Standardizing immunophenotyping for the Human Immunology Project

Abstract

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.

Figure 1. A typical flow cytometry experiment

Sample preparation from blood often involves Ficoll gradient separation of mononuclear cells, and sometimes cryopreservation, before staining with fluorescent antibody conjugates. Each of these steps can introduce variability in the assay results. Instrument setup involves setting voltage gains for the photomultiplier tubes (PMTs) so as to achieve optimal sensitivity. To the extent that this is not standardized, it becomes a source of variability as well. Data acquisition involves passing the stained cells through a laser beam and recording the fluorescence emission from all of the bound antibody conjugates. Here, the main variable is the type of instrument, including the lasers and optical filters used. This is followed by data analysis, in which cell populations of interest are defined and reported on, which is another significant source of variation. Ref., reference; SD, standard deviation.

Figure 2. Identification of immune cell subsets by eight-colour antibody staining

The figure shows the cell populations that can be identified using the markers targeted by each of the five antibody cocktails of the Human Immunophenotyping Consortium (HIPC) phenotyping panel shown in TABLE 2. CCR, CC-chemokine receptor; CXCR3, CXC-chemokine receptor 3; DC, dendritic cell; NK, natural killer; PBMC, peripheral blood mononuclear cell; T_H, T helper; T_Reg, regulatory T.

Figure 3. The importance of antibody choice

The staining patterns of two commercially available clones of human CD38-specific antibody are very different, despite the fact that both antibodies were conjugated to allophycocyanin (APC) by the same vendor, and were used to stain peripheral blood mononuclear cells (PBMCs) from the same healthy subject under identical conditions. V450, violet 450. Data courtesy of Angelique Biancotto, National Heart, Lung and Blood Institute, USA.