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Updated: Jun 30, 2026
Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay
Published on: September 16, 2012
Cross-platform immunophenotyping of human peripheral blood mononuclear cells with four high-dimensional flow
Alexander Heubeck1, Adam Savage1, Katherine Henderson1
1Allen Institute for Immunology, Seattle, Washington, USA.
Insights
We developed four high-parameter flow cytometry panels for deep immunophenotyping of human peripheral blood mononuclear cells (PBMC). These panels enable comprehensive immune cell analysis across different lineages and activation states.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- High-dimensional flow cytometry is crucial for multi-omic studies of the human immune system.
- Deep immunophenotyping requires robust and comprehensive cell surface marker panels.
Purpose of the Study:
- To present four high-parameter flow cytometry panels for deep immunophenotyping of human peripheral blood mononuclear cells (PBMC).
- To provide guidelines for designing flow cytometry experiments for immune profiling.
Main Methods:
- Development of four 25+ color flow cytometry panels targeting 64 cell surface markers.
- Integration of common lineage bridging markers for inter-panel quality control.
- Validation of panels on both BD Symphony A5 and Cytek Aurora cytometers using nine PBMC samples.
Main Results:
- The four panels enabled deep immunophenotyping of T, B, NK, and myeloid lineages, including activation and memory states.
- Comparable high-quality data were obtained from both conventional and spectral cytometers.
- The panels demonstrated suitability for longitudinal or cross-sectional immune profiling.
Conclusions:
- The presented modular panel set and instrument performance metrics offer a valuable resource for researchers in human immunology.
- These panels facilitate standardized and reproducible deep immunophenotyping of PBMCs.
- The findings support the use of spectral cytometry for high-dimensional immune profiling.
Abstract:
Immunophenotyping using high dimensional flow cytometry is a central component of human immune system multi-omic studies. We present four high parameter flow cytometry panels for deep immunophenotyping of human peripheral blood mononuclear cells (PBMC). This set of four 25+ color panels include 64 cell surface markers to resolve broad immune compartment populations, as well as activation and memory of specific T, B, natural killer (NK), and myeloid lineages. Common lineage bridging markers are integrated into each panel to allow for inter-panel quality control through major lineage frequency verification. These panels were developed using a five laser BD Symphony A5 conventional cytometer and successfully transferred to a five laser Cytek Aurora spectral cytometer capable of acquiring the panels. Nine representative PBMC samples were stained with the four phenotyping panels and acquired on both instruments to evaluate population frequency and visual staining patterns for gating between the systems. Both instruments produced comparable high quality flow cytometry data and supported our decision to acquire samples on the spectral cytometer moving forward. This modular set of panels and instrument performance metrics provide guidelines for designing flow cytometry experiments suitable for longitudinal or cross-sectional immune profiling.
