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. 2017 Mar 10;7:43873.
doi: 10.1038/srep43873.

Standardized and Flexible Eight Colour Flow Cytometry Panels Harmonized Between Different Laboratories to Study Human NK Cell Phenotype and Function

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Free PMC article

Standardized and Flexible Eight Colour Flow Cytometry Panels Harmonized Between Different Laboratories to Study Human NK Cell Phenotype and Function

John P Veluchamy et al. Sci Rep. .
Free PMC article

Abstract

Advancements in multi-colour fluorescence activated cell sorting (FACS) panel warrant harmonized procedures to obtain comparable data between various laboratories. The intensifying clinical exploration of Natural Killer (NK) cell-based immunotherapy demands standardized and harmonized NK cell FACS panels and acquisition protocols. Eight colour FACS panels were designed to study human NK cell phenotype and function within peripheral blood mononuclear cells (PBMC). The panels were designed around fixed backbone markers and channels, covering antigens for non-NK lineage exclusion (CD3, TCRγδ, CD19, CD14, SYTOX® Blue) and NK cell selection (CD45, CD56, CD16), complemented with variable drop-in markers/channels to study NK cell phenotype (NKG2A, NKG2C, NKG2D and KIR2D) or NK cell function and activation (CD25, NKp44 and CD107a). Harmonized FACS set-up and data analysis for three different flow cytometers has been established, leading to highly comparable and reproducible data sets using the same PBMC reference samples (n = 6). Further studies of NK cells in fresh or cryopreserved PBMC samples (n = 12) confirmed that freezing and thawing of PBMC samples did not significantly affect NK phenotype or function. In conclusion, our data demonstrate that cryopreserved PBMC samples analysed by standardized FACS panels and harmonized analysis protocols will generate highly reliable data sets for multi-center clinical trials under validated conditions.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Experimental design to compare variability between flow cytometers.
Flow chart of the experimental set up outlining the establishment and verification of the NK cell phenotype and function panels to compare variability between flow cytometers. Following selection of backbone and drop-in antibodies, reference samples were used for optimizing and checking fluorochrome intensities using appropriate single stains and fluorescence minus one (FMO) controls. Further, the data generated from three instruments were analysed using KALUZA® software and results were tabulated using Graphpad Prism for statistical differences for validation using the same reference sample.
Figure 2
Figure 2. Comparison of FACS panels antibody fluorochrome intensities between three flow cytometers.
All antibodies in the NK cell phenotype and function panel were tested for their fluorochrome intensities using the same staining and acquisition protocols on three different flow cytometers. Single stains from backbone antigens were evaluated for their antigen expression levels. PBMC were gated on NK cells for detecting NK receptors and NK functional antigens in the drop-in channels. One representative set of histograms (n = 3) is shown for each marker tested at three different centers using three different flow cytometers. Antigen expression is expressed as percentage of positive cells for backbone antibodies in (A) and for NK cell drop ins in (B).
Figure 3
Figure 3. Evaluation of NK cell phenotypes and function marker expression between different flow cytometers.
Expression levels for drop-in antigens in the NK cell phenotype (A) and function panel (B) were analysed and compared across different centers using the same reference samples (n = 6). NKG2A, NKG2C, NKG2D, KIR2D and CD16 expression in BD LSRFortessaTM, BD FACSCantoTM II and MACSQuant® Analyzer 10 were measured for NK phenotype panel (A). Similarly, for NK cell function panel analysis, three time points of reference sample PBMC were stimulated with A431 cells and their percentage of NK cells positive for CD107a, CD25 and NKp44 levels were measured (B). Statistical analysis was performed using the non-parametric Kruskal-Wallis test.
Figure 4
Figure 4. Experimental design to compare different PBMC conditions.
The experimental set up describes the work flow to compare NK phenotype and function changes under different PBMC (non-stimulated and stimulated) conditions. PBMC from 12 healthy donors were stained with NK cell phenotype and NK function panel cocktails. Data was acquired for 6 donors using BD LSRFortessaTM and for other 6 donors in MACSQuant® Analyzer 10. Further, FCS files from LSRFortessaTM were analysed on KALUZA® and for MACSQuant® Analyzer 10 in MACSQuantifyTM 2.8. Data obtained from both devices were combined and examined for statistical significance between fresh and cryopreserved NK cells.
Figure 5
Figure 5. Comparison of NK cell phenotypes between fresh and cryopreserved NK cells.
NK cell phenotypes between freshly isolated and cryopreserved thawed PBMC samples were compared. NK cells were either activated or non-activated with cytokines (IL-2+IL-15) and target cells (A431) alone or targets coated with cetuximab (CET). Expression levels of NKG2A (A), NKG2C (B), NKG2D (C), KIR2D (D) were compared for the following conditions: i) NK only ii) NK+A431 and iii) NK+A431+CET conditions. NK only conditions are depicted as open rectangles, followed by NK+A431 with grey shades and NK+A431+CET conditions represented as black rectangles. Columns represent data from12 donors, from each donor the mean of triplicate values was used; with bars showing SEM. Data are from independent experiments performed in triplicates from 12 PBMC donors (6 donors: BD LSRFortessa +6 donors: MACSQuant). Statistical analysis was performed using the Wilcoxon test.
Figure 6
Figure 6. Comparison of NK cell function between fresh and cryopreserved NK cells.
NK cell functions between freshly isolated and cryopreserved thawed PBMC samples were compared. NK cells were either activated or non-activated with cytokines (IL-2+IL-15) and target cells (A431) alone or targets coated with cetuximab (CET). Expression levels of CD107a (A), CD16 (B), NKp44 (C), CD25 (D) were compared for the following conditions: i) NK only ii) NK+A431 and iii) NK+A431+CET conditions. NK only conditions are depicted as open rectangles, followed by NK+A431 with grey shades and NK+A431+CET conditions represented as black rectangles. Columns represent data from12 donors, from each donor the mean of triplicate values was used; with bars showing SEM. Data are from independent experiments performed in triplicates from 12 PBMC donors (6 donors: BD LSRFortessa+6 donors: MACSQuant). Statistical analysis was performed using the Wilcoxon test.

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