[Microbeads and flow cytometry: how and why put the "-metry" in immuno-cytometry?]

Area of Science:

• Immunophenotyping
• Cellular biology
• Quantitative analysis

Context:

• Flow cytometers are primarily used as cell counters in immunophenotyping.
• These instruments possess the capability to measure fluorescence intensity, enabling molecule counting on individual cells.
• The biological significance of membrane receptors is often tied to the number of accessible molecules.

Purpose:

• To review essential features for reliable quantitation of cellular antigens.
• To advocate for the use of real units (molecules per cell) instead of arbitrary units.
• To highlight the importance of calibration tools for accurate measurements.

Summary:

• This paper discusses reliable methods for quantifying cellular antigens using flow cytometry, expressing results as molecules per cell.
• It emphasizes the need for standardized calibration tools beyond fluorescent beads, focusing on immunological calibration systems.
• The review covers staining reagents, data analysis software, and instrument quality control for reproducible results.

Impact:

• Facilitating a shift from arbitrary units to absolute quantification in clinical biology.
• Enhancing the reliability and reproducibility of immunophenotyping across different platforms and laboratories.
• Broadening access to advanced clinical biology insights through the generalization of quantitative immunocytometry.