow measurement ranges (such as, IgA, IgG, C3, C4, PA and TRF) and performed after the data were logarithmic transformed for those with wide ranges (IgM, β2M, Cystatin C, RF, ASO, CRP/HsCRP, cTnI, CK-MB, MYO, TSH, PSA, AFP, CEA, CA125,CA153and CA199).Results1. Correlation and comparabilityBased on the correlation results and scatter plots, the measurands were divided into3groups:(1) Measurands with good correlations(R<mTEa) among most of the methods, includingIgA, IgG, IgM, C3, C4, PA, β2M, Cystatin C, TRF, CRP/HsCRP, CK-MBand MYO, in which IgA, IgM, PA and CRP/HsCRP had consistent results among different methods. The rest7measurands showed systematic biases in one or more methods and the results were inconsistent.(2)Measurands with R> mTEa for most of the methods, includingASO, cTnI, CK-MB, TSH, PSA, AFP, CEA, CA125and CA153, in which CK-MB and PSA, but not the rest of the7measurands, had consistent results among methods.(3) Measurands with poor correlations among most of the methods (R> mTEa),including RF and CA199. However, for RF, correlations were desirable among imported B2, D3and domestic Dalthough results were inconsistent. For measurands CA199, correlations and comparabilities were desirable amongimported B4, D2, E and domestic Alssays.2. Commutability of potential reference materials Human fresh frozen serum pools(HSP) were commutablefor all pair comparisons for22evaluated measurands. International reference materials,ERM470,471and474, were commutableamong all pair comparisonsfor IgA, IgG, IgM, C4, PA, Cystatin C, TRF, andCRP, but not for C3. However, SRM2921only showed commutability for certain combinations of methods.NIBSC81/565wascommutable when diluted with saline and bovine serum albumin,but lack of commutability when diluted with human or swine serum. PSA reference materialNIBSC96/670, diluted with saline and bovine serum albumin, did not showcommutability for some combinations. EQA materials were commutableinall pair comparisonsforlgM, PA, B2M, TRF, CRP, MYO, TSH, AFP, CEA and CA153assays. Purified materials were commutable for PA, β2M and CA125but not for PSA. Swine sera collected from an abattoir or model swineswith acute myocardial infarction (AMI)were incommutable for IgG, IgM, cTnI, CK-MB and MYO. Moreover, swine serashowed no reactivity to IgA, C3, C4and PA assay kits. Rat sera collected form AMI rats showed markedly decreasedcommutability for cTnI and MYO among certain combinations, and it showed no reactivity to all CK-MB kits. Highconcentration human sera diluted with swine sera were commutable only for β2M and RF, but not forPA, Cystatin C, CRP/HsCRP, TSH, PSA andCA125.Pleural effusion diluted with human or swine sera showed commutability for CA125.ConclusionsThe comparabilities of protein results were variable depending on different measurands. Some proteins hadconsistent results among most of the methods, some showed significant calibration biases, while some others had analytical methods that not so specific.Different reference materials showed different commutabilities. HSP was commutable for all the evaluated measurands and methods. Other prepared materials and animal sera were lack of commutability in varying degrees. Therefore, according to different situations, selections of proper reference materials, and establishmentof effective quality monitoring and improving mechanisms, will be important tasks in the standardization of protein measurements in the future.更多还原