Immunophenotyping by multi-parametric flow cytometry is the cornerstone technology for enumeration and characterization of immune cell populations in health and disease. Standardized procedures are essential in order to allow for inter-individual comparisons in the context of population based or clinical studies. The Milieu Intérieur Consortium used standardized and automated procedures for immunophenotyping of human whole blood samples. This approach provides robust laboratory protocols for affordable, semi-automated eight-color cytometric immunophenotyping that can be used in population-based studies and clinical trial settings.
The Milieu Intérieur Consortium has established 8 cytometry panels.
To enable detection, enumeration and phenotyping of major leukocyte populations present in circulation – PMNs, T cell, B cells, NK cells, monocytes and DCs – we designed four 8-color cytometry panels.
- The “lineage” panel covered the major cell populations, providing a reference for comparison with other consortia and served as an internal control for other panels cytometry_panel_lineage_panel.jot_.zip
- The “PMN” panel enabled the classification of neutrophils (CD16+ FceRIa- cells), basophils (FceRIa+ CD16-) and eosinophils (CDw125+) cytometry_panel_pmn_panel.jot_.zip. Activation status of neutrophils was assessed by CD62L expression, and used as a marker of healthy donor status.
- The “T cell” panel was designed to classify CD4+ and CD8+ naïve (TnaÏve), central memory (TCM), effector memory (TEM) and EMRA+ T cells (TEMRA) subsets, utilizing the relative expression levels of CD27, CD45RA and CCR7. By combining anti-CD8a and anti-CD8b antibodies within the same panel, we were able to distinguish CD8aa, CD8ab and CD4 CD8aa T cells cytometry_panel_t_cell_panel.jot_.zip
- The “DC” panel delineates three principle subsets of dendritic cells in peripheral blood: plasmacytoid dendritic cells (pDCs), BDCA-1+ and BDCA-3+ conventional dendritic cells (herein referred to as cDC1 and cDC3, respectively) cytometry_panel_dc_panel.jot_.zip
This work establishes a foundation for defining reference values in healthy human donors. Our approach provides robust laboratory protocols for affordable, semi-automated eight-color cytometric immunophenotyping that can be used in population-based studies and clinical trial settings.
Whole blood (2mL) was washed by mixing fresh whole blood and PBS at a 1:1 ratio, followed by centrifugation at 500g for 5min at 18–22oC (room temperature). Washed blood and pre-mixed liquid reagents were loaded onto the Freedom Evo 150 liquid handling system. The supernatant was aspirated and discarded, followed by the addition of fresh PBS taking it to the same final volume as input whole blood. Antibody premixes were prepared, shortly spun (about 20s) and 100ml of the resuspended cells were aliquoted into tubes containing the pre-mixed antibody cocktail. The samples were shortly vortexed and incubated 20min in the dark at room temperature (RT). In samples stained with the PMN and DC panels 1 mL of 1x viability dye solution was added, followed by incubation for 30min in the dark at 4°C. Thereafter, 1 ml of cold PBS (4°C) was added to the tubes, which were centrifuged for 5 min at 500g and the supernatant aspirated. All samples, irrespective of the panel used, were resuspended in 2000μl of 1x RBC lysing solution, shortly vortexed and incubated 15 min at RT protected from light. After centrifugation for 5 min at 500g, the supernatant was aspirated, the samples were resuspended in 240μl PBS and immediately acquired on the cytometer.
Please find the staining_protocol_for_cytometry_manualsemi-automated.pdf.
All clinical laboratory tests use automation in sample processing and attempts have been made to implement automation in genomic assays (DNA/RNA extractions, genotyping, microarray assays etc). We decided to take advantage of automation in sample preparation for cellular immunophenotyping. To achieve this, we implemented our protocol using the EVO150 liquid handling platform (Tecan). The premix of antibodies was prepared manually on a daily basis, and all other steps for the staining protocol were performed using the liquid handling platform, with the exception of centrifugation. The pipetting scripts for the platform were created to enable staining of 4 to 12 samples, in parallel, in 96-deep well plates.
Pease find attached tecan_scripts.esc_.zip
The lineage panel
For the characterization of major leukocyte populations, we first identified CD45+ hematopoietic cells, followed by exclusion of doublets (Figure 2A). Subsequently, B cells were gated as CD19+ CD16-, and T cells were identified as CD19- cells followed by CD3+ staining, then analyzed for the expression of CD4 and CD8 (Figure 2B). Within the CD3- cells, NK cells were identified as CD56+ and analyzed for their expression of CD16 and CD56. In the population of CD56- cells, CD16hiSSClow cells were selected in order to segregate monocytes from neutrophils. Further gating identified CD14+CD16int monocytes and CD14lowCD16hi monocytes. Neutrophils were defined as CD16hi SSChi(Figure 2B).
Please find gating_strategy_lineage_panel.pdf.
The PMN panel
To characterize granulocytes populations, doublets were first excluded (Figure 3A) and neutrophils were identified as CD16hiCDw125- live cells. We also assessed the expression of CD62L within this cell population as a marker of activation (Figure 3B). Basophils and eosinophils were gated within the CD16low/- cells as FceRIa+CD203c+ and CDw125+, respectively (Figure 3C). Of note, we highlight a difference in the staining of different subpopulations of PMN for fixable viability dye (FVD) (Figure 3A-C), using saponin treated cells as a positive control for dead cells (Figure 3D).
Please find gating_strategy_pmn_panel.pdf.
The T Cell panel
T cells were identified as CD3+ cells (Figure 4A). Upon exclusion of doublets (Figure 4A), CD4+ and CD8b+ were gated and analyzed. We characterized naïve (TN), central memory (TCM), effector memory (TEM) and effector memory expressing RA (TEMRA) subpopulations of both T cell subsets, based on their expression of CD45RA and CD27 [17, 20] (Figure 4B). TN and TCM cells have also been defined by the expression of CCR7. We therefore assessed the expression of CCR7 by these cell populations. The activation status was determined by HLA-DR expression. In addition, the CD4+ T cells population expressing CD8aa was identified (Figure 4B).
Please find gating_strategy_t_cell_panel_.pdf.
The DC Panel
To characterize DCs, we first gated on HLA-DR+ CD14- and excluded dead cells doublets, and CD3+, CD19+ or CD14+ lineage positive cells using a cocktail of reagents (Figure 5A). pDC, cDC1 and cDC3 populations were identified as BDCA4+BDCA2+ (CD304+CD303+), BDCA1+ (CD1c+) and BDCA3+ (CD141+), respectively (Figure 5B). The activation status of the three DC subsets was assessed by their expression of HLA-DR and the costimulatory molecule CD86 (Figure 5C). The position of gates to define cDC subsets was determined using HLA-DR-CD14- cells as a negative control (Figure 5D).
Please find the gating_strategy_dc_panel.pdf.