Standardized and flexible eight colour flow cytometry panels harmonized between different laboratories to study human...

Area of Science:

• Immunology
• Cell Biology
• Biotechnology

Background:

• Multi-color fluorescence-activated cell sorting (FACS) requires harmonization for inter-laboratory data comparability.
• Natural Killer (NK) cell immunotherapy research necessitates standardized FACS panels and acquisition protocols.

Purpose of the Study:

• To design and validate harmonized 8-color FACS panels for human NK cell phenotyping and functional analysis.
• To establish standardized FACS setup and data analysis protocols for reproducible results across different flow cytometers.
• To assess the impact of cryopreservation on NK cell phenotype and function.

Main Methods:

• Development of 8-color FACS panels with fixed backbone markers (lineage exclusion, NK cell selection) and variable markers (phenotype, function).
• Standardization of FACS setup and data analysis across three distinct flow cytometers.
• Analysis of NK cell phenotype and function in fresh and cryopreserved peripheral blood mononuclear cells (PBMC).

Main Results:

• Highly comparable and reproducible data sets were obtained using reference PBMC samples (n=6) with the harmonized protocols.
• Cryopreservation and thawing of PBMC samples did not significantly alter NK cell phenotype or function (n=12).
• Validated protocols enable reliable analysis of NK cells in both fresh and cryopreserved samples.

Conclusions:

• Standardized FACS panels and harmonized analysis protocols generate reliable data for multi-center clinical trials.
• Cryopreserved PBMC are suitable for analysis, simplifying logistics in multi-center studies.
• The developed protocols facilitate consistent and reproducible NK cell characterization in clinical research.