A streamlined whole blood CyTOF workflow defines a circulating immune cell signature of COVID‐19

Abstract

Mass cytometry (CyTOF) represents one of the most powerful tools in immune phenotyping, allowing high throughput quantification of over 40 parameters at single‐cell resolution. However, wide deployment of CyTOF‐based immune phenotyping studies are limited by complex experimental workflows and the need for specialized CyTOF equipment and technical expertise. Furthermore, differences in cell isolation and enrichment protocols, antibody reagent preparation, sample staining, and data acquisition protocols can all introduce technical variation that can confound integrative analyses of large data‐sets of samples processed across multiple labs. Here, we present a streamlined whole blood CyTOF workflow which addresses many of these sources of experimental variation and facilitates wider adoption of CyTOF immune monitoring across sites with limited technical expertise or sample‐processing resources or equipment. Our workflow utilizes commercially available reagents including the Fluidigm MaxPar Direct Immune Profiling Assay (MDIPA), a dry tube 30‐marker immunophenotyping panel, and SmartTube Proteomic Stabilizer, which allows for simple and reliable fixation and cryopreservation of whole blood samples. We validate a workflow that allows for streamlined staining of whole blood samples with minimal processing requirements or expertise at the site of sample collection, followed by shipment to a central CyTOF core facility for batched downstream processing and data acquisition. We apply this workflow to characterize 184 whole blood samples collected longitudinally from a cohort of 72 hospitalized COVID‐19 patients and healthy controls, highlighting dynamic disease‐associated changes in circulating immune cell frequency and phenotype.

Convalescent COVID-19 Patients Without Comorbidities Display Similar Immunophenotypes Over Time Despite Divergent Disease Severities

“…The immune signatures after infection with SARS-CoV-2 represent immune correlates of viral clearance and clinical recovery and most likely also of protection from reinfection in correlation with symptom scores. High-dimensional data analyses including single-cell RNAseq and CyTOF, which are currently further elucidating the intricacies of COVID-19 associated immune signatures are expected reveal more robust insights into the cellular activation states and T cell exhaustion and might serve as cellular biomarkers ( 88 , 89 ). Although most single-cell studies with clinical material suffer the caveats of limited patient numbers and patient cohort stratification, they provide in-depth insights into the transcriptional regulation of the cellular target population or allow correlations with complex cellular subgroups that are identified based on differential gene expression patterns ( 23 , 90 ).…”

Shift of lung macrophage composition is associated with COVID-19 disease severity and recovery

“…Whole blood samples for CyTOF were taken from CPT tubes and directly stained with a lyophilized antibody panel using Fluidigm MaxPar Direct Immune Profiling Assay (MDIPA) tubes for 30 mins at RT. Stained whole blood samples were then stabilized and fixed with Prot1 Proteomic Stabilizer for 10 mins at RT before storage at -80°C as previously described (55). CPT tubes were then centrifuged at 1800 rcf for 15 mins to separate plasma and PBMC.…”

Downregulation of exhausted cytotoxic T cells in gene expression networks of multisystem inflammatory syndrome in children

“…CyTOF validation. CyTOF analysis was performed as previously described 68 . Briefly, fresh whole blood samples were stained using the Fluidigm MaxPar Direct Immune Profiling Assay and then fixed and frozen using SmartTube Prot1 buffer.…”