The growing field of advanced cell therapies, including CAR-T and modified NK cells, demands comprehensive phenotypic and functional characterisation. In this context, multiplex assays have become essential tools for obtaining multidimensional data in a single analysis, enabling simultaneous assessment of functional, activation, and viability parameters.
However, optimising these assays requires a robust strategy that correctly combines marker panels, calibration beads, and compensation parameters to ensure both reproducibility and sensitivity.
The foundation of any effective multiplex assay lies in a rational marker panel design. In the context of cell therapy development, the goal is to simultaneously assess:
Expression of activation markers (CD69, CD25, CD137)
Presence of exhaustion molecules (PD-1, TIM-3, LAG-3)
Production of intracellular cytokines (IFN-γ, TNF-α, IL-2)
Cell viability and proliferation status
To achieve optimal resolution, fluorochromes with minimal spectral overlap should be selected according to the laser configuration and dynamic range of the cytometer used. Combining bright fluorochromes (such as PE or APC) for low-expression markers and stable fluorochromes (FITC, PerCP-Cy5.5) for highly expressed markers enhances overall assay performance.
Calibration beads are indispensable for converting fluorescence signals into standardised quantitative units, ensuring comparability between experiments, days, and instruments.
Their use in multiplex assay optimisation serves two primary purposes:
Adjusting detector linearity and sensitivity, by establishing calibration curves for each fluorescence channel.
Controlling inter-instrument variability, ensuring that shifts in gain or optical alignment do not affect biological interpretation.
When control beads and compensation beads are properly combined, the system achieves complete harmonisation, facilitating reliable comparison of multiple experiments under consistent conditions.
The real power of multiplex assays is realised when functional markers, calibration beads, and advanced gating strategies are integrated to provide a comprehensive view of cellular behaviour.
By using multidimensional analysis software such as FlowJo, Cytobank, or SpectroFlo, researchers can integrate date on mean fluorescence intensity (MFI), subpopulation frequency, and functional correlations to generate unique immunophenotypic signatures for each treatment.
A well-optimised multiplex assay can simultaneously reveal:
Changes in activation or exhaustion profiles of CAR-T cells
Cytotoxic capacity against target antigens
All this can be achieved in a single experimental acquisition, reducing both sample consumption and analysis time.
To ensure the highest levels of reproducibility and sensitivity in multiplex assays, we recommend:
Validate each antibody individually before integrating it into the full panel.
Use daily control beads to adjust PMT voltage and gain settings.
Implement FMO (Fluorescence Minus One) controls to correctly define gating boundaries.
Record all instrument settings and compensation parameters for each run.
These practices not only ensure analytical reliability but also enhance traceability in GMP or quality control environments.
The optimisation of multiplex assays for cell therapies is a critical step towards the comprehensive characterisation of advanced cell-based products. The intelligent integration of markers, fluorochromes, and calibration beads provides a truly multidimensional insight into cellular states, driving both basic research and quality control inadvances therapy manufacturing.