Preparation And Evaluation Of Candidate Reference Materials For Coagulation Factor â…§ And â…¨ Testing

Objective:The detecting of FⅧ and FIX is mainly used in the diagnosis of hemophilia A and B. In addition, it can be used in auxiliary diagnosis of hemorrhagic disease and thrombotic disease. At present, there is no international reference method of FⅧ and FⅨ 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. This research investigated the current status and problems in the detecting of FⅧ and FⅨ in domestic clinical laboratories. We prepared FⅧ and FⅨ candidate reference materials and evaluated the uniformity, stability and applicability to provide basis for the harmonization and quality improvement of the detecting of FⅧ and FⅨ.Methods:1. Collected information and investigated current status and problems in the detecting of FⅧ、FⅨ in domestic clinical laboratories through questionnaires. Quality control materials were distributed to domestic clinical laboratories to realize the actual situation of the detecting of FⅧ and FⅨ. Different evaluation criterias were used to analyze the results to explore suitable evaluation criterion.2. Researched different preparation methods and the key factors in preparation process to choose appropriate preparation method and the best experimental condition. Fresh frozen plasma was used to prepara two lots (normal and pathological value) candidate reference materials.3. The homogeneity and stability of the preparations were evaluated according to ISO Guide35and CNAS-GL03. The preparations were also used as internal quality control materials and were provided to54clinical laboratories, then collected feedback results for statistical analysis.Results:1. The amount of samples was less than30per month in72%of laboratories.39%of laboratories did not run internal quality control, and about21%of laboratories just performed the normal level quality control. Individual laboratories showed a high cumulative CV of internal quality control. Most laboratories used reference range provided by the reagent manufacturer but not verified. The frequencies of calibration were different and only34%of laboratories performed calibration in a different assay batch. The results of detecting quality control materials showed the results of different reagents groups vary significantly. Part of the laboratories lacked comparability among results, especially when detected abnormal concentration level quality control materials, the highest results and the lowest results differed more than2times. The grading evaluation results of20%of laboratories were unqualified and they should be treated as focus of quality improvement. The criterion (group median±30%) was more applicable to evaluate the results of FⅧ and FⅨ and the pass rates of each group were about70%. The criterion should be adjusted according to the data returned.2. The low temperature precipitation combined with chemical adsorption was adopted to separate FⅧ and FⅨ from frozen plasma. The dissolved sediment liquid, liquid of chemical adsorption and the raw material were mixed in proportion and adjust concentration levels. Then the preparations were partial shipmented and were kept under freezing condition. Finally we prepared two lots candidate reference materials.3. Homogeneity evaluation of the candidate reference materials showed that there was no statistically significant difference between the groups (P>0.05). A linear regression based on stability evaluation indicated that the linear trend was insignificant (P>0.05). The uncertainty range of the homogeneity and stability was0.9%～2.7%and0.7%～2.2%, respectively. The inter-day imprecisions of the two lots candidate reference materials were5.8%、9.0%for FⅧ:C and6.2%、9.0%for FⅨ:C, respectively. The inter-day imprecisions of the two batchs manufactural quality control materials were8.4%,5.4%for FⅧ:C and6.4%、8.1%, respectively. The investigation results showed that the candidate reference materials can be used for external quality assessment of FⅧ and FⅨ when distributed by cold-chain transport way. The results of the candidate reference materials were partly comparable with the manufactural quality control materials.Conclusion:According to the survey, the main problem exsited in detecting FⅧ and FⅨ was the key links in quality control lack standardization, and the results of different reagent groups lack comparability. It is necessary to formulate guidelines of detecting FⅧ and FⅨ and establish the external quality assessment program for FⅧ and FⅨ assay. An acceptable method was set and two lots of FⅧ and FⅨ candidate reference materials with different levels were prepared. The candidate reference materials prepared with frozen plasma were stable and homogenous, can be used in internal quality control and external quality assessment when distributed by cold-chain transport way.