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Updated: May 5, 2026
Examination of Thymic Positive and Negative Selection by Flow Cytometry
Published on: October 9, 2012
OMIP-112: 42-Parameter (40-Color) Spectral Flow Cytometry Panel for Comprehensive Immunophenotyping of Human
Laurien A Waaijer1, Bram van Cranenbroek1, Hans J P M Koenen1
1Department of Laboratory Medicine, Radboud University Medical Center, Nijmegen, the Netherlands.
Insights
This study presents a novel 42-parameter spectral flow cytometry panel for comprehensive human immune cell profiling from limited patient blood samples. The optimized panel identifies over 80 immune subsets, aiding disease research and therapy monitoring.
Area of Science:
- Immunology
- Biotechnology
- Single-cell analysis
Background:
- Understanding the human immune system is crucial for disease research.
- Spectral flow cytometry (SFM) offers deep single-cell profiling capabilities.
- High-dimensional data analysis in SFM presents significant challenges.
Purpose of the Study:
- To develop and optimize a high-dimensional SFM panel for comprehensive immune cell profiling.
- To enable detailed identification of innate and adaptive immune cell subsets.
- To facilitate disease understanding and therapy response monitoring through immune profiling.
Main Methods:
- Optimization of a 42-parameter SFM panel using peripheral whole blood.
- Inclusion of 40 commercially available fluorochromes, one stacked fluorochrome, and an autofluorescent parameter.
- Utilized co-stimulatory, checkpoint, activation, homing, and maturation markers for deeper phenotyping.
Main Results:
- Successfully identified innate and adaptive immune cells, including neutrophils, T cells, B cells, NK cells, and ILCs.
- Identified over 80 distinct immune cell subsets within a single measurement using FlowSOM and manual annotation.
- The 42-parameter panel is the first optimized on peripheral whole blood and demonstrates superior complexity compared to existing 40-color panels.
Conclusions:
- The developed high-dimensional SFM panel provides a powerful tool for detailed immune profiling.
- This panel enables the identification of a broad spectrum of immune cell subsets from limited patient samples.
- The optimized panel can be instrumental in advancing disease research and monitoring therapeutic interventions.
Abstract:
Profiling the human immune system is essential to understanding its role in disease, but it requires advanced and novel technologies. Spectral flow cytometry (SFM) enables deep profiling at the single-cell level. It is able to detect many fluorescent parameters within one measurement; therefore, it is vastly useful when patient material is limited. However, designing and analyzing these high-dimensional datasets remains complex. We optimized a 42-parameter panel (40 commercially available fluorochromes, one stacked fluorochrome and an autofluorescent (AF) parameter) that enables the identification of innate and adaptive immune cell composition. It is the first 42-parameter panel that is optimized on peripheral whole blood, and it outperforms other published OMIPs of 40 colors in terms of complexity. With this panel, we are able to identify neutrophils, basophils, eosinophils, monocytes, dendritic cells, CD4 T cells, CD8 T cells, regulatory T cells, mucosal-associated invariant T (MAIT) cells, γδ T cells, B cells, NK cells, dendritic cells, and innate lymphoid cells (ILCs). Furthermore, with the utilization of co-stimulatory, checkpoint, activation, homing, and maturation markers, this panel enables deeper phenotyping. Within one measurement, more than 80 distinct immune cell subsets were identified by FlowSOM and annotated manually. In conclusion, with this high-dimensional SFM panel, we aim to generate immune profiles to understand disease and monitor therapy response.
