Panel Design and Optimization for High‐Dimensional Immunophenotyping Assays Using Spectral Flow Cytometry

Abstract

Technological advances in fluorescence flow cytometry and an ever‐expanding understanding of the complexity of the immune system have led to the development of large (20+ parameters) flow cytometry panels. However, as panel complexity and size increase, so does the difficulty involved in designing a high‐quality panel, accessing the instrumentation capable of accommodating large numbers of parameters, and analyzing such high‐dimensional data. A recent advancement is spectral flow cytometry, which in contrast to conventional flow cytometry distinguishes the full emission spectrum of each fluorophore across all lasers, rather than identifying only the peak of emission. Fluorophores with a similar emission maximum but distinct off‐peak signatures can therefore be accommodated within the same flow cytometry panel, allowing greater flexibility in terms of panel design and fluorophore detection. Here, we highlight the specific characteristics of spectral flow cytometry and aim to guide users through the process of building, designing, and optimizing high‐dimensional spectral flow cytometry panels using a comprehensive step‐by‐step protocol. Special considerations are also given for using highly overlapping dyes, and a logical selection process for optimal marker‐fluorophore assignment is provided. © 2020 by John Wiley & Sons, Inc.

New cytometry tools for immune monitoring during cancer immunotherapy

“…Establishing functional SFC unmixing controls requires more time and care upfront than CFC compensation, because it is optimal to use cells identically treated to the sample, rather than beads, as slight discrepancies in signal signatures between the controls and the sample can cause unmixing of the sample to fail. Fortunately, on newer machines, the controls normalize to the detector gains during the daily instrument set‐up so that it is not necessary to re‐acquire the controls alongside the sample for every assay, which may save the analyst significant time when repeatedly performing the same high‐dimensional panel (Ferrer‐Font et al, 2020). It will take time for cytometrists to learn the nuances of SFC assays and fully determine the technical and operational advantages and disadvantages; for now, it is likely that SFC will complement rather than replace CFC technology.…”

“…Fortunately, on newer machines, the controls normalize to the detector gains during the daily instrument set-up so that it is not necessary to re-acquire the controls alongside the sample for every assay, which may save the analyst significant time when repeatedly performing the same high-dimensional panel (Ferrer-Font et al, 2020). It will take time for cytometrists to learn the nuances of SFC assays and fully determine the technical and operational advantages and disadvantages; for now, it is likely that SFC will complement rather than replace CFC technology.…”

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“…Related to the staining protocol: It is recommended that for the selection of the fluorochrome that will be coupled to the mAb, the intensity of expression of the molecule to be evaluated is considered; thus, with regard to molecules with abundant expression, it is preferable to use low-bright fluorochromes such as those of the blue laser (488 nm) and red (633 nm), while for molecules with minimal or unknown expression, it is preferable to use bright fluorochromes such as those excited by the violet laser (405 nm) [36]. However, each commercial house establishes its own rules for the use of its mAbs and fluorochromes, so it is essential to read their recommendations.…”

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“…For example, fluorochromes such as APC and Alexa Fluor® 647, or PerCP-Cy®5.5 and PerCP-eFluor® 710 (Fig. 1C) can be used in the same panel if other factors such as SS, antigen coexpression, and antigen density are taken into account in the panel design (16,17). These new possibilities will enable the expansion of multicolor immunophenotyping panels to the 35-40 color range with currently available fluorochromes.…”

“…While, in the cases presented in this article, this did not significantly impact the overall results, as panels become more complex the reduction in SS and concomitant increase in resolution will play a major role in achieving fully resolvable populations as panels move into the 30-40 color range. Careful panel design becomes extremely critical to ensure data quality is adequate to identify all populations of interest (17).…”