How many parameters does the largest published flow cytometry panel have?

The largest published flow cytometry panel has 50 parameters.

The OMIP-102, published in Cytometry Part A, presents a 50-fluorochrome spectral flow cytometry panel developed for in-depth characterization of the human immune system, with particular emphasis on T cells and dendritic cells.

Article:OMIP-102: 50-color phenotyping of the human immune system with in-depth assessment of T cells and dendritic cells.

However, the aim of this work went far beyond simply exploring the limits of spectral flow cytometry.

The authors developed an optimized, tested, and validated panel, aiming to maximize the amount of information obtained from a single sample while maintaining sufficient resolution to identify different cell populations and states of cellular differentiation and activation. The panel was initially optimized using human PBMCs and includes markers for the major immune cell populations.

Perhaps one of the most interesting aspects of the article is precisely how this panel was built.

During its development, the authors used and discussed several tools to support decision-making in the design of high-dimensional panels.

Tools used:

• Similarity index between fluorochromes;

• Assessment of fluorochrome brightness;

• Computational selection of fluorochrome combinations;

• Assessment of unmixing-dependent spreading error;

• Relationship between marker co-expression and fluorochrome selection;

• FMO controls to assess panel resolution.

The authors started with a manual selection of 40 fluorochromes and used an algorithmic approach to identify combinations that allowed them to reach 50 colors. The study also provides examples of reagents that were tested but ultimately excluded during optimization, making the supplementary material particularly valuable for those working with complex panel design.

Reference:

Konecny AJ, Mage PL, Tyznik AJ, Prlic M, Mair F. OMIP-102: 50-color phenotyping of the human immune system with in-depth assessment of T cells and dendritic cells. Cytometry. 2024;105(6):430–436. DOI: 10.1002/cyto.a.24841.