Multi‐site reproducibility of a human immunophenotyping assay in whole blood and peripheral blood mononuclear cells preparations using CyTOF technology coupled with Maxpar Pathsetter, an automated data analysis system

Abstract

High‐dimensional mass cytometry data potentially enable a comprehensive characterization of immune cells. In order to positively affect clinical trials and translational clinical research, this advanced technology needs to demonstrate a high reproducibility of results across multiple sites for both peripheral blood mononuclear cells (PBMC) and whole blood preparations. A dry 30‐marker broad immunophenotyping panel and customized automated analysis software were recently engineered and are commercially available as the Fluidigm® Maxpar® Direct™ Immune Profiling Assay™. In this study, seven sites received whole blood and six sites received PBMC samples from single donors over a 2‐week interval. Each site labeled replicate samples and acquired data on Helios™ instruments using an assay‐specific acquisition template. All acquired sample files were then automatically analyzed by Maxpar Pathsetter™ software. A cleanup step eliminated debris, dead cells, aggregates, and normalization beads. The second step automatically enumerated 37 immune cell populations and performed label intensity assessments on all 30 markers. The inter‐site reproducibility of the 37 quantified cell populations had consistent population frequencies, with an average %CV of 14.4% for whole blood and 17.7% for PBMC. The dry reagent coupled with automated data analysis is not only convenient but also provides a high degree of reproducibility within and among multiple test sites resulting in a comprehensive yet practical solution for deep immune phenotyping.

The use and limitations of single‐cell mass cytometry for studying human microglia function

“…One of such approaches is CytoNorm (77), which includes the use of anchor samples, accounting for the different biological settings used in the study, and an algorithm that corrects for batch-to-batch variability. Some studies have already proven the potential reproducibility of the technique, showing multi-site PBMC and whole-blood comparability (5,44). However, this workflow or the other normalisation protocols has never been applied to huMG studies.…”

Mass Cytometry—A Tool for the Curious: Networking in Berlin

“…In the Basics and Reagents workshop Michael Leipold, Antonio Cosma, Marjolijn Hameetman (LUMC, Leiden), Henrik Mei, and Axel Schulz (moderator) reviewed and discussed the current needs in the field, that is, to achieve technically consistent data across a large number of samples and measurements, expanding the measurement capacity of mass cytometry, and, to ease the setup of mass cytometry assays. Concerning the latter, benefits of novel pre‐made antibody panels suitable to characterize the most common immune cell populations and designed for easy assay handling were discussed. The easy access to such commercially available panels can help speed up standard immune profiling by mass cytometry especially when combined with proprietary software for analyzing this assay: While this strategy may pave the way toward using mass cytometry in clinical practice, its customization is limited.…”

Issue Highlights – November 2020

“…However, the perennial problem that 'a standardized flow cytometric panel' remains an oxymoron presents its usual resistance to any widespread adaptation of any particular dried inflexible panel. One solution to that obstacle is to increase the number of markers utilized and provide for drop-in reagents, such as described in the 30-marker study by Bagwell, et al, 2020. Let me conclude again with the wish that 2021 finds us all as soon as possible on the other side of the world-wide pandemic that has been so disastrous in countless ways. Our unique technology combined with the medical and scientific prowess we wield should be helpful in doing just that.…”