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Analyzing Platelet Subpopulations by Multi-color Flow Cytometry
Published on: June 10, 2025
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Fluorochrome choices for multi-color flow cytometry.
Juan Flores-Montero1, Tomas Kalina2, Alba Corral-Mateos1
1Cancer Research Center (IBMCC-CSIC/USAL-IBSAL), Cytometry Service (NUCLEUS) and Department of Medicine, University of Salamanca, Salamanca, Spain; Centro de Investigación Biomédica en Red de Cáncer (CIBER-ONC) CB16/12/00400, Instituto de Salud Carlos III, Madrid, Spain.
Journal of Immunological Methods
|June 11, 2019
Summary
Selecting fluorochromes is crucial for multi-color flow cytometry antibody panels. This study presents strategies for evaluating fluorochromes to optimize panel design for diagnosing blood cancers and immune deficiencies.
Area of Science:
- Immunology
- Biotechnology
- Analytical Chemistry
Background:
- Multi-color flow cytometry requires careful fluorochrome selection for accurate leukocyte subpopulation analysis.
- The growing number of available fluorochromes necessitates robust strategies for panel design.
Purpose of the Study:
- To illustrate EuroFlow strategies for evaluating fluorochrome compatibility and guiding selection for multi-color antibody panels.
- To identify optimal fluorochrome combinations for enhanced fluorescence intensity and reduced spectral overlap.
Main Methods:
- Evaluation of 75 samples (bone marrow and blood) using EuroFlow strategies.
- Analysis of fluorochrome profiles, brightness, fluorescence overlap, stability, and conjugation reproducibility.
- Comparison of candidate fluorochromes for violet and red laser detectors.
Main Results:
- Identification of optimal fluorescence profiles for Brilliant Violet (BV)421, BV510, Allophycocyanin (APC) hilite 7 (H7), and APC C750.
- Guided selection of appropriate fluorochrome conjugates (e.g., BV605, BV650, PE CF594, AF700, APC AF700) for multi-color panels.
- Proposed sets of 8-, 10-, and 12-color fluorochrome combinations as reference frameworks.
Conclusions:
- The presented fluorochrome evaluation approach is effective for designing multi-color antibody panels.
- This strategy supports the diagnosis, classification, and monitoring of hematological malignancies and primary immunodeficiencies.
- Standardized fluorochrome combinations provide a valuable reference for initial antibody panel design.
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