Establish Evaluation Methods Using Flow Cytometry Monoclonal Antibody IVD Performance

Objective:Establishing linear range reagents, precision, accuracy and specificity of monoclonal antibody in vitro diagnostic of flow cytometry performance evaluation methods. Using the existing system, improve performance assessment methods in antibody production, the performance of in vitro diagnostic reagents antibody class have a better understanding, and thus can be summed up monoclonal antibody IVD in vitro diagnostic reagents trial performance evaluation methods.Methods:1. the linear range of the methods:(1) screening for positive and negative control cells to CD19and CD33monoclonal antibody by flow cytometry;(2) Select the nine levels, each level four replicate solution by analyzing the corresponding concentrations to assess the percentage of positive cells diluted cell concentration linear range;(3) Screening positive and negative cell lines with a constant total number of cells to be mixed in two different ratios of the cells, the proportion of the number of positive cells and negative cells from0%to100%, and the percentage of positive cells recognized reasonable linear range2. Evaluation methods to assess the accuracy:(1) Cells with known target value, labeled reagent, repeat the test three times, the detection values observed are consistent with the target value to get the accuracy;(2) Line methodological comparison, the use of reagents and control reagents tested simultaneously detect40unknown samples, the detection results of the two reagents do paired samples T-test and correlation analysis to obtain the accuracy of the reagent to be tested.3. Assess precision detection method:(1) The use of bone marrow samples, detecting20consecutive days, twice a day is completed by two full runs, each run intervals of not less than three hours, the results of between-run precision and within run peceision to obtain reagents precision;(2)Use commercially QC blood detected20consecutive days, twice a day is completed by two full runs, each run intervals of not less than three hours, the results of between-run precision and within run peccision to obtain reagents precision;(3) The use of bone marrow samples detected eight consecutive days, they were a complete run four times a day, at intervals of not less than three hours, the results of between-run precision and within run peccision to obtain reagents total precision4. Specific detection:(1) Hemolysis, man-made varying degrees of bone marrow hemolysis, compare the bone marrow with hemolysis and without hemolysis, deviation analysis of test results to determine whether there is interference hemolysis reagent;(2)Drugs, according to the method of daunorubicin and dexamethasone used for drug dilution, added to the untreated patient’s bone marrow, evaluation and daunorubicin and dexamethasone for the existence of cross-reactive reagents;(3) Protein, a patient with multiple myeloma bone marrow was replaced with PBS, test the bone marrow which was replaced with PBS, to determine whether the protein reagent influential。Result:1.(1) REH and HSB2are chosenas quality control for the CD19-APC, HL60and CEM as CD33-PE QC cells;(2) By measuring the concentration of nine mixed cell, CD19-APC cell concentration per person diluted to the linear range6-0.25x106, CD33-PE per person linear range of cells diluted to6～0.5×106;(3) Positive cells and negative cells increased from0%to100%, and confirmed that CD19-APC and CD33-PE percentage of the linear range of0%to100%.2.(1) The results of CD19-APC detect commercialization quality control of blood is within the target range, and CD33-PE to detect the commercialization the quality control of blood, the result exceeded the target range;(2) Methodological comparison, the results of T-test of CD19-APC and CD33-PE with their control, P values were greater than0.05, the correlation coefficients were greater than0.95.3.(1)20days precision using bone marrow samples tested, the percentage of positive results within run precision is less than10%, the fluorescence of between-run precision is less than10%, while the deviation of the fluorescence values greater than10%;(2) Blood detection is the use of quality control and within the precision between-run precision are less than10%;(3)8days bone marrow testing, within run precision results were less than10%, while in the between-run precision, the positive percentage deviation is greater than10%, deviation fluorescence is less than10%。4.(1) Hemolysis, hemolysis occurs, the test deviation is greater than10%, and more critical hemolysis, the larger influence is;(2) Drugs, when added to dexamethasone, detect deviations is less than10%, dexamethasone had no effect on detection reagents, while adding daunorubicin, detect deviations is greater than10%, daunorubicin impact of reagents;(3) Protein, before and after the replacement of plasma, the test results deviation is less than10%, the protein had no effect on the use of such agents。Conclusion:1. Screening of suitable cell lines which can be used as quality control cell lines, to assess monoclonal antibody agents. QC cells can use to assess the linear of monoclonal antibody IVD, in order to truly reflect the performance of reagents, the linear range of the dilution and percentage of positive cells should be given.2. Commercialization blood with a target value of quality control, only use by a small part of the reagent, and imitations of manufacturers, transport storage conditions and stability restrict the scope of application, applications methodology is broader than that.3. Bone marrow samples were unable to meet the long-time testing needs, a small range of quality control of blood can fit the test, we need to find better ways to evaluate the precision of reagent.4. Man-made destruction and and drugs or other components to the sample, the reagent can be evaluated for specificity, the selected types should be broad enough to reagents truer.