The Nobel Prize-deserving concept of blocking inhibitory pathways in T cells, to unleash their anti-tumoral capacity, became one of the pillars of cancer treatment in the last decade and has resulted in durable clinical responses for multiple cancer types. Currently, two of the most important goals in cancer immunotherapy are to understand the mechanisms resulting in failure to checkpoint blockade and to identify predictive immunological biomarkers that correlate to treatment response, disease progression or adverse effects. The identification and validation of biomarkers for routine clinical use is not only critical to monitor disease or treatment progression, but also to personalize and develop new therapies. To achieve these goals, powerful research tools are needed. Flow cytometry stands as one of the most successful single-cell analytical tools used to characterize immune cell phenotypes to monitor solid tumors, hematological malignancies, minimal residual disease or metastatic progression. This technology has been fundamental in diagnosis, treatment and translational research in cancer clinical trials. Most recently, the need to evaluate simultaneously more features in each cell has pushed the field to implement more powerful adaptations beyond conventional flow cytometry, including Full Spectrum Flow Cytometry (FSFC). FSFC captures the full emission spectrum of fluorescent molecules using arrays of highly sensitive light detectors, and to date has enabled characterization of 40 parameters in a single sample. We will summarize the contributions of this technology to the advancement of research in immunotherapy studies and discuss best practices to obtain reliable, robust and reproducible FSFC results.

Flow cytometry facilitates the detection of multiple cell parameters simultaneously with a high level of resolution and throughput, enabling in-depth immunological evaluations. High data resolution in flow cytometry depends on multiple factors, including the concentration of reagents used in the staining protocol, and reagent validation and titration should be the first step in any assay optimization. Titration is the process of finding the concentration of the reagent that best resolves a positive signal from the background, with the saturation of all binding sites, and minimal antibody excess. The titration process involves the evaluation of serial reagent dilutions in cells expressing the antigen target for the tested antibody. The concentration of antibody that provides the highest signal to noise ratio is calculated by plotting the percentage of positive cells and the intensity of the fluorescence of the stained cells with respect to the negative events, in a concentration–response curve. The determination of the optimal antibody concentration is necessary to ensure reliable and reproducible results and is required for each sample type, reagent clone and lot, as well as the methods used for cell collection, staining, and storage conditions. If the antibody dilution is too low, the signal will be too weak to be accurately determined, leading to suboptimal data resolution, high variability across measurements, and the underestimation of the frequency of cells expressing a specific marker. The use of excess antibodies could lead to non-specific binding, reagent misuse, and detector overloading with the signal off scale and higher spillover spreading. In this publication, we summarized the titration fundamentals and best practices, and evaluated the impact of using a different instrument, sample, staining, acquisition, and analysis conditions in the selection of the optimal titer and population resolution.

The need for more in-depth exploration of the human immune system has moved the flow cytometry field forward with advances in instrumentation, reagent development and user-friendly implementations of data analysis methods. The increase in the number of markers evaluated simultaneously requires a careful selection of highly overlapping dyes to avoid introducing detrimental spread and compromising population resolution. In this manuscript, we present the strategy used in the development of a high-quality human 45-color panel which allows for comprehensive characterization of major cell lineages present in circulation including T cells, gamma delta T cells, NKT-like cells, B cells, NK cells, monocytes, basophils, dendritic cells, and ILCs, as well as more in-depth characterization of memory T cells. The steps taken to ensure that each marker in the panel was optimally resolved are discussed in detail. We highlight the outstanding discernment of cell activation, exhaustion, memory, and differentiation states of CD4+ and CD8+ T cells using this 45-color panel, enabling an in-depth description of very distinct phenotypes associated with the complexity of the T cell memory response. Furthermore, we present how this panel can be effectively used for cell sorting on instruments with a similar optical layout to achieve the same level of resolution. Functional evaluation of sorted specific rare cell subsets demonstrated significantly different patterns of immunological responses to stimulation, supporting functional and phenotypic differences within the T cell memory subsets. In summary, the combination of flow cytometry full spectrum technology, careful assay design and optimization, results in high resolution multiparametric assays. This approach offers the opportunity to fully characterize immunological profiles present in peripheral blood in the context of infectious diseases, autoimmunity, neurodegeneration, immunotherapy, and biomarker discovery. PURPOSE AND APPROPRIATE SAMPLE TYPES: This 45-color flow cytometry-based panel was developed as an expansion of the previously published OMIP-069 [1] and serves as an in-depth immunophenotyping of the major cell subsets present in human peripheral blood. The goal of this panel is to maximize the amount of high-quality data that can be acquired from a single sample, not only for more in-depth characterization of the immune system, but also to address the issue of limited sample availability. The panel’s development included identifying fluorochromes that could improve the performance of the original 40-color panel and expanding the number of markers for deeper delineation of memory status of T cell subpopulations. To increase the number of markers, it was critical that any expansion did not negatively impact the resolution and quality of the data. To achieve this, the fluorochrome combinations were carefully characterized to ensure optimal resolution of each marker. The panel allows for deep characterization of the major cell lineages present in circulation (CD4 T cells, CDS T cells, regulatory T cells, yo T cells, NKT-like cells, B cells, NK (Natural Killer) cells, monocytes, and dendritic cells), while also providing an in-depth characterization of the T cell compartment, with a combination of activation, inhibitory, exhaustion, and differentiation markers. The panel supports deep exploration of the memory status of CD4 ⁺ T cells, CDS ⁺ T cells, and NKT-like cells. The steps taken in the optimization of the panel ensured outstanding resolution of each marker within the multicolor panel and unequivocal identification of each cell subset. This panel design and optimization will enhance the ability to characterize immunological profiles present in peripheral blood in the context of oncology, infectious diseases, autoimmunity, neurodegeneration, immunotherapy, and biomarker discovery. The panel was developed using fresh and cryopreserved human peripheral blood mononuclear cells (PBMCs) from healthy adults. We have not tested the panel on whole blood or biopsies; hence it is anticipated that the panel might require further optimization to be used with other sample types.

Spectral flow cytometry has revolutionized traditional single-cell profiling to a new era of high-dimensional analysis, allowing for unprecedented deep phenotyping and more precise cell characterization, thereby significantly enhancing our multiplexing capability. The recent application of this technology in clinical settings has been redefining the landscape of clinical diagnostic panels and immune monitoring, particularly for hematologic malignancies, immunological disorders, and drug discovery. Emerging technologies like ghost cytometry, LASE, and imaging flow cytometry are advancing cytometry by improving sensitivity, throughput, and spatial resolution. In this review, we discuss the requirements, challenges, and considerations for spectral applications in clinical diagnostic laboratories and pharmaceutical/contract research organization (CRO) settings. We discuss how these recent innovations are set to push the boundaries of diagnostic accuracy and analytical power, heralding a new frontier in clinical cytometry with the potential to dramatically enhance patient care and treatment outcomes.