Study On The Harmonization Of HLA-B27 Typing And CD34~+ Cell Enumeration By Flow Cytometry

Part 1 Study on the harmonization of HLA-B27 typing by flow cytometryObjective1.To investigate the domestic current status and problems of Human leukocyte antigen B27(HLA-B27)typing by flow cytometry in clinical laboratories,and to provide basis for the harmonization of HLA-B27 typing and the quality improvement of clinical laboratory detection.2.Evaluate the comparability of HLA-B27 typing results among different flow measurement systems,and understand the performance differences and problems of different flow measurement systems,to provide reference for the selection of HLA-B27 measurement systems and to promote the harmonization of HLA-B27 typing.3.A large number of whole blood quality control materials with good homogeneity and stability were prepared,which suitable for HLA-B27 typing by flow cytometry,to assist clinical laboratories to implement quality improvement measures.Methods1.Current status of HLA-B27 typing by flow cytometry:Questionnaires and quality control materials were distributed to collect information about HLA-B27 typing by flow cytometry in domestic clinical laboratories,including the instruments and reagents used in laboratories,quality control and testing results of quality control materials,etc.Collect and sort out the questionnaire return results,and use the number and percentage of laboratories to describe the laboratory’s compliance with the quality control requirements;The quality control materials testing results were grouped according to measurement system,and the agreement between the test results of each group and the target value of quality control materials was calculated.2.Evaluation of the comparability of HLA-B27 typing results among different flow measurement systems:A total of 109 clinical specimens were tested using BD,Beckman Coulter and Mindray flow measurement systems,respectively,and the results of the three flow measurement systems were compared.For specimens whose test results were in the indeterminate zone and whose test results were disagreement with those of any two flow measurement systems,sequencing analysis was performed to confirm the test results of the specimens.3.Study on batch preparation conditions and evaluation of whole blood quality control materials for HLA-B27 typing:(1)Study on batch preparation conditions:①Confirm the HLA-B27 fluorescence intensity in mixed blood specimens:the maximum fluorescence intensity difference of mixed specimens was determined by mixing and detecting the specimens with increasing HLA-B27 fluorescence intensity difference until double peaks appeared in HLA-B27 histogram of flow cytometry.②Confirm the blood type in mixed blood specimens:specimens with the required fluorescence intensity were collected and mixed with same blood type and different blood type,respectively.The stable conditions of specimens mixed with same blood type or mixed with different blood type were compared.(2)Preparation and evaluation of quality control materials:According to the above experimental results,the optimal batch preparation conditions and scheme was determined,suitable volunteers who meet the requirements were screened,and three lots of HLA-B27 whole blood quality control materials were prepared by blood sampling,fixation,mixing and packaging,and each lot was divided into 650 pieces.①Homogeneity evaluation:10 samples were selected from quality control materials at medium intervals,and each sample was tested twice.If the test results were consistent with the expected results,the homogeneity was considered to be good.②Long-term stability evaluation:13 time points were selected for detection according to the principle of "dense before sparse",and each lot of quality control material was tested twice in each time point.If the test results were consistent with the expected results,the stability was considered to be good.Results1.The results of current status investigation:(1)Questionnaire investigation results:A total of 54 laboratories(100%)returned questionnaire information and quality control materials test results;24 laboratories(44.4%),19 laboratories(35.2%)and 11 laboratories(20.4%)used BD instruments,Beckman Coulter instruments and Mindray instruments,respectively,and the matching rate of instrument and reagent was 98.1%(53/54);In 52 laboratories(96.3%),specimens were tested within 48 hours after collection,and 36 laboratories(66.7%)stored specimens at room temperature(18-22℃)prior to testing;6 laboratories(11.1%)adjusted the concentration of specimens according to the white blood cell count before staining;35 laboratories(64.8%)stopped testing after collecting 2000 lymphocytes for each specimen;2 laboratories(3.7%)used whole blood quality control material to perform internal quality control.(2)Test results of quality control materials:The target value of 202101 quality control material is negative,and the target value of 202102 quality control material is positive.The test results of 202101 quality control material in 54 laboratories(100.0%)were negative;the test results of 202102 quality control material in 48 laboratories(88.9%)were positive,in 4 laboratories(7.4%)were indeterminate zone,in 2 laboratories(3.7%)were negative.2.Comparability evaluation results:A total of 50 specimens were positive and 42 specimens were negative by the three flow measurement systems.Sequencing analysis was performed on 17 specimens whose results were in the indeterminate zone and disagree between any two flow measurement systems,and the sequencing result of these 17 specimens were HLA-B27 genotype.In 17 specimens,the results of BD flow measurement system were positive;the results of Beckman flow measurement system were 1 positive,14 indeterminate and 2 false negative;the results of Mindray flow measurement system were 15 positive and 2 indeterminate.3.Preparation conditions and evaluation results of quality control materials:(1)Confirmation of batch preparation conditions:The detection was performed on BD flow cytometry,when the HLA-B27 fluorescence intensity difference between the two samples before mixing increased to 8,double peaks appeared in HLA-B27 histogram of flow cytometry.The time point and degree of hemolysis and increased cell debris were similar after mixing the same blood type and different blood type.(2)Evaluation results of quality control materials:The homogeneity test results of lot 202201,202202 and 202203 are consistent with the expected results,so the homogeneity can be considered to meet the requirements.The homemade quality control materials were kept at 2-8℃,and the test results of three lots of quality control materials were consistent with the expected results during 0～102 days of long-term stability monitoring.Conclusion1.Clinical laboratories have poor compliance with some quality control requirements,such as the cell concentration should be adjusted according to the white blood cell count before staining the specimen,whole blood quality control material should be used for internal quality control before detection.Besides,the testing results of HLA-B27 positive specimens with different systems vary greatly.Therefore,it is necessary to carry out laboratory external quality assessment activities to evaluate the state-of-art of HLA-B27 typing by flow cytometry and the agreement of test results among different laboratories.2.The laboratory should verify the determinate value and indeterminate zone provided by the manufacturer are applicable to the population in this area before carrying out HLA-B27 typing by flow cytometry.3.The homogeneity of the homemade whole blood quality control materials of HLAB27 typing met the requirements,and they could be stably stored at 2～8℃ for 102 days.Part 2 Study on the verification of CD34+ cell enumeration by flow cytometryObjective To investigate the analytical performance verification protocols and performance specifications of CD34+cell enumeration by flow cytometry.Methods According to Clinical Laboratory Standards Institute(CLSI)documents and National Health Standard of China,the performance verification of CD34+cell enumeration was designed and implemented using BD FACSCanto Ⅱ cytometry and its corresponding reagent kit.(1)Precision:Four quality assessment materials were tested three times and five times daily for five days,the coefficient of variation(CV)of intra-run and inter-run precision were calculated,and the assessment criterion was set according to the package inserts and published literature.(2)Linearity:Six pooled samples with different concentrations were prepared in the low and high range,respectively.Each sample was tested three times in a single run.The slope and R2 of linear regression of mean of measured value and theoretical value,as well as bias,were calculated,and the assessment criterion was set according to National Health Standard of China.(3)Carryover:A high-value sample was tested three times followed by a low-value sample also in three times,the carryover was calculated,and the assessment criterion was set according to package inserts.(4)Trueness:The samples were the same as precision,each sample was tested five times daily for five days.The robust mean value of "BD instrument+BD reagent group" in the results of national external quality assessment(EQA)was taken as the target value,the bias between the mean of measured value and the target value was calculated,and the assessment criterion was set according to the 50 percent of the total allowable error.(5)Accuracy:Each sample was tested once,the bias between the measured value and the target value was calculated,and the total allowable error was set according to the assessment criterion of EQA.Results The CVs of intra-run precision of 3×5 and 5×5 scheme were 3.0%～9.0%and 2.5%～6.5%,the CVs of inter-run precision were 3.8%～8.7%and 2.8%～10.5%,respectively.The slopes of linearity regression equation of low range(3.6-123.6/μL)and high range(83.9～1043.8/μL)were 0.9932 and 1.0136,R2 were 0.9996 and 0.9987,and the biases were-7.15%～1.16%.The carryover of percent and absolute count were 0.07%and 0.00%.When percent count≤0.2%or absolute count≤20/μL,the absolute biases of trueness were in the range of ±0.006%or ±0.5/μL,the absolute biases of accuracy were in the range of ±0.02%or ±0.9/μL;when percent count>0.2%or absolute count>20/μL,the relative biases of trueness were in the range of±5.65%,the relative biases of accuracy were in the range of±8.19%.Conclusion The verification results of CD34+ cell enumeration for precision,linearity,carryover,trueness and accuracy met the assessment criteria set in this study,which can be taken as a reference of performance verification for clinical laboratories.