Cytokine quantification is a cornerstone of immunology, oncology, and advanced cell therapies. In complex clinical scenarios such as cytokine release syndrome (CRS) associated with CAR-T therapies or bispecific antibodies, the ability to measure multiple analytes with high sensitivity using microvolume samples has become a critical requirement. In this context, advanced multiplexing technologies have rapidly evolved, with platforms such as Luminex, Olink, and Simoa standing out.
Modern assays increasingly demand more information from limited sample volumes, particularly in longitudinal studies, pediatric settings, or preclinical research. Multiplexing enables the simultaneous analysis of dozens of cytokines, reducing sample consumption, assay time, and experimental variability. However, not all technologies provide the same balance between sensitivity, dynamic range, and robustness to matrix effects.
Luminex technology, based on fluorescently coded microspheres, has long been a standard in multiplex cytokine assays. It allows simultaneous detection of multiple cytokines in a single well, offering a solid balance between cost, flexibility, and throughput. Nevertheless, its limit of detection may be insufficient for low-abundance cytokines that are clinically relevant in the early stages of CRS.
In contrast, Olink employs a Proximity Extension Assay (PEA) approach, where dual antibody recognition is coupled with PCR-based signal amplification. This technology excels in high sensitivity, strong performance in microvolume samples, and reduced susceptibility to matrix interference. However, its closed format and reliance on predefined panels may limit experimental customization.
At the high end of sensitivity, Simoa (Single Molecule Array) enables cytokine detection at femtomolar concentrations by reading individual molecules. This ultra-sensitive capability is particularly relevant for early CRS biomarkers, although it is often accompanied by greater instrumental complexity and lower multiplexing capacity compared to other platforms.
In the context of cytokine release syndrome, early detection of mediators such as IL-6, IL-1β, IFN-γ, and TNF-α can be decisive for clinical decision-making. Assays must achieve very low limits of detection while maintaining accuracy and reproducibility. This is where technologies like Olink and Simoa offer clear advantages over more conventional multiplex platforms, particularly during the early phases of CRS.
A common challenge in real-world clinical studies is sample quality. Hemolysis and lipemia can introduce significant interferences in cytokine quantification, affecting both signal intensity and assay specificity. For this reason, validation using compromised samples is a critical step in any robust analytical workflow.
Advanced multiplex platforms should be evaluated not only under ideal conditions but also in the presence of complex biological matrices, ensuring that results remain interpretable and comparable. Strategies such as internal controls, recovery studies, and interference testing are essential to guarantee data reliability, especially in translational research setting.
Advanced multiplexing for cytokine quantification in microvolumes has become an indispensable tool in immunology and cell therapy research. The choice between Luminex, Olink, and Simoa should be guided by the required limit of detection, number of analytes, sample availability, and clinical context of the study. In demanding applications such as CRS assays, sensitivity, robustness to sample quality issues, and reproducibility are key factors for generating clinically meaningful data.