Standardizing Flow Cytometry Immunophenotyping Analysis from the Human ImmunoPhenotyping Consortium

Abstract

Standardization of immunophenotyping requires careful attention to reagents, sample handling, instrument setup, and data analysis, and is essential for successful cross-study and cross-center comparison of data. Experts developed five standardized, eight-color panels for identification of major immune cell subsets in peripheral blood. These were produced as pre-configured, lyophilized, reagents in 96-well plates. We present the results of a coordinated analysis of samples across nine laboratories using these panels with standardized operating procedures (SOPs). Manual gating was performed by each site and by a central site. Automated gating algorithms were developed and tested by the FlowCAP consortium. Centralized manual gating can reduce cross-center variability, and we sought to determine whether automated methods could streamline and standardize the analysis. Within-site variability was low in all experiments, but cross-site variability was lower when central analysis was performed in comparison with site-specific analysis. It was also lower for clearly defined cell subsets than those based on dim markers and for rare populations. Automated gating was able to match the performance of central manual analysis for all tested panels, exhibiting little to no bias and comparable variability. Standardized staining, data collection, and automated gating can increase power, reduce variability, and streamline analysis for immunophenotyping.

Computational flow cytometry: helping to make sense of high-dimensional immunology data

“…A reference dataset is constructed and new samples, analysed with the same panels, can be mapped onto this reference to classify a patient. Also in larger community-wide efforts, such as the Human Immunology Project Consortium (HIPC) 25 or the Milieu Interieur project 26 , the design of standardized panels is of key importance. To facilitate sharing and dissemination of optimized panels, the cytometry community has recently introduced a new series of protocols to improve reproducibility, referred to as optimized multicolour immunofluorescence panels (OMIPs) 27 .…”

The 10,000 Immunomes Project: Building a Resource for Human Immunology

“…Transformation and compensation were done using the preprocessing.batch function in MetaCyto (Hu et al, 2017). The cell definitions from the Human ImmunoPhenotyping Consortium (Finak et al, 2016) were used to identify 24 types of immune cells using the searchClster.batch function in MetaCyto. Specifically, each marker in each cytometry panels was bisected into positive and negative regions.…”

“…A reference “immunome” might reasonably include flow cytometry, gene expression, human leukocyte antigen (HLA) type, cytokine measurements, clinical assessments, and more. Furthermore, standardized protocols for measurement and conventions for naming cell types and cytokines are only currently being developed, and adherence is inconsistent (Finak et al., 2016). For the experimental or clinical immunologist, the cost of generating the necessary data from scratch—or the temporal and computational costs associated with standardizing and harmonizing data from publicly available cohorts across platforms, time points, and institutions—is prohibitive without significant resources.…”

Development of a Modular Assay for Detailed Immunophenotyping of Peripheral Human Whole Blood Samples by Multicolor Flow Cytometry

“…Recently, Finak et al [19] reported that in multi-centric studies the intra-site variability is generally low, but the inter-site variability is a major problem, which also was confirmed by others [18,20,127]. This predominantly was due to unintended deviations in preparation and analyses between the different sites even though detailed SOPs (standard operating procedures) were provided [19].…”