本文選題：抗凝血酶　切入點：蛋白C　出處：《北京協(xié)和醫(yī)學院》2015年碩士論文　論文類型：學位論文

【摘要】：目的：抗凝血酶、蛋白C、蛋白S活性檢測可用于遺傳性易栓癥的診斷與分型,也可用于獲得性易栓癥的診斷及預后判斷。目前抗凝血酶、蛋白C、蛋白S活性檢測無國際公認的參考方法,國際標準品不易獲得,不同檢測系統(tǒng)間結果差異不明確,且目前國內無抗凝蛋白檢測規(guī)范化操作指南,影響了其在臨床的應用價值。本研究通過問卷對目前國內抗凝血酶、蛋白C、蛋白s活性檢測現(xiàn)狀及存在的問題進行調查；嘗試研制候選參考物質并對其均勻性、穩(wěn)定性、互通性進行評價并定值；對檢測系統(tǒng)的性能驗證、不同檢測系統(tǒng)結果的比對、校準模式等結果一致化相關問題進行研究,為促進抗凝血酶、蛋白c、蛋白s活性檢測結果一致化及質量改進提供依據(jù)。方法：1.檢測現(xiàn)狀的調查研究：通過發(fā)放調查問卷,收集國內開展AT、PC、 PS檢測項目的臨床實驗室相關檢測信息,包括使用儀器、試劑、方法學信息,性能驗證,標準曲線的建立及參考區(qū)間的設定,室內質控和室間質評情況等,與國外相關指南文件的要求進行比較,明確檢測現(xiàn)狀和存在問題,并針對性地提出改進措施建議。2.候選參考物質的研制與評價：選擇合適的原料和制備方法,利用新鮮血漿制備2個批次3-5個批號的候選參考物質。依據(jù)ISO Guide35《標準物質/標準樣品的定值——通用原則和統(tǒng)計原理》,CNAS-GL03《能力驗證樣品均勻性和穩(wěn)定性評價指南》,NCCLS EP14-A《基質效應評價指南》的要求,對候選參考物質的均勻性、穩(wěn)定性、互通性進行評價。依據(jù)ISO Guide35選取不同檢測系統(tǒng)的8家實驗室,以NIBSC凝血標準物質LOT4為溯源標準進行定值。將候選參考物質試應用于室內質控,同時檢測進口商品質控物作為比較。3.檢測結果一致化相關問題研究：①參考國外文獻及指南文件,結合廠家建議及專家意見制訂性能驗證方案,對Stago STA-R evolution全自動血凝儀AT、 PC、PS活性檢測的批內精密度、日間精密度、準確度、攜帶污染率、線性進行驗證；②依據(jù)EP9-A2利用臨床標本對3種常用AT、PC、PS活性檢測系統(tǒng)的檢測結果進行系統(tǒng)間的比對,明確系統(tǒng)間檢測結果的差異并進行校準模式探討。結果：1.檢測現(xiàn)狀的調查研究：調查結果顯示國內的抗凝蛋白檢測以活性檢測為主,AT活性檢測實驗室最多(102家),PC和PS活性檢測實驗室數(shù)量較少(36家和31家),僅個別實驗室開展抗凝蛋白抗原檢測。各實驗室普遍采用儀器法進行抗凝蛋白活性檢測,最常用的檢測系統(tǒng)為希森美康(Sysmex)、思塔同(Stago)和沃芬(instrument labortary, IL)及其配套試劑；各實驗室定標頻率不同,對于AT、PC和PS分別有40.2%、33.3%和32.3%的實驗室在質控失控時定標,另有16.1%的實驗室在每批次PS活性檢測前定標；各實驗室使用的參考區(qū)間不盡相同,僅59.8%的實驗室對參考區(qū)間進行驗證,對于PS僅5家(占16.1%)實驗室使用按性別區(qū)分的參考區(qū)間；約42%的實驗室未進行儀器性能驗證；約15%的實驗室不常規(guī)開展室內質控；對于AT、PC、PS分別有85.7%、75.0%和71.0%的實驗室未參加任何形式的室間質評活動。2.候選參考物質的研制：采用新鮮冰凍血漿為原料,緩沖液稀釋法調整AT、 PC、PS活性水平,分裝成1.0ml/支置于-80℃冷凍條件下保存,最終制備出第1批次3個批號,第2批次3-5個批號(批號分別為AT/PC/PS201401～201403, AT201501～201503,PC/PS201501～201505)活性水平范圍可控的候選參考物質。候選參考物質均勻性良好,評價結果顯示差異無統(tǒng)計學意義(P0.05)；復融后在室溫和冷藏條件下3種抗凝蛋白活性穩(wěn)定時間分別為24h和24h、8h和12h、3h和12h；長期穩(wěn)定性評價線性回歸分析顯示AT和PC活性檢測各批次各濃度水平候選參考物質在24周觀察期內穩(wěn)定,正常濃度PS活性檢測候選參考物質可穩(wěn)定19周,異常低濃度PS活性檢測候選參考物質可穩(wěn)定15周(P0.05)；各批號候選參考物質在3種檢測系統(tǒng)間均具有互通性；AT201501～AT201503批號候選參考物質的定值結果分別為：101.4±7.53、72.3±6.25和41.5±4.98；PC201501～PC201505批號候選參考物質的定值結果分別為：96.3±6.45、116.1±6.88、77.3±3.58、47.5±3.62和29.1±2.72；PS201501～PS201505批號候選參考物質的定值結果分別為：95.1±20.62、103.2±20.71、60.6±8.37、33.7±9.63和18.2±9.58。室內質控試應用結果顯示,候選參考物質和商品質控品AT、PC和PS檢測結果CV分布范圍分別為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%,均小于廠家規(guī)定的批間不精密度。3.檢測結果一致化相關問題研究：①性能驗證結果顯示AT、PC、PS活性檢測批內及日間精密度均在廠家要求范圍內；準確度驗證與NIBSC凝血標準物質靶值間的偏倚分別為-1.09%、9.78%和-3.09%；攜帶污染率分別為1.37%、0.0%和2.15%；線性回歸方程相關系數(shù)R2均0.99,AT活性檢測在23-128%區(qū)間內線性驗證通過,PC活性檢測在30-150%區(qū)間內線性驗證通過,PS活性檢測在25-126%區(qū)間內線性驗證通過。②可比性研究結果顯示對于AT活性檢測,兩兩檢測系統(tǒng)間檢測結果相關性和可比性可接受；對于PC活性檢測,兩兩檢測系統(tǒng)間檢測結果相關性和可比性好；對于PS活性檢測,第1批次可比性評價結果IL與Sysmex兩系統(tǒng)間檢測結果相關性可接受,Stago與上述兩系統(tǒng)間檢測結果相關性好,兩兩檢測系統(tǒng)間檢測結果可比性可接受,第2批次可比性評價IL與Sysmex兩系統(tǒng)間檢測結果相關性可接受,Stago與上述兩系統(tǒng)間檢測結果相關性差,兩兩檢測系統(tǒng)間檢測結果可比性差。使用LOT4模擬定標曲線將比對樣本檢測結果進行轉化后,AT活性檢測IL與Sysmex系統(tǒng)間差異略減小,Stago與另外兩系統(tǒng)間差異增大；PC活性檢測IL與另外兩系統(tǒng)間差異略減小,Stago與Sysmex系統(tǒng)間差異增大；PS活性檢測2次系統(tǒng)間可比性結果均呈增大趨勢。結論：1.問卷調查結果顯示與國外相關技術指南的要求進行比較,國內實驗室在抗凝蛋白活性檢測的關鍵技術環(huán)節(jié)質量控制方面仍有較大的改進空間,應對實驗室檢測人員進行系統(tǒng)化技術操作、定標、參考區(qū)間驗證、性能驗證及質量控制培訓,并開展室間質量評價。2.利用新鮮冰凍血漿制備AT,PC和PS候選參考物質,其均勻性和穩(wěn)定性良好,在3種主流檢測系統(tǒng)間具有互通性,并完成了8家實驗室包含3種主流檢測系統(tǒng)的聯(lián)合定值。候選參考物質與商品質控物室內質控試應用CV及變化趨勢具有可比性,表明其可以應用于日常室內質控。3.①對我室規(guī)范操作檢測系統(tǒng)進行性能驗證,驗證結果符合廠家及相關文件的要求,性能驗證方案可為其他檢測系統(tǒng)的性能驗證提供參考。②對于AT活性檢測具體的不同試劑間診斷敏感性差異還需進一步選擇有明確AT Ⅱ型Cambridge缺陷和Ⅱ型HBS缺陷患者的血漿加以比對；對于PC活性檢測3種系統(tǒng)的精密度,可比性及相關性均較好；PS的評價結果存在批間差異,利用LOT4的模擬定標曲線校準檢測結果的模式可能對于改善系統(tǒng)間檢測結果的一致性并無幫助。
[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 鍏鋒湁鍙瘮鎬，

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