Preparation And Evaluation Of Candidate Reference Materials For Von Willebrand Factor Testing And Study On Consistency Of VWF:Ag Testing Results

Objective:The detection of von Willebrand Factor (vWF) is mainly used in the diagnosis and characteristic of the illness type of von Willebrand disease. In addition, it can be used in auxiliary diagnosis of hemophilia. However, there is no international reference method for vWF testing and the international standard material is diffcult to acquire. So the comparability of results among different laboratories is too poor to satisfy the clinical needs. In order to facilitate the clinical laboratories to implement quality improvement and to promote the harmonization of the vWF detection, the questionnaire survey was performed to investigate the domestic status and existing problems of vWF detection. VWF candidate reference materials were prepared and evaluated the homogeneity, stability, commutability, characterization and application of internal quality control. Some related issues of vWF detection hamonization were studied, such as performance verification and calibration of measurement system.Methods:1. The investigation of current status of vWF testing:The information of vWF detection in domestic clinical laboratories were collected by designing and issuing questionnaires, including basic information of laboratories, measurement sytem, quality control and reference intervals. Comparative analysis was performed between the status of domestic laboratory and the requirements of foreign standards. Then the measurements for improvement were put forward. 2. Preparation and evaluation of the candidate reference materials:The key factors in preparation process were studied to determined the best experimental condition. The homogeneity and stability of the preparations were evaluated according to ISO Guide35 and CNAS-GL03. The commutability between STAGO and IL instruments was evaluated according to the industry standard of WS/T 356-2011. Then the candidates were characterized by several laboratories according to ISO Guide35 with traceability to the Secondary Coagulation Standard established by NIBSC (SSCLOT4). The preparations were also used as internal quality control materials and the coefficient of variations were compared with that of commercial quality controls.3. Issues of vWF detection hamonization:The performance verification of measurement system were performed which included the trueness, the within-run impricision, inter-day precision, carryover and linear evaluation. The comparability between two measurement systems were evaluated using the fresh patient samples. The simulate calibration on two systems with the secondary coagulation standard was carred out by the standards of NIBSC.Results:1. The investigation of current status of vWF testing:The amount of samples was less than 50 per month in 65.5% of laboratories.20.7% of laboratories did not run internal quality control, and about 16.7%-20.0% of laboratories just tested the normal level quality control materials.44.8% of laboratories ran performance verification. The reference interval of vWF:Ag range from 40.6%-75% to 122%-176%. Two laboratories set their reference interval according to blood type group. Most laboratories used reference interval provided by the reagent manufacturer without verification.2. Preparation and evaluation of the candidate reference materials:The candidates were prepared from fresh frozen plasma (FFP) by low temperature centrifugation. The mixture of FFP and cryosupernatant on certain proportion were distributed into 1 mL per tube and kept in -80℃. In this study, two batches of candidate reference materials were prepared (the first batch preparation’s lots were vWF201401, vWF201402 and vWF201403 and vWF:Ag concentration range of 37.0%～83.5%; the second batch of preparation’s lots were vWF201501, vWF201502, vWF201503, vWF201504 and vWF201505 and vWF:Ag concentration range of 14.0%～133.5%, vWF:A range 6.3%～ 118.3%). The evaluation of homogeneity, stablity, commutabity, characterization and application of internal quality control was completed for vWF:Ag detection. The evalution of homogeneity and stablity(for 16 weeks) was fiished for vWF:A detection. The candidate reference materials were of good homogeneity for vWF:Ag and vWF:A detection (P>0.05). The long-term stability evaluation of the first baches of candidate reference materials showed no significant trend changes (P>0.05) for VWF:Ag detection in 44 weeks and the uncertainty of long term stability (ults) were 0.79%～1.20%. The long-term stability of the second batch of candidate reference material (vWF:Ag and vWF:A) continued to observe. The candidate reference materials for vWF:Ag testing showed commutable across the two measurement systems of STAGO and IL. The assigned value was 12.2%～138.9% for the second batch of candidate reference materials and the uncertainty of characterisation (uchar) was 0.06%～0.09%. The combined uncertainty (uc) was 1.05%～-2.17% and the expanded uncertainty U (k=2) was 2.1%～4.4%. Internal quality control test showed the CV of inter-day imprecision were 1.3%～2.3%for candidate reference material and the CV were 1.2%～2.6% for commercial control materials.3. Issues of vWF detection hamonization:The resutls of performance verification showed that the bias was -0.99%～3.96%; the intra-assay precision and inter-assay precision were 1.1%～1.8% and 2.9%～3.6%; the carryover was 0; the slope of linear evaluation was 1.003 and r2 was equal to 1.000; the bias of the measured value and theoretical value of each dilution was within 10%. In the comparability analysis, the correlation coefficient of two systems was 0.953 and the bias ranged from -20.8%～ 23.7%. The relative bias between two systems before and after calibration were-20.8%～23.7% and -9.7%～13.3%. The absolute bias between two systems before and after calibration were -30.5%～20.9% and -26.7%～24.3%.Conclusion:1. The main problem from the questionnaire survey is that quality control of vWF detection is not standardized because of the lack of the related detection standards/ guidelines,.2. This study determined the best preparation method for vWF candidate reference materials and different levels of reference materials could be prepared. The homogeneity, stability and commutabity of the materials are good and the materials could be used for internal quality control.3. Performance verification scheme of the measurement system used in this study can be taken for example to carry out the performance verification for other systems. The correlation is good for different measurement systems while the bias remains big. Although the measurement systems have been calitrated by the same international standards, the difference between the systems is still large. Further studies were needed to implore.