| Objective:The immune system can maintain homeostasis and resist pathogen invasion.Immune monitoring and immune status assessment play an important role in the diagnosis and treatment of a variety of immune-related diseases.The detection of regulatory T cells(Tregs)based on flow cytometry(FCM)has been paid increased attention as an important indicator of immune function,and its wide application provides an effective reference for the diagnosis,treatment and prognosis assessment of a variety of diseases.The performance verification of test scheme in clinical laboratory is an important link to ensure the quality of test,and the establishment of standardized performance verification of test is an important premise.Most clinical FCM-based tests use the self-combination of multiple single-branch antibodies and lack performance verification.In addition,the lack of a cell reference material,the difficulty in stable preservation of peripheral blood,the difficulty in evaluating between-run precision and accuracy,and a unified gating strategy contribute to the complexity of validating cell-based FCM approaches.Currently,there is no standardized performance verification scheme for Tregs detection based on FCM.In addition,based on the establishment of the performance verification scheme,consistency comparison of multiple reagent platform detection can provide basis for the mutual recognition of results.The consistency of Tregs detection based on different reagent platforms has not been reported.The purpose of this study was to conduct a standardization study,establish a gating strategy for Tregs and determine the optimal antibody concentration,verify the performance of the three reagent platforms(sample stability,accuracy,within-run precision,between-run precision,and reportable range)of Tregs detection based on FCM,and analyze the consistency of different reagent platforms,so as to provide a basis for the standardization of Tregs detection.It also provides reference for the performance verification of immunosurveillance projects based on FCM.Methods:In this study,a gated strategy for the detection of Tregs was first established by FCM,then the antibody was titrated and the titration curve was drawn.The highest SNR(signal to noise ratio)value was selected as the optimal concentration of the antibody.The samples were selected to be stored at room temperature,and the 2 h detection results were taken as the baseline to calculate the percentage difference of Tregs at 24 h,48 h,72 h and 96 h,respectively,to evaluate the sample stability after collection.Two different levels of commercial quality control products(IMMUNO-TROL Cells and IMMUNO-TROL Low Cells)were used as alternative reference materials to establish target ranges for Tregs to assess accuracy.For within-run precision,samples with high,medium and low Tregs percentage levels were repeated 11times and CV were calculated for the next 10 times.For between-run precision,the percentage of Tregs was measured once a day using two different levels of commercial quality control products for 20 consecutive days to calculate the CV.The samples of 5healthy people were collected in triplicate for reporting range,and CD3+CD4+T cell populations were obtained,respectively.The stop count was set as 10000,5000,2500,1000,500,250,100 and CV was calculated for the three results.Finally,the Tregs detection results of 23 samples were compared and analyzed.Bland-Altman analysis and linear regression analysis were used to compare the consistency of the three reagent platforms(BD,Quanto Bio and Cellgene).Results:This study established a Tregs gating strategy controlled by fluorescence minus one(FMO).Good resolution can be obtained from CD3+CD4+T cell populations by using hand-placed polygon gating,thereby effectively distinguishing Tregs cell populations.The optimal antibody concentrations of CD45-APC-Cy7,CD3-Per CP,CD4-FITC,CD25-APC and CD127-PE provided by BD reagent platform were 50μg/m L,6.25μg/m L,1.5μg/m L,6μg/m L and 12.5μg/m L,respectively.And we found that the optimal concentration of antibody was different in three reagent platforms.For the stability analysis of samples at room temperature,antibodies of BD,Quanto Bio and Cellgene were used to label Tregs.The mean percentage difference of samples from baseline to 72 h after collection was less than 20%,but there was significant instability at96 h after collection,indicating that whole blood samples of Tregs could be stably stored at room temperature for 72 h.In addition,we evaluated the stability of the samples at 4℃and found that the mean percentage difference from baseline to 48 h after collection was less than 20%,indicating that room temperature was more suitable for preservation of the Tregs test samples.In this study,two different levels of commercial quality control products IMMUNO-TROL Cells and IMMUNO-TROL Low Cells were used for accuracy assessment for the first time.The results showed that IMMUNO-TROL Cells and IMMUNO-TROL Low Cells could clearly identify Tregs cell populations.Through20 consecutive days of Tregs detection on quality control products,The target range of Tregs was established,and the coincidence rate of another 10 Tregs tests using the three reagent platform antibodies within the target range was≥80%.The within-run precision,between-run precision of the Tregs labeled with antibodies from the three reagent platforms were acceptable,and the coefficient of variation was less than 10%,which met the requirements for clinical testing.For the reportable range,antibody labeling of Tregs using BD and Cellgene observed that 80%of samples met the CV requirement of less than 20%when 1000 cells were collected in the CD3+CD4+T cell gate.Antibody labeling Tregs from Quanto Bio showed that when 500 cells were collected in the CD3+CD4+T cell gate,80%of the samples met the requirement of CV less than 20%,but insufficient CD3+CD4+T cells would increase the difficulty in accurately identifying Tregs cell groups according to the established strategy of drawing the gate.At least 1000CD3+CD4+T cells should be obtained to determine the percentage of Tregs in peripheral blood.In addition,the Bland-Altman curve of the differences between the three reagent platforms shows that 95.65%of the measured differences are within the 95%confidence interval of the mean differences.Based on the percentage of Tregs using antibodies derived from BD,there was a close correlation with the detection results of antibodies derived from Quanto Bio(R~2=0.9680)and Cellgene(R~2=0.9735),respectively.Similarly,the detection results of Tregs using antibodies derived from Quanto Bio and Cellgene showed that there was a strong correlation,R~2>0.95(R~2=0.9679).Through correlation analysis,the detection values of antibody labeled Tregs of the three reagent platforms had good consistency.Conclusions:This study established a performance verification scheme for the detection of peripheral blood Tregs for the first time.The results of the detection of peripheral blood Tregs by the three reagent platforms have good methodological performance and consistency,providing a practical solution for clinical verification of the performance of immune surveillance projects based on FCM. |
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