抗凝蛋白活性参考物质研制及结果一致化相关问题研究

发布时间：2018-03-12 06:33

  本文选题：抗凝血酶　切入点：蛋白C　出处：《北京协和医学院》2015年硕士论文　论文类型：学位论文

【摘要】：目的：抗凝血酶、蛋白C、蛋白S活性检测可用于遗传性易栓症的诊断与分型,也可用于获得性易栓症的诊断及预后判断。目前抗凝血酶、蛋白C、蛋白S活性检测无国际公认的参考方法,国际标准品不易获得,不同检测系统间结果差异不明确,且目前国内无抗凝蛋白检测规范化操作指南,影响了其在临床的应用价值。本研究通过问卷对目前国内抗凝血酶、蛋白C、蛋白s活性检测现状及存在的问题进行调查；尝试研制候选参考物质并对其均匀性、稳定性、互通性进行评价并定值；对检测系统的性能验证、不同检测系统结果的比对、校准模式等结果一致化相关问题进行研究,为促进抗凝血酶、蛋白c、蛋白s活性检测结果一致化及质量改进提供依据。方法：1.检测现状的调查研究：通过发放调查问卷,收集国内开展AT、PC、 PS检测项目的临床实验室相关检测信息,包括使用仪器、试剂、方法学信息,性能验证,标准曲线的建立及参考区间的设定,室内质控和室间质评情况等,与国外相关指南文件的要求进行比较,明确检测现状和存在问题,并针对性地提出改进措施建议。2.候选参考物质的研制与评价：选择合适的原料和制备方法,利用新鲜血浆制备2个批次3-5个批号的候选参考物质。依据ISO Guide35《标准物质/标准样品的定值——通用原则和统计原理》,CNAS-GL03《能力验证样品均匀性和稳定性评价指南》,NCCLS EP14-A《基质效应评价指南》的要求,对候选参考物质的均匀性、稳定性、互通性进行评价。依据ISO Guide35选取不同检测系统的8家实验室,以NIBSC凝血标准物质LOT4为溯源标准进行定值。将候选参考物质试应用于室内质控,同时检测进口商品质控物作为比较。3.检测结果一致化相关问题研究：①参考国外文献及指南文件,结合厂家建议及专家意见制订性能验证方案,对Stago STA-R evolution全自动血凝仪AT、 PC、PS活性检测的批内精密度、日间精密度、准确度、携带污染率、线性进行验证；②依据EP9-A2利用临床标本对3种常用AT、PC、PS活性检测系统的检测结果进行系统间的比对,明确系统间检测结果的差异并进行校准模式探讨。结果：1.检测现状的调查研究：调查结果显示国内的抗凝蛋白检测以活性检测为主,AT活性检测实验室最多(102家),PC和PS活性检测实验室数量较少(36家和31家),仅个别实验室开展抗凝蛋白抗原检测。各实验室普遍采用仪器法进行抗凝蛋白活性检测,最常用的检测系统为希森美康(Sysmex)、思塔同(Stago)和沃芬(instrument labortary, IL)及其配套试剂；各实验室定标频率不同,对于AT、PC和PS分别有40.2%、33.3%和32.3%的实验室在质控失控时定标,另有16.1%的实验室在每批次PS活性检测前定标；各实验室使用的参考区间不尽相同,仅59.8%的实验室对参考区间进行验证,对于PS仅5家(占16.1%)实验室使用按性别区分的参考区间；约42%的实验室未进行仪器性能验证；约15%的实验室不常规开展室内质控；对于AT、PC、PS分别有85.7%、75.0%和71.0%的实验室未参加任何形式的室间质评活动。2.候选参考物质的研制：采用新鲜冰冻血浆为原料,缓冲液稀释法调整AT、 PC、PS活性水平,分装成1.0ml/支置于-80℃冷冻条件下保存,最终制备出第1批次3个批号,第2批次3-5个批号(批号分别为AT/PC/PS201401～201403, AT201501～201503,PC/PS201501～201505)活性水平范围可控的候选参考物质。候选参考物质均匀性良好,评价结果显示差异无统计学意义(P0.05)；复融后在室温和冷藏条件下3种抗凝蛋白活性稳定时间分别为24h和24h、8h和12h、3h和12h；长期稳定性评价线性回归分析显示AT和PC活性检测各批次各浓度水平候选参考物质在24周观察期内稳定,正常浓度PS活性检测候选参考物质可稳定19周,异常低浓度PS活性检测候选参考物质可稳定15周(P0.05)；各批号候选参考物质在3种检测系统间均具有互通性；AT201501～AT201503批号候选参考物质的定值结果分别为：101.4±7.53、72.3±6.25和41.5±4.98；PC201501～PC201505批号候选参考物质的定值结果分别为：96.3±6.45、116.1±6.88、77.3±3.58、47.5±3.62和29.1±2.72；PS201501～PS201505批号候选参考物质的定值结果分别为：95.1±20.62、103.2±20.71、60.6±8.37、33.7±9.63和18.2±9.58。室内质控试应用结果显示,候选参考物质和商品质控品AT、PC和PS检测结果CV分布范围分别为3.02～7.39%和4.28～9.37%；3.11-4.88%和3.95-5.22%；3.53-5.88%和5.72-5.74%,均小于厂家规定的批间不精密度。3.检测结果一致化相关问题研究：①性能验证结果显示AT、PC、PS活性检测批内及日间精密度均在厂家要求范围内；准确度验证与NIBSC凝血标准物质靶值间的偏倚分别为-1.09%、9.78%和-3.09%；携带污染率分别为1.37%、0.0%和2.15%；线性回归方程相关系数R2均0.99,AT活性检测在23-128%区间内线性验证通过,PC活性检测在30-150%区间内线性验证通过,PS活性检测在25-126%区间内线性验证通过。②可比性研究结果显示对于AT活性检测,两两检测系统间检测结果相关性和可比性可接受；对于PC活性检测,两两检测系统间检测结果相关性和可比性好；对于PS活性检测,第1批次可比性评价结果IL与Sysmex两系统间检测结果相关性可接受,Stago与上述两系统间检测结果相关性好,两两检测系统间检测结果可比性可接受,第2批次可比性评价IL与Sysmex两系统间检测结果相关性可接受,Stago与上述两系统间检测结果相关性差,两两检测系统间检测结果可比性差。使用LOT4模拟定标曲线将比对样本检测结果进行转化后,AT活性检测IL与Sysmex系统间差异略减小,Stago与另外两系统间差异增大；PC活性检测IL与另外两系统间差异略减小,Stago与Sysmex系统间差异增大；PS活性检测2次系统间可比性结果均呈增大趋势。结论：1.问卷调查结果显示与国外相关技术指南的要求进行比较,国内实验室在抗凝蛋白活性检测的关键技术环节质量控制方面仍有较大的改进空间,应对实验室检测人员进行系统化技术操作、定标、参考区间验证、性能验证及质量控制培训,并开展室间质量评价。2.利用新鲜冰冻血浆制备AT,PC和PS候选参考物质,其均匀性和稳定性良好,在3种主流检测系统间具有互通性,并完成了8家实验室包含3种主流检测系统的联合定值。候选参考物质与商品质控物室内质控试应用CV及变化趋势具有可比性,表明其可以应用于日常室内质控。3.①对我室规范操作检测系统进行性能验证,验证结果符合厂家及相关文件的要求,性能验证方案可为其他检测系统的性能验证提供参考。②对于AT活性检测具体的不同试剂间诊断敏感性差异还需进一步选择有明确AT Ⅱ型Cambridge缺陷和Ⅱ型HBS缺陷患者的血浆加以比对；对于PC活性检测3种系统的精密度,可比性及相关性均较好；PS的评价结果存在批间差异,利用LOT4的模拟定标曲线校准检测结果的模式可能对于改善系统间检测结果的一致性并无帮助。
[Abstract]:Objective: antithrombin, protein C, protein S activity can be used in the diagnosis of hereditary thrombophilia and type, also can be used to get to the diagnosis and prognosis of embolism. The antithrombin, protein C, protein S activity was detected without reference to the internationally recognized methods, the international standard is not easy to obtain, different detection the difference between the system is not clear, and the current domestic non operation guide specification for the testing of anticoagulant proteins, affect its clinical application. This research through the questionnaire on the current domestic antithrombin, protein C, protein S activity was detected to investigate the status quo and problems of development; the candidate reference material and the uniformity, stability of try interoperability, evaluation and value; to verify the performance of the detection system, the detection results of different alignment system, calibration model results studied related issues, to promote the anti thrombin protein, C The activity of S protein, consistent with results and provide the basis for quality improvement. 1. detection methods: investigation situation: through the issuance of questionnaires, collected to carry out AT, PC, PS detection project related information detection in clinical laboratories, including the use of instruments, reagents, methods of information, performance verification, establishment of standard curve and reference interval setting, internal quality control and external quality control etc., comparing with foreign related document requirements, clear detection situation and existing problems, and puts forward the improvement measures of.2. development and evaluation of candidate reference materials: selection of raw materials and preparation methods, the use of fresh plasma preparation 2 batches of 3-5 batches of candidate reference materials. According to the ISO Guide35< standard / standard sample value -- General principles and statistical principle > CNAS-GL03<, the homogeneity and stability of samples used The Evaluation Guide >, NCCLS EP14-A< matrix effect evaluation guide >, the candidate reference material uniformity, stability, evaluation of interoperability. Based on 8 different Guide35 ISO laboratory detection system, using NIBSC coagulation standard substance LOT4 as traceability standard was determined. The candidate reference material for internal quality control test. Simultaneous detection of imported goods quality control materials as compared.3. results consistent related issues: the research references and guidance documents, combined with the manufacturer's recommendations and expert opinion for performance verification scheme, the Stago STA-R evolution automatic blood coagulation analyzer AT, PC, PS activity detection within batch precision, precision, accuracy and the contamination rate, linear to verify; according to EP9-A2 using clinical specimens of 3 kinds of commonly used AT, PC, PS activity detection system the ratio between the system, clear the system The detection results of the differences and discuss the calibration mode. Results: 1. study the status quo: the survey results show that the detection of anticoagulant protein detection in domestic activity detection, AT activity detection laboratory at most (102), the laboratory detection of PC and PS activity number (36 and 31), only individual laboratory testing of anticoagulation protein antigen. Each laboratory commonly used instrument to detect anticoagulant protein activity, the most commonly used detection system for Sysmex (Sysmex), Scarlett tower (Stago) and walfen (instrument labortary, IL) and reagents; each laboratory calibration of different frequencies, for AT, PC and PS were 40.2% and 33.3%. 32.3% out of control in laboratory calibration, and another 16.1% of the laboratory in the detection of each batch of PS activity before calibration; the laboratory reference interval is not the same, only 59.8% of the laboratory reference interval of inspection For the PS card, only 5 (16.1%) laboratory using gender differentiated reference interval; about 42% of the laboratory without instrument performance verification; about 15% of the laboratories do not routinely carried out indoor quality control; for AT, PC, PS were 85.7%,.2. candidate reference materials development activities 75% and 71% did not participate in the laboratory any form of EQA: using fresh frozen plasma as raw material, the buffer solution method to adjust AT, PC, PS activity, 1.0ml/ branch in -80 C packed under the condition of freezing preservation, was prepared from first batches of 3 batches, second batches of 3-5 batches (batch number: AT/PC/PS201401 - 201403, AT201501 ~ 201503, PC/PS201501 ~ 201505) candidate reference material of controllable range. The activity level of the candidate reference material of good uniformity, the evaluation results showed no statistically significant difference (P0.05); after melting at room temperature and refrigerated conditions of 3 kinds of anti Protein activity and stability time were 24h and 24h, 8h and 12h, 3H and 12h; long-term stability evaluation of linear regression analysis showed that AT and PC activity detection in each batch of the levels of candidate reference material in the 24 week observation period, the normal concentration of PS and activity detection of candidate reference materials can be stable for 19 weeks, abnormal low concentration PS activity detection of candidate reference materials can be stable for 15 weeks (P0.05); the number of candidate reference materials in 3 kinds of detection system with interoperability; AT201501 ~ the fixed value of AT201503 number candidate reference materials respectively: 101.4 + 7.53,72.3 + 6.25 and 41.5 + 4.98; PC201501 ~ the fixed value of PC201505 number of candidate reference material respectively: 96.3 + 6.45116.1 + 6.88,77.3 + 3.58,47.5 + 3.62 and 29.1 + 2.72; PS201501 ~ the fixed value of the reference material of PS201505 batch candidate respectively: 95.1 + 20.62103.2 + 20.71,60.6 + 8.37,33.7 + 9.63 and 18.2 + 9.58. internal quality control test results showed that the candidate reference material and commodity quality control products AT, PC and PS detection results of CV distribution range was 3.02 ~ 7.39% and 4.28 ~ 9.37%; 3.11-4.88% and 3.95-5.22%; 3.53-5.88% and 5.72-5.74%, were less than specified by the manufacturer of inter batch fine density of.3. test results consistent correlation question: verification results show the performance of AT, PC, PS activity detection of intra and inter day precisions were in the range of accuracy requirements of manufacturers; verification and NIBSC coagulation standard material target value between bias were -1.09%, 9.78% and -3.09%; carry pollution rate were 1.37%, 0% and 2.15%; linear regression equation the coefficient of R2 was 0.99, AT activity detection linear in the range of 23-128% verification by PC activity detection linear in the range of 30-150% verification by PS activity detection linear in the range of 25-126% is verified by the comparison research. The results show that for AT activity detection, the correlation between results and comparability of acceptable 22 detection system; to detect the activity of PC, the correlation between results and good comparability between the 22 detection system; to detect the activity of PS, first batches of comparability of evaluation results of IL and Sysmex between the two systems the correlation between results of acceptable. The results of Stago and the correlation between these two systems is good, can the test results of the 22 detection system than acceptable, second batches of comparable IL and Sysmex evaluation system between the two detection results of acceptable correlation, Stago test results and the correlation between the two systems, 22 detection system detection results in poor comparability. LOT4 simulation of calibration curve will sample detection results are transformed, the difference of AT activity detection of IL and Sysmex system is slightly reduced, Stago increased with the addition of two system the difference between IL and PC; activity detection Another difference between the two systems is reduced, and the difference between Stago Sysmex system increased; the activity of PS was detected in 2 system comparable results were increased. Conclusion: the 1. survey results show that the guide and foreign relevant technical requirements, quality control of key technology of domestic laboratory detection in anticoagulation protein there is still much room for improvement, with laboratory personnel to operate technology, system calibration, reference interval verification, performance verification and quality control training, and carry out the external quality assessment of.2. using fresh frozen plasma preparation of AT, PC and PS of candidate reference material, its good uniformity and stability, with interoperability in 3 kinds of detection system, and completed the combined value of 8 laboratories contains 3 kinds of mainstream detection system. A candidate reference material and commodity quality control material internal quality control test and the trend of application of CV 鍏锋湁鍙�姣旀�，

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