Standardization of Transfer across Labs between Flow Cytometers for Detection of Lymphocytes in Japanese Encephalitis Vaccinated Children

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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 cytomete....

Results

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.......

Discussion

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 needed^9,10,11. He.......

Materials

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