Numerous consortia today are working to achieve standardized approaches to important topics for research and clinical applications. The work they do to achieve reproducible results is critical to good research results and reliable clinical diagnosis and outcomes. In this series, we will highlight standardization efforts from The Human Immune Phenotyping Consortium (HIPC), the EuroFlow Consortium, and the ONE Study. These studies contain detailed panels and strategies to improve the reproducibility of flow cytometry.
The Human Immune Phenotyping Consortium
Established in 2010, The Human Immune Phenotyping Consortium (HIPC) (https://www.immuneprofiling.org) was developed by the Federation of Clinical Immunology Societies (FOCIS) to address standardization across flow cytometry assays.
This program has expanded to provide centralized research resources for the comprehensive understanding of the human immune system.
In their 2016 Nature publication, the HIPC describes their standardization study. Identified sources of experimental variability include: combinations of markers and fluorochromes, sample handling, instrument type and set up, gating and analysis strategies, and ways in which data are reported.
To control for markers and fluorochromes, the HIPC immunophenotyping experts developed five standardized panels as pre-configured, lyophilized reagents in 96-well plates consisting of eight-color antibody cocktails to phenotype major immune cell subsets in peripheral blood mononuclear cells (PBMC). The plates along with lyophilized control PBMCs and a consensus detailed staining protocol were distributed to nine international laboratories.
Data collected from this study were analyzed manually at each site. FCS files were sent to a central site where they were analyzed using both manual and automated gating. Central manual gating was found to significantly reduce the variability across datasets. Larger more easily identified subsets such as CD3+, CD4+ T-cells had less variability compared to smaller, dimmer subsets. In addition, subsets that required multiple successive gates had higher CVs. Automated gating algorithms gave similar results to central gating except in the cases of rare subsets and poorly resolved populations. In some cases, these differences could be attributed to subtle differences in the manual gating of upstream subsets.
This paper also contains detailed panels and a table describing which populations of cells could be reliably detected by automated gating in their study. Information provided may help laboratories to set up their own SOPs especially for the detection of rare and dim subsets.
Finak, Greg, et al. "Standardizing flow cytometry immunophenotyping analysis from the human immunophenotyping consortium." Scientific reports 6 (2016). www.nature.com/