This article was originally published here
Cytometry A. 2021 December 24. doi: 10.1002/cyto.a.24528. Online for print.
INTRODUCTION: Pediatric SARS-CoV-2 infection is often mild or asymptomatic and the immune responses of children are underexposed compared to adults. Here we present and evaluate the performance of a two-panel flow cytometry-based approach (16 and 17 parameters) for immunophenotypic analysis of cryopreserved infant PBMC samples after SARS-CoV-2 infection. The panels have been optimized based on previous SARS-CoV-2 related studies for the immune system of children.
METHODS: PBMC samples from seven SARS-CoV-2 seropositive children as of early 2020 and five age-matched healthy controls were stained for analysis of T cells (Panel T), B and innate immune cells (Panel B). The performance of the panels was evaluated in two parallel approaches, namely classical manual gating of known subpopulations and unbiased clustering using the R-based algorithm PhenoGraph.
RESULTS: Using manual gating, we clearly identified 14 predefined subpopulations of interest in Panel T and 19 populations in Panel B in low volume pediatric samples. PhenoGraph found 18 clusters within the T cell panel and 21 clusters within the innate and B cell panel that can be unmistakably annotated. Combining the data from the two panels and analysis approaches, we found expected differentially abundant clusters in SARS-CoV-2 seropositive children compared to healthy controls, which enhances the value of these two panels for the analysis of the immune response to SARS-CoV-2. 2 underlined.
CONCLUSION: We have established a two-panel flow cytometry approach that can be used with limited amounts of cryopreserved pediatric samples. Our workflow enabled fast, comprehensive and robust pediatric immunophenotyping with comparable performance in manual gates and unbiased clustering. These panels can be adapted for large multi-center cohort studies to investigate the pediatric immune response to emerging virus variants in the ongoing and future pandemics. This article is copyrighted. All rights reserved.
PMID:34953025 | DOI:10.1002/cyto.a.24528