Frozen CyTOF panel enables robust and consistent longitudinal assessment of the SARS-CoV-2 immune landscape

Abstract During the Coronavirus Disease 2019 (COVID-19) outbreak, a robust and consistent strategy was required for assessing the immune landscape. Our rapid research response to COVID-19 was enabled by use of standardized assays. The assays included a 33-marker mass cytometry (CyTOF) panel that characterized major immune cells subsets in fresh whole blood including both innate and adaptive cells. Intraassay variation, determined by staining of the same fresh whole blood sample independently by two technicians on the same day, was CV<20% for populations with a frequency >5%. However, technical consistency was crucial to accurately capture and compare immune changes. Thus, we tested the stability of an antibody cocktail stored at −80C to limit inherent variability in daily panel preparation. Using lyophilized whole blood and cryopreserved R848-stimulated PBMC as controls encompassing the breadth of cell subsets and markers, we assessed the panel performance 7 times (at days 0, 16, 33, 42, 56, 72 and 96 after freezing). We found stability of staining over time: variability of the frozen cocktail was comparable to repeated cocktail preparations as seen in rare plasmablasts (CV<25% frozen vs 13.1% repeat fresh), but also CD4 T cells (CV<13% vs 10.6%), and neutrophils (CV<4% vs 9.8%). Applying the highly-consistent frozen cocktail to study of 59 COVID-19 subjects revealed changes in common subsets (CD4 central memory T cells increased >30% with severe disease), but also in rare plasmablasts (increased 6% of CD19), and more subtle neutrophil changes (increased 15% of CD45). Thus, freezing a high-content CyTOF panel cocktail provides a reliable method to control variability of staining in assays where sample collection is variable.