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Standardizing immunophenotyping for the Human Immunology Project

A Corrigendum to this article was published on 18 May 2012

Key Points

  • Standardized immunophenotyping assays are a requisite for accomplishing the proposed Human Immunology Project, which involves the comprehensive elucidation of the metrics of healthy versus diseased or perturbed human immune systems.

  • The variables inherent in flow cytometry immunophenotyping are largely known, and include reagent choice, sample handling, instrument setup and data analysis; strategies to mitigate each of these variables are available.

  • Several groups, including the Human Immunophenotyping Consortium, are standardizing reagent panels for flow cytometry.

  • Together with the adoption of such standard panels, an infrastructure for aggregating and mining results will be needed.

  • Availability of such panels and the data-mining infrastructure should result in more rapid biomarker discovery for immunologically relevant diseases.


The heterogeneity in the healthy human immune system, and the immunological changes that portend various diseases, have been only partially described. Their comprehensive elucidation has been termed the 'Human Immunology Project'. The accurate measurement of variations in the human immune system requires precise and standardized assays to distinguish true biological changes from technical artefacts. Thus, to be successful, the Human Immunology Project will require standardized assays for immunophenotyping humans in health and disease. A major tool in this effort is flow cytometry, which remains highly variable with regard to sample handling, reagents, instrument setup and data analysis. In this Review, we outline the current state of standardization of flow cytometry assays and summarize the steps that are required to enable the Human Immunology Project.

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Figure 1: A typical flow cytometry experiment.
Figure 2: Identification of immune cell subsets by eight-colour antibody staining.
Figure 3: The importance of antibody choice.


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The authors thank all the contributors to the HIPC/FITMaN immunophenotyping panel, in particular S. Heck, F. Nestle, A. Biancotto, S. Gupta, M. Malipatlolla, L. Devine, R. Montgomery and D. Hafler. The mention of any commercial products in this manuscript does not imply endorsement by the US Government or the US National Institutes of Health.

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Correspondence to Holden T. Maecker.

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Holden T. Maecker is a stockholder of BD Biosciences, which is a manufacturer of flow cytometers and reagents.

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Immune phenotypes

Measurable aspects of the immune system, such as the proportions of various cell subsets or measures of cellular immune function.


Sequential filters that are applied to a set of flow cytometry data to focus the analysis on particular cell subsets of interest.

Human Immunology Project

The comprehensive mapping of immune phenotypes in healthy and diseased human populations.

Density gradient separation

The isolation of mononuclear cells from blood or other sources by centrifugation over a density gradient, which usually consists of a carbohydrate polymer solution (Ficoll).


The processing and storage of cells at sub-zero temperatures under conditions that preserve their viability for later assays.

Lineage cocktail

A mixture of antibodies specific for various lineage-specific markers, such as CD3 (for T cells), CD14 (for monocytes) and CD19 and CD20 (for B cells).

Antibody capture beads

Microparticles conjugated with immunoglobulin-specific antibodies. These beads can be stained with fluorescent antibodies to create single-colour controls for flow cytometry instrument setup and compensation.

Phosphoepitope flow cytometry

(Also known as 'phospho-flow'). A technique that uses antibodies specific for phosphorylated versions of proteins to analyse cell signalling by flow cytometry.

Mass cytometry

Flow cytometry using antibodies tagged with heavy metal ions, which are detected by mass spectrometry (as opposed to classical flow cytometry, which uses antibodies tagged with fluorophores and optical detection).

Optical spillover

The presence of signals from fluorescent antibody staining in multiple detectors of a cytometer, resulting in a loss of resolution sensitivity.

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Maecker, H., McCoy, J. & Nussenblatt, R. Standardizing immunophenotyping for the Human Immunology Project. Nat Rev Immunol 12, 191–200 (2012).

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