| Objective(1)To characterize and evaluate the homemade reference material of lymphocyte subsets(percent and absolute counts)and apply homemade reference material to the performance verification of testing system and internal quality control(IQC)of lymphocyte subsets enumeration using flow cytometry,and to lay the foundation for large-scale promotion and application in clinical laboratories.(2)To investigate the current status and problems of CD34+hematopoietic stem cell enumeration in clinical laboratories and provide suggestions for the development of quality improvement programs.Methods(1)Evaluation and application study of lymphocyte subsets homemade reference material:①Characterization of homemade reference material:according to the ISO Guide 35 and JJF1343-2012 guideline documents,the homogeneity and stability of the homemade reference material Lym202001 and Lym202002 were evaluated;percent and absolute counts were characterized by 9 clinical laboratories.②Performance verification;A.Precision verification:referring to the ICSH and ICCS and CLSI H62 guideline documents,intra-assay precision used 1 homemade reference material,2 quality control materials and 3 fresh clinical specimens,within an experimental batch,each specimen was labeled 3 times and each tube was repeatedly acquired 3 times;inter-assay precision referred to the IQC application.The CVs of the precision test results were compared with the manufacturer’s instructions and the imprecision performance specifications calculated based on biological variation data;B.Linearity verification:referring to the EP6-A guideline document,specimens with different concentration gradients were prepared using homemade reference material in concentrated dilutions,and the verification results were evaluated using WS/T 420-2013 and EP6-A evaluation methods;C.Trueness verification:referring to the EP15-A3 guideline document,the 2 batches of homemade reference material were repeatedly labeled 5 times within the same experimental batch,and a total of five batches of experiments were conducted,which passed verification if the total mean value of the test results was within the verification interval,and was compared with the bias performance specifications calculated based on biological variation data。③IQC application:referring to the WS/T 641-2018 document,used 1 batch of homemade reference material and 1 batch of quality control material,within an experimental batch,each specimen was repeatedly acquired 3 times,and the assay was run twice a week for 2 months to compare the IQC application effects of both.(2)Study of the current status of CD34+cell enumeration and preliminary evaluation of reference material:①Survey on the current status of the assay:101 laboratories participating in the national external quality assessment(EQA)program of CD34+cell enumeration were surveyed.Questionnaires and reference material were distributed to collect information on assay methodology and testing results.Quality control requirements for CD34+ cell enumeration were determined concerning international guidelines,and the compliance of the surveyed laboratories was analyzed.Testing results were analyzed in groups and compared with CAP quality assessment data.②Evaluation of the homogeneity and longterm stability monitoring of reference material:the homogeneity of the reference material was evaluated concerning ISO Guide 35 and JJF1343-2012,and the long-term stability was monitored,once a week from week 0 to 12,and once every two weeks after week 12,to evaluate the stability period of the reference material.Results(1)Evaluation and application study of lymphocyte subsets homemade reference material:①Characterization of homemade reference material:Lym202001 and Lym202002 had good homogeneity of each assay parameter and were stable for 27 weeks(6 months)under storage conditions of 2-8℃.The percent counts(%)of CD3+,CD3+CD4+,CD3+CD8+,CD3-CD19+and CD3-CD16/56+of Lym202001 were determined as 62.8±3.4,15.4±1.4,41.7±3.7,19.1 ±2.3 and 17.9±3.0,respectively,and the absolute counts(/μL)were determined as 649±92,158±19,428±45,199±41 and 186±46,respectively;The percent counts(%)of CD3+,CD3+CD4+,CD3+CD8+,CD3-CD19+ and CD3CD16/56+of Lym202002 were determined as 76.6±3.8,38.2±1.9,33.3±2.9,6.6± 1.0 and 13.8±2.3,respectively,and the absolute counts(/μL)were determined as 892±130,439±78,387±55,75± 19 and 157±43,respectively.②Performance verification:The CVs of the intra-assay precision and inter-assay precision were less than the manufacturer’s instructions or the imprecision performance specification based on the desirable level of biological variation;the R2 of the linear regression equation was greater than 0.995 for each assay parameter for the linearity verification,and the linearity errors and assay imprecision were within the allowed range for each parameter,indicating that the results of each subpopulation assay were linear within the verified range;the mean values of each assay parameter were within the verification interval for the trueness verification.③Internal quality control application results:The CV of the results of each parameter of the homemade reference material is close to the variation level of the imported quality control material,and both are within the allowable imprecision range of calculated based on the BV data.(2)Study of the current status of CD34+cell enumeration and preliminary evaluation of reference material:①A total of 97 laboratories returned questionnaire information and 99 laboratories returned results of reference material.The questionnaire survey data showed high compliance rates with quality control requirements such as gating protocols,pipetting methods,and the number of events acquired:92.8%,83.9%,and 82.5%respectively.The laboratories have relatively low compliance rates such as the use of whole blood quality control materials for IQC,selection of erythrocyte lysing reagents,sample processing method,whether to report absolute count results,and quality control of flow cytometry:5.2%,28.9%,39.2%,46.4%,and 55.7%,respectively.Testing results showed that the CV of percent counts was similar to the CAP quality assessment data,but the CV of absolute counts was greater than the CAP data.②Evaluation of the homogeneity and long-term stability monitoring of the reference material:the homogeneity of the reference material dispensed in this investigation met the requirements,and the long-term stability monitoring results showed that the reference material can be stable for 20 weeks under the storage conditions of 2-8℃.Conclusion(1)The homemade reference material for lymphocyte subsets in this study has good homogeneity and a stability period of up to 6 months.After percent and absolute counts are characterized in several clinical laboratories,it can be used in the performance verification parameters of lymphocyte subsets assay,such as precision,linearity,and trueness,to provide a reliable and stable basis for performance evaluation.The homemade reference material has a good effect on IQC and can be used in the daily IQC of lymphocyte subsets assay in clinical laboratories.(2)Clinical laboratories have poor compliance with some quality control requirements and the variability of absolute count results between laboratories is not satisfactory,so it is recommended that laboratories strengthen the training related to the quality control of CD34+cell enumeration,especially the training of absolute counting.The CD34+cell reference material has good homogeneity and a long stability period and is feasible for IQC and EQA of CD34+ cell enumeration. |
PDF Full Text Request