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Clinical flow cytometry is a critical tool for immune-oncology research because it enables the measurement of different cell types and their functional status. This type of flow cytometry data is critical to evaluating therapeutic effects of experimental therapies in animal models as well as assessing clinical responses. Clinical flow cytometry requires the development of validated panels, which are comprised of fluorescently labeled antibodies that bind to specific molecules on different cell subsets. Here we highlight essential considerations for the development and validation of a clinical flow cytometry panel.
Hardware Considerations
The development of any flow cytometry panel begins with understanding the parameters of your flow cytometer[1]. Flow cytometry is based on the method of staining cells with fluorochrome-conjugated antibodies, which are excited by specific light wavelengths emitted by lasers within the cytometer. Excitation of these fluorescent dyes causes emission of photons at discrete wavelengths that are detected and used to identify cells bound by these antibodies. Flow cytometers typically have two or more lasers as well as optical filters and detectors that are optimized to detect different light wavelengths. The design of any clinical flow cytometry panel is limited by the wavelengths that can be detected by a flow cytometer and the limitations caused by spectral overlap between fluorescent dyes that emit photons at similar wavelengths. There are several widely used fluorochromes that are conjugated to antibodies for flow cytometry, including FITC, phycoerythrin (PE), and allophycocyanin (APC). Advances in fluorochrome technology include the development of fluorochrome conjugates like Cy5.5-PE and the use of quantum dots like QDot800, and these newer fluorochromes have enabled the development of panels with more colors.
Cell Specifications
The primary goal of most flow cytometry studies for immune-oncology research is to monitor the immune system status longitudinally before and during treatment[2]. To this end, certain cell subsets are more critical to monitor in terms of treatment efficacy or adverse events. Most clinical flow cytometry panels for immuno-oncology studies track multiple T cell subsets, including T helper 1 (Th1), T helper 2 (Th2), and regulatory T (Treg) cells. Other lymphocyte subsets, including B cells, natural killer (NK) cells, NKT cells, and mucosal-associated innate-like T cells (MAIT), are also sometimes included, as well as myeloid cell subsets, including monocyte and dendritic cell (DC) subtypes, neutrophils, and eosinophils. Beyond identification of different cell types, flow cytometry panels can also be used to characterize functions of cells based on expression of different markers, including activation, memory, and exhaustion. Flow cytometry has become instrumental in evaluating responses to immune checkpoint blockade treatments, particularly when screening tumor-infiltrating lymphocytes for expression of molecules like PD-1 and CTLA-4 to predict treatment efficacy[3]. Another key consideration when defining a panel is to determine the type of sample being stained, whether it is whole blood, peripheral blood mononuclear cells, or dissociated tumor tissues, as sample processing and storage can alter the detection of some cell types.
Clinical flow cytometry panel design does not differ greatly from panel design for basic research in terms of identifying reagents and optimizing protocols. The main difference is the need for assay validation for clinical flow cytometry protocols. Validation assures that flow cytometry assays carried at different times or on different cytometers will meet predetermined standards of sensitivity, specificity, accuracy, precision, and limits of detection. Assay validation is a continuous process to assure that different batches of fluorochrome-conjugated antibodies are performing within these standards, and different users can produce reliable data regardless of location or time of experiment. Assay validation for clinical flow cytometry can be challenging given the inherent variability of working with human samples, and commercially available antibodies do not always perform consistently given differences in dye chemistry[4]. Most clinical flow cytometry protocols are validated externally to assure that assays meet regulatory requirements needed for clinical trials.
The above considerations are essential to developing a clinical flow cytometry panel, and these issues may require the guidance or involvement of clinical flow cytometry experts and access to a validated cytometer. Seeking external support for clinical flow cytometry panel development and validation can assure that your flow cytometry data will be accurate, consistent, and will satisfy regulatory requirements.
[1] Maecker HT, McCoy JP, Nussenblatt R. Standardizing immunophenotyping for the Human Immunology Project [published correction appears in Nat. Rev. Immunol. 2012 Jun;12(6):471]. Nat. Rev. Immunol. 2012;12(3):191-200.
[2] Pitoiset F, Cassard L, El Soufi K, Boselli L, Grivel J, Roux A, Klatzmann D, Chaput N, Rosenzwajg M. Deep phenotyping of immune cell populations by optimized and standardized flow cytometry analyses. Cytometry A. 2018 Aug;93(8):793-802.
[3] Nishino M, Ramaiya NH, Hatabu H, Hodi FS. Monitoring immune-checkpoint blockade: response evaluation and biomarker development. Nat. Rev. Clin. Oncol. 2017;14(11):655-668.
[4] Kalina T, Lundsten K, Engel P. Relevance of Antibody Validation for Flow Cytometry. Cytometry A. 2020 Feb;97(2):126-136. doi: 10.1002/cyto.a.23895.
