Study On The Optimum Selection And Biological Variability Of Anther Species Determination Method

The thesis contains two parts. In the first part of study, we conduct a comprehensive performance evaluation of two lupus anticoagulant assays (dRWT and SCT) based on two different test principles on automated coagulation analyzers. Platelet of 50 poor platelet plasma were counted to validate the centrifuge procedure, and the stability of the room temperature stayed sample were evaluated by comparison of 25 plasma results tested at the time of sample collected,2hours, four hours and 8 hours respectively. According to the CLSI EP5-A2, EP6-A2, EP9-A2 and WS/T 406-2012 Standardizations, all the projects were detected in the apparatus to evaluate the within-run and between-run precision, carryover rate, method comparison and reference range (RI)/Cut-off value validation also had been performed. The platelet were 0-9×109/L of the 50 platelet poor plasma, and meet the requirement of three guidelines. The difference of the results at different point of time were insignificant. The within-run precision of dRWT were 0.79%-1.40%, and the within-run precision of SCT were 1.16%-2.35%. The between-run precision of dRVVT and SCT were 2.76%-3.53% and 6.23%-7.02%, respectively. The precision of normalized ratio were less than the other two expressions. The carryover rate of the two tests were less than 3.00%. The RI/Cut-off value of dRVVT and SCT established by the insert were 1.24 and 1.16 respectively. The correlation coefficent of the two tests were greater than 0.900, and there is no significant difference of normalized ratio between two tests. In this portion of research, the results indicated that the existing sample processing program fulfilled the guidelines, and the two lupus anticoagulant assays based on different principles have a good performance in precision, carryover rate, and method comparison, which is suitable for the detection of clinical specimens.The objective of the second portion of this part was to establish the RI/Cut-off values of different lupus anticoagulant tests and different result expressions (ICA,%C, and normalized ratio) based on three guidelines, and to evaluate the sensitivity and specificity of the RI/Cut-off values, as well as the correlation between test combination and clinical manifestation. Lupus anticoagulant were tested by three APTT reagents, dRWT and SCT respectively, and 1:1 mixing tests before incubation were studied in all the recruited subjects.135 healthy subjects were used to establish the RI/Cut-off values as the guidelines recommended,99th percentile (ISTH 2009) and 97.5th percentile (BCSH 2009, CLSI 2014) for detail. The group consisted of 121 autoimmune diseases (antiphospholipid syndrome, systemic lupus erythematosus) were used to evaluate the sensitivity of different RI/Cut-off values.40 patients with congenital coagulation factor deficient (FD), and 49 patients under oral anticoagulant (OAT) were studied to evaluate the specificity. The diagnostic performance of different test combinations was analyzed as well. Sensitivity of three APTT ICA were less than 55.0%, and the sensitivity of silica based APTT was higher than ellagic acid-based APTT. The sensitivity of three dRVVT result expressions were 70.3%～93.1%, while sensitivity of three SCT result expressions were 55.4%-79.2%. Except the ICA of APTT-Actin and SCT (RI/Cut-off value as the 97.5th percentile), the specificity of other tests were more than 90.0%. The positive likelihood ratio of silica based APTT were higher ellagic acid-based APTT. The sensitivity of ellagic acid-based APTT ICA were less than 20.0% in the weak positive lupus anticoagulant. APTT-SS were prolonged in APS, SLE, FD and OAT group, and the positive rate of dRVVT normalized ratio and combinations were the highest in APS. The positive rate of mixing test of APTT-SS in FD was 7.5%. And the positive rate of SCT were 0 in FD, OAT and HS group. The traditional three step procedure and updated normalized ratio were correlated with thrombotic risk (OR 4.400-5.667), lupus anticoagulant positivity determined by traditional screen-mixing-confirm showed a higher thrombotic risk than normalized ratio. The activators and phospholipid construction affect the sensitivity of APTT reagent to lupus anticoagulant. The sensitivity of silica based APTT was higher than ellagic acid-based APTT, which is in accordance with ISTH 2009 guideline, every laboratory should establish local RI/Cut-off value.1:1 mixing test can cause false negative result of weak lupus anticoagulant because of dilution effect. The traditional three step procedure and updated normalized ratio were correlated with thrombotic risk, and it should take the pre-analysis factor into account when interpreting the lupus anticoagulant test result.The results of lupus anticoagulant, antithrombin, protein C, and protein S testing, and the values of von Willebrand factor antigen are important in diagnosis and therapeutic monitoring of thrombosis and hemostasis diseases. Thus far, no published study has focused on the biological variations in LA testing, and only a few studies have examined the biological variations of antithrombin, protein C, protein S and von Willebrand factor. With the latest fully automated instruments and improved reagents. In the second part of study, the analytical, within-subject and between-subject biological variations were estimated for these five coagulant parameters in a cohort of 25 apparently healthy subjects. Blood specimens were collected at 8:00 AM,12:00 PM, and 4:00 PM on days 1,3, and 5. The analytical variation (CVA) values of all the parameters were less than 3%. The within-subject variation (CVw) and between-subject variation (CVG) values of the lupus anticoagulant normalized ratio were 4.64% and 6.83%, respectively. No significant differences were observed in the intra-day and inter-day biological variations of lupus anticoagulant tests, or in antithrombin, protein C, protein S and von Willebrand factor values. Additionally, the utility of the conventional population based reference intervals of the five coagulation parameters was evaluated by the index of individuality (II), and data on CVw and CVAwere used to calculate the reference change value (RCV) to identify the significance of changes in serial results from the same individual.