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* These authors contributed equally
In this study, a method was developed to facilitate the transfer of experimental settings and analysis templates between two flow cytometers in two laboratories for the detection of lymphocytes in Japanese encephalitis-vaccinated children. The standardization method for the flow cytometer experiments will allow research projects to be effectively conducted in multiple centers.
An increasing number of laboratories need to collect data from multiple flow cytometers, especially for research projects performed across multiple centers. The challenges of using two flow cytometers in different labs include the lack of standardized materials, software compatibility issues, inconsistencies in instrument setup, and the use of different configurations for different flow cytometers. To establish a standardized flow cytometry experiment to achieve the consistency and comparability of experimental results across multiple centers, a rapid and feasible standardization method was established to transfer parameters across different flow cytometers.
The methods developed in this study allowed the transfer of experimental settings and analysis templates between two flow cytometers in different laboratories for the detection of lymphocytes in Japanese encephalitis (JE)-vaccinated children. A consistent fluorescence intensity was obtained between the two cytometers using fluorescence standard beads to establish the cytometer settings. Comparable results were obtained in two laboratories with different types of instruments. Using this method, we can standardize analysis for evaluating the immune function of JE-vaccinated children in different laboratories with different instruments, diminish the differences in data and results among flow cytometers in multiple centers, and provide a feasible approach for the mutual accreditation of laboratory results. The standardization method of flow cytometer experiments will ensure the effective performance of research projects across multiple centers.
The standardization of flow cytometry is useful for the comparability of results obtained from different cytometers across different laboratories and study centers, and conducive to the mutual recognition of results to improve work efficiency. An increasing number of scenarios require standardization. During the drug development process, flow cytometry standardization is important, as a developed and validated assay will support the whole drug development process from preclinical to clinical analysis. Flow cytometric methods are frequently transferred between the pharmaceutical industry and collaborating laboratories1. Moreover, it is essential to obtain comparable data from multicentric clinical studies. For example, a standardization workflow was developed in the Systemic Autoimmune Diseases Multicenter Clinical Research Project to obtain comparable data from multicentric flow cytometry2.
The standardization of flow cytometry methods is challenging. The challenges experienced across labs are attributed to the lack of standardized materials, software compatibility issues, inconsistencies in instrument setup, and the use of different configurations among different flow cytometers and divergent gating strategies between the centers3,4. Therefore, it is important to conduct a gap analysis between laboratories. Sample access, quality systems, personnel qualifications, and instrument configuration must be reviewed to ensure that the requirements are met.
At present, children vaccinated with the Japanese encephalitis (JE) vaccine have a significantly reduced incidence of JE5. Monitoring peripheral blood immune cells can help understand the changes in cell-mediated adaptive immunity after vaccination, and the correlation between the changes in peripheral blood lymphocyte subsets and the effects of vaccination. Due to the limited stability of whole blood samples, evaluations of vaccine efficacy are often performed in multiple centers. For this analysis, we defined naïve CD8+ or CD4+ T cells as CD27+ CD45RA+, central memory T cells (TCM) as CD27+ CD45RA-, effector memory T cells (TEM) as CD27- CD45RA-, and terminally differentiated effector memory T cells (TEMRA) as CD27- CD45RA+. CD19+ B cells can be separated into populations that express CD27 versus IgD6,7, naïve B cells express CD27n memory B cells (mBCs) can be identified based on the expression of IgD6, and regulatory T cells (Tregs) can be identified as CD4+CD25++CD127low 8. To establish a standardized flow cytometry experiment to achieve the consistency and comparability of experimental results in multiple centers, a rapid and feasible standardization method was established to facilitate the transfer of protocols across different flow cytometers for the detection of lymphocytes in the whole blood of JE-vaccinated children. Six healthy children (2 years old) were recruited from Beijing Children's Hospital, Capital Medical University. After receiving a prime and boost vaccination with a live-attenuated JE SA14-14-2 vaccine less than 6 months prior, peripheral blood samples were collected from the volunteers. Highly comparable data were obtained from different instruments following standardized procedures, which is helpful for multicenter assessments.
The study was approved by the Ethics Committee of Beijing Children's Hospital, Capital Medical University (Approval Number: 2020-k-85). Informed consent of human subjects was waived as only residual samples after clinical testing were used in this study. Two labs are involved in this study. The transferring lab is where the standardized method was developed using one flow cytometer. The cytometer in this lab is hereinafter referred to as cytometer A. The test method lab is the laboratory that receives methods using another flow cytometer, and the cytometer in this lab is hereinafter referred to as cytometer B.
1. Peripheral blood sample collection and cell preparation
2. Preparing compensation beads and single-stained control
3. Using identical configuration names on the different cytometers in two labs
4. Standardizing experiment using cytometer A in the transferring lab
5. Transferring the experimental template to cytometer B in the test method lab
NOTE: The experimental template includes instrument settings, the analysis template, and the target value template of median fluorescence intensity (MFI).
6. Consistency between the experimental results obtained on the two cytometers
Figure 1 shows a global worksheet of the target value template for the CST bright beads. Using an FSC/SSC plot, a polygon gate is drawn to select the CST bright beads. Histogram plots of 10 fluorescence channels were obtained for the CST bright beads: FITC, PE, BB700, PE-Cy7, APC, R718, APC-H7, BV421, V500, and BV605. The target value for each parameter is displayed by showing the median within the histogram gates in Table 2. The screenshots of templates and parameter s...
Immunophenotyping of peripheral blood lymphocyte subsets can help understand the changes in cell-mediated adaptive immunity after vaccination in children. In clinical applications, unexpected situations occur, such as a failure to detect samples in a timely manner or the replacement of a flow cytometer; therefore, rapid standardized methods that facilitate transfers between flow cytometers in different labs are needed9,10,11. He...
The authors declare no conflicts of interest.
RW was supported by Beijing Natural Science Foundation, China (No. 7222059), National Natural Science Foundation of China (No. 82002130), XZ was supported by the CAMS Innovation Fund for Medical Sciences (No. 2019-I2M-5-026).
Name | Company | Catalog Number | Comments |
BD CompBeads Anti-Mouse Ig, κ/Negative Control Compensation Particles Set | BD | 552843 | compensation |
BD FACSCanto | BD | FACSCanto | flow Cytometry A in the transferring lab |
BD FACSDiva CS&T Research Beads | BD | 655051 | define flow cytometer baseline and track cytometer performance |
BD Horizon BV421 Mouse Anti-Human CD127 | BD | 562436 | Fluorescent antibody |
BD Horizon BV605 Mouse Anti-Human CD27 | BD | 562656 | Fluorescent antibody |
BD Horizon V500 Mouse Anti-Human CD45 | BD | 560777 | Fluorescent antibody |
BD LSRFortessa | BD | LSRFortessa | flow Cytometry B in the test method lab |
BD OptiBuild BB700 Mouse Anti-Human CD19 | BD | 745907 | Fluorescent antibody |
BD OptiBuild R718 Mouse Anti-Human CD8 | BD | 751953 | Fluorescent antibody |
BD Pharmingen APC Mouse Anti-Human CD45RA | BD | 550855 | Fluorescent antibody |
BD Pharmingen APC-H7 Mouse Anti-Human CD3 | BD | 560176 | Fluorescent antibody |
BD Pharmingen FITC Mouse Anti-Human CD4 | BD | 566320 | Fluorescent antibody |
BD Pharmingen PE Mouse Anti-Human CD25 | BD | 555432 | Fluorescent antibody |
BD Pharmingen PE-Cy7 Mouse Anti-Human IgD | BD | 561314 | Fluorescent antibody |
Brilliant Staining Buffer Plus | BD | 566385 | Staining Buffer |
Centrifuge | Eppendorf | 5810 | Cell centrifugation |
Centrifuge Tube | BD Falcon | BD-35209715 | 15 mL centrifuge tube |
CS&T IVD Beads | BD | 662414 | standard beads to setup cytometer settings in different flow cytometer |
Lysing Solution 10x Concentrate | BD | 349202 | lysing red blood cells |
Phosphate-buffered Saline (PBS) | Gibco | 10010-023 | PBS |
Round-bottom test tube | BD Falcon | 352235 | 5 mL test tube |
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