| Objective:Antithrombin, protein C, protein S activity testing is mainly used in the diagnosis and classification of hereditary thrombophilia and also can be used in the diagnosis and prognosis of acquired thrombophilia. At present, there is no international reference method of AT, PC and PS activity testing and the international standard materials are difficult to be acquired. In addition, the differences between different measuring methods were not known and there was no standard operation guideline of testing in domestic so that the clinical application values of the activity testing of these three anticoagulant proteins were imparied. This study investigated the current status and problems of AT, PC and PS activity testing in domestic clinical laboratories. The candidate reference materials for AT, PC and PS activity testing were prepared and the uniformity, stability, commutability of the candidate reference materials were evaluated. They were also characterized and applied in internal quality control. Some issues related to the harmonization of the result of AT, PC and PS activity testing were also discussed which including the performance validation、the comparability study and the method of calibration in order to promote the harmonization and qulity improvement of AT, PC and PS activity testing.Methods:1. The investigation of the current status of AT, PC and PS activity testing: The informations of AT, PC and PS testing in dometic clinical laboratories were collected and compared with the requirements in foreign guidelines to realize the actual situation and problems of AT, PC and PS activity testing and propose improvement mesurments accordingly.2. The preparation and evaluation of candidate reference materials:Two batches of different lots of candidate reference materials were prepared with fresh plasma after a detailed study about the selection of suitable raw materials and the preparation methods. According to ISO Guide35 Reference material-General and statistical principles for certification, CNAS-GL03 the proficiency testing samples homogeneity and stability evaluation guideline and NCCLS EP14-A Evaluation of Matrix Effects-Approved Guideline, the homogeneity, stability and commutability of the candidate reference materials were evaluated. According to ISO Guide 35,8 laboratories with different kinds of detection systems were selected in characterization study using NIBSC coagulation standard material as a standard for tracibility. The candidate reference materials were also used for internal quality control, and manufacture quality control materials were detected at the same time as comparison.3.Issues related with testing result harmonization:① Referencing to foreign literatures and guidelines, as well as the manufacturers and the experts opinions, the performance verification of AT, PC and PS activity testing were proceeded, including intra-batch precision, between-run precision, accuracy, carryover and linear range verification. ②The comparability of test results of the AT, PC, PS activity among three main testing systems were evaluated using the clinical specimens according to EP9-A2. Clarified the differences between detection systems and proposed possible calibrate model to decrease bias between different detection systems.Results:1. Questionnaire survey showed that activity testing is the main assay of anticoagulant proteins testing in demenstic clinical laboratories. The testing of AT, PC and PS activity were performed in 102,36 and 31 laboratories respectively. Automated cogulation analyzer was usesed in all laboratories, which mainly from the manufactures Sysmex, Stago and IL. The frequences of calibration were very different. There were 40.2%,33.3% and 32.3% laboratories which run recalibration for AT, PC and PS respectively when the inter-quality were out of control, while another 16.1% of the laboratories which also run calibration before each batch of PS activity testing. Different reference intervals were used in the laboratories which only 59.8% of the laboratory verified the reference intervals. For PS, only 5 (16.1%) laboratories using gender dependent reference intervals. About 42% of the laboratories didn’t go through instrument performance verification. About 15% of the laboratories do not routinely carried out internal quality control. For AT, PC and PS, there were respectively 85.7%, 75% and 71% of the laboratories did not participate in any form of external quality assessment.2. Candidate reference materials were prepared from fresh frozen plasmas and the buffer dilution method was used to adjust the AT, PC and PS activity levels. Plasma was packed into 1.0ml/bottle and were freezing stored in -80℃.Finally two batches of activity level controllable candidate reference materials were made, which the first batch included 3 lots and the second batch included 3～5 lots (lots numbers were AT/PC/PS201401～201403, AT201501～201503, PC/PS201501～201505).All candidate reference materials had good homogeneity, the evaluation results showed there were no statistically significant differences (P>0.05).The stability times of 3 kinds of anticoagulant proteins activity after melting at room temperature and refrigerated conditions were 24h and 24h,8h and 12h,3h and 12h respectively. Linear regression analysis of long-term stability evaluation showed that the candidate reference materials were stable in the 24 week observation period for AT and PC, and can be stable in 19 weeks and 15 weeks for the normal and abnormal low concentration of PS activity testing respectively (P>0.05). All candidate reference materials were commutable for the three measurement systems. The values of AT201501 to AT201503 candidate reference materials were 101.4±7.53,72.3±6.25 and 41.5±4.98 respectively. The values of PC201501 to PC201505 were 96.3±6.45, 116.1±6.88,77.3±3.58,47.5±3.62 and 29.1±2.72 respectively. The values of PS2015 01 to PS201505 were 95.1±20.62,103.2±20.71,60.6±8.37,33.7±9.63 and 18.2±9.58 respectively.Intemal quality control application results showed that the CVs of AT,PC and PS of candidate reference materials and manufacture quality control substances detection were respectively 3.02±7.39% vs 4.28～ 9.37%,3.11～4.88% vs 3.95～ 5.22% and 3.53～5.88% vs 5.72～5.74%, which were all less than the given manufacturer between-batch imprecision.3.① Prformance verification results showed that the intra-batch precision and between-run precision of AT, PC and PS activity testing all can meet the manufacture’s requirements. Accuracy verification results showed that the testing bias from NIBSC standard coagulation substance target values were-1.09%,9.78% and-3.09% respectively. The carryover rates were 1.37%,0.0% and 2.15% respectively. All the square of correlation coefficient from the linear regression were> 0.99, the linearation were validated for AT,PC and PS which the ranges were23～128%,30～ 150% and 25～126% respectively. ② Comparisons among 3 kinds of testing systems showed that for AT activity testing results, correlation and comparability between two systems were both acceptable, while for PC activity testing results, correlation and comparability between two systems were both good. For PS activity testing results, the first batch of comparison showed acceptable correlation between IL and Sysmex and good correlation between Stago and the other two systems with acceptable comparability, while the second batch showed acceptable correlation between IL and Sysmex and bad correlation between Stago and the other two systems with unacceptable comparability. Datas transform by new calibration curve, which came from LOT4 detection results, showed that AT and PC bias between IL and Sysmex as well as PC bias between IL and other two systems decreased a little, other bias all increased.Conclusion:1.The questionnaire survey results showed that compared with the requirements of foreign relevant technical guidelines, there were still large room for improvement in the key technology procedures of quality control for anticoagulant proteins activity testing in domestic laboratories. Systematic personnel training should are needed on technical operation, calibration, reference intervals verification, performance verification and quality control and external quality assessment should be taken in.2. AT, PC and PS candidate reference materials prepared with fresh frozen have good homogeneity, stability and commutability among the three mainstream testing systems. Each lot of candidate reference materials was characterized by ally eight laboratories using three kinds of main detection systems. The CV and the changing trends of the candidate reference materials are comparable with the commodity quality control materials, which can be applied to daily internal quality control.3.① The Performance of the testing in our laboratory was validated which fit the requriements of manufacture and relevant guidelines. The schems for performance validation can be used as a reference in other labrotories.② To investigate the different diagnostic sensitivities of different AT activity detection reagents, we need further select clearly diagnosed AT type Ⅱ Cambridge defects and type Ⅱ HBS defects patients plasma and do more comparison study.There are good precision, comparison and correlation for the results of PC activity testing using three kinds of testing systems. There are large CVs between batchs for PS activity testing. New calibration curve prepared by LOT4 has no help decreasing bias between different detection systems. |
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