A 19-color single-tube full spectrum flow cytometry assay for the detection of measurable residual disease in acute myeloid leukemia.

Multiparameter flow cytometry (MFC) has emerged as a standard method for quantifying measurable residual disease (MRD) in acute myeloid leukemia. However, the limited number of available channels on conventional flow cytometers requires the division of a diagnostic sample into several tubes, restricting the number of cells and the complexity of immunophenotypes that can be analyzed. Full spectrum flow cytometers overcome this limitation by enabling the simultaneous use of up to 40 fluorescent markers. Here, we used this approach to develop a good laboratory practice-conform single-tube 19-color MRD detection assay that complies with recommendations of the European LeukemiaNet Flow-MRD Working Party. We based our assay on clinically-validated antibody clones and evaluated its performance on an IVD-certified full spectrum flow cytometer. We measured MRD and normal bone marrow samples and compared the MRD data to a widely used reference MRD-MFC panel generating highly concordant results. Using our newly developed single-tube panel, we established reference values in healthy bone marrow for 28 consensus leukemia-associated immunophenotypes and introduced a semi-automated dimensionality-reduction, clustering and cell type identification approach that aids the unbiased detection of aberrant cells. In summary, we provide a comprehensive full spectrum MRD-MFC workflow with the potential for rapid implementation for routine diagnostics due to reduced cell requirements and ease of data analysis with increased reproducibility in comparison to conventional FlowMRD routines.

Fluorochrome-dependent specific changes in spectral profiles using different compensation beads or primary cells in full spectrum cytometry.

Full spectrum flow cytometry is a powerful tool for immune monitoring on a single-cell level and with currently available machines, panels of 40 or more markers per sample are possible. However, with an increased panel size, spectral unmixing issues arise, and appropriate single stain reference controls are required for accurate experimental results and to avoid unmixing errors. In contrast to conventional flow cytometry, full spectrum flow cytometry takes into account even minor differences in spectral signatures and requires the full spectrum of each fluorochrome to be identical in the reference control and the fully stained sample to ensure accurate and reliable results. In general, using the cells of interest is considered optimal, but certain markers may not be expressed at sufficient levels to generate a reliable positive control. In this case, compensation beads show some significant advantages as they bind a consistent amount of antibody independent of its specificity. In this study, we evaluated two types of manufactured compensation beads for use as reference controls for 30 of the most commonly used and commercially available fluorochromes in full spectrum cytometry and compared them to human and murine primary leukocytes. While most fluorochromes show the same spectral profile on beads and cells, we demonstrate that specific fluorochromes show a significantly different spectral profile depending on which type of compensation beads is used, and some fluorochromes should be used on cells exclusively. Here, we provide a list of important considerations when selecting optimal reference controls for full spectrum flow cytometry.

Within- and between-subject biological variation estimates for the enumeration of lymphocyte deep immunophenotyping and monocyte subsets.

OBJECTIVES: This study aimed to deliver biological variation (BV) estimates for 25 types of lymphocyte subpopulations subjected to deep immunophenotyping (memory T/B cells, regulatory T cells, etc.) and classical, intermediate, and nonclassical monocyte subsets based on the full spectrum flow cytometry (FS-FCM) and a Biological Variation Data Critical Appraisal Checklist (BIVAC) design. METHODS: Samples were collected biweekly from 60 healthy Chinese adults over 10 consecutive two-week periods. Each sample was measured in duplicate within a single run for lymphocyte deep immunophenotyping and monocyte subset determination using FS-FCM, including the percentage (%) and absolute count (cells/µL). After trend adjustment, a Bayesian model was applied to deliver the within-subject BV (CVI) and between-subject BV (CVG) estimates with 95 % credibility intervals. RESULTS: Enumeration (% and cells/µL) for 25 types of lymphocyte deep immunophenotyping and three types of monocyte subset percentages showed considerable variability in terms of CVI and CVG. CVI ranged from 4.23 to 47.47 %. Additionally, CVG ranged between 10.32 and 101.30 %, except for CD4+ effector memory T cells re-expressing CD45RA. No significant differences were found between males and females for CVI and CVG estimates. Nevertheless, the CVGs of PD-1+ T cells (%) may be higher in females than males. Based on the desired analytical performance specification, the maximum allowable imprecision immune parameter was the CD8+PD-1+ T cell (cells/µL), with 23.7 %. CONCLUSIONS: This is the first study delivering BV estimates for 25 types of lymphocyte subpopulations subjected to deep immunophenotyping, along with classical, intermediate, and nonclassical monocyte subsets, using FS-FCM and adhering to the BIVAC design.

Multicompartmental analysis of the murine pulmonary immune response by spectral flow cytometry.

Studies of pulmonary inflammation require unique considerations due to the complex structure and composition of the lungs. The lungs have multiple compartments and diverse immune cell populations, with inherently high autofluorescence, and are involved in the host response to pulmonary pathogens. We describe a protocol that accounts for these factors through a novel combination of methodologies-in vivo compartmental analysis and spectral flow cytometry with a broad panel of antibodies. In vivo compartmental analysis enables the precise localization of immune cells within the marginated vasculature, lung interstitium, nonlavageable airways, and lavageable airways of the lungs, as well as the pulmonary lymph nodes. Spectral flow cytometry with a broad panel of antibodies supports an unbiased exploratory approach to investigating diverse immune cell populations during pulmonary inflammation. Most importantly, spectral flow uses cellular autofluorescence to aid in the resolution and identification of immune cell populations. This methodology enables the acquisition of high-quality data compatible with informed gating and dimensionality reduction algorithms. In addition, our protocol emphasizes considerations for compartmentalization of the inflammatory response, spectral flow panel design, and autofluorescence spectra analysis. These methodologies are critical for increasing the rigor of pulmonary research. We apply this protocol for the precise characterization and localization of leukocytes in the pulmonary host response to influenza A virus in C57BL/6J mice. In particular, we demonstrate that this protocol improves the quantification and localization of alveolar macrophages within the airways. The methodology is modifiable and expandable to allow for further characterization of leukocyte populations of special interest.NEW & NOTEWORTHY We describe a novel combination of methodologies that incorporates dual in vivo compartmental analysis using intravascular and intratracheal CD45 labeling, a broad panel of antibodies for identifying lymphoid and nonlymphoid cells, and spectral flow cytometry that uses cellular autofluorescence to aid in resolving and identifying immune cell populations. This methodology allows precise localization of immune cells in the lavageable airways, nonlavageable airways, interstitial lung tissue, and marginated in the lung vasculature.

OMIP-086: Full spectrum flow cytometry for high-dimensional immunophenotyping of mouse innate lymphoid cells.

This 25-parameter, 22-color full spectrum flow cytometry panel was designed and optimized for the comprehensive enumeration and functional characterization of innate lymphoid cell (ILC) subsets in mouse tissues. The panel presented here allows the discrimination of ILC progenitors (ILCP), ILC1, ILC2, NCR+ ILC3, NCR- ILC3, CCR6+ lymphoid tissue-inducer (LTi)-like ILC3 and mature natural killer (NK) cell populations. Further characterization of ILC and NK cell functional profiles in response to stimulation is provided by the inclusion of subset-specific cytokine markers, and proliferation markers. Development and optimization of this panel was performed on freshly isolated cells from adult BALB/c lungs and small intestine lamina propria, and ex vivo stimulation with phorbol 12-myrisate 13-acetate, ionomycin, and pro-ILC activating cytokines.

OMIP-095: 40-Color spectral flow cytometry delineates all major leukocyte populations in murine lymphoid tissues.

High-dimensional immunoprofiling is essential for studying host response to immunotherapy, infection, and disease in murine model systems. However, the difficulty of multiparameter panel design combined with a lack of existing murine tools has prevented the comprehensive study of all major leukocyte phenotypes in a single assay. Herein, we present a 40-color flow cytometry panel for deep immunophenotyping of murine lymphoid tissues, including the spleen, blood, Peyer's patches, inguinal lymph nodes, bone marrow, and thymus. This panel uses a robust set of surface markers capable of differentiating leukocyte subsets without the use of intracellular staining, thus allowing for the use of cells in downstream functional experiments or multiomic analyses. Our panel classifies T cells, B cells, natural killer cells, innate lymphoid cells, monocytes, macrophages, dendritic cells, basophils, neutrophils, eosinophils, progenitors, and their functional subsets by using a series of co-stimulatory, checkpoint, activation, migration, and maturation markers. This tool has a multitude of systems immunology applications ranging from serial monitoring of circulating blood signatures to complex endpoint analysis, especially in pre-clinical settings where treatments can modulate leukocyte abundance and/or function. Ultimately, this 40-color panel resolves a diverse array of immune cells on the axes of time, tissue, and treatment, filling the niche for a modern tool dedicated to murine immunophenotyping.

Development of a 27-color panel for the detection of measurable residual disease in patients diagnosed with acute myeloid leukemia.

Acute myeloid leukemia (AML) measurable residual disease (MRD) evaluated by multiparametric flow cytometry (MFC) is a surrogate for progression-free and overall survival in clinical trials and patient management. Due to the limited number of detection channels available in conventional flow cytometers, panels used for assessing AML MRD are typically split into multiple tubes. This cripples the simultaneous and correlated assessment of all myeloblast measurements. In response, we prototyped a single-tube 27-color MFC assay for the evaluation of AML MRD, incorporating all recommended markers. Marrow aspirates from 22 patients were processed for analysis using full spectrum flow cytometry (FSFC). The signal resolution of each marker was compared between samples stained with single antibody vs. the fully stained panel. The analytical accuracy for quantifying hematopoietic cells between our established 8-color assay and the new 27-color method were compared. Variations within an operator and between separate operators were assessed to evaluate the assays reproducibility. The limited of blank (LOB), limit of detection (LOD), and lower limit of quantification (LLOQ) of the 27-color method were empirically determined using limiting dilution experiments. The stability of antibody cocktails over a period of 120 h was also studied using cryopreserved marrow cells. The stain indices for all antibodies were lower in the fully stained panel compared to cells stained with one antibody but clear separations between negative and positive signals were achieved for all antibodies. Our results demonstrated a high concordance between the established 8-color method and the new 27-color assay for enumerating myeloblasts and MRD interpretation within and between operators. The data further showed that the single-tube 27-color assay easily achieved the minimum required detection sensitivity of 0.1%. When antibodies were combined, however, expression intensity of some antigens deteriorated significantly when stored. Our single-tube 27-color panel is a suitable, high sensitivity flow cytometric approach that can be used for AML MRD testing, which improves the correlation of aberrant antigens and detection of asynchronous differentiation patterns. Based on the stability study, we recommend the full panel be made prior to staining.

Full spectrum flow cytometry and mass cytometry: A 32-marker panel comparison.

High-dimensional single-cell data has become an important tool in unraveling the complexity of the immune system and its involvement in homeostasis and a large array of pathologies. As technological tools are developed, researchers are adopting them to answer increasingly complex biological questions. Up until recently, mass cytometry (MC) has been the main technology employed in cytometric assays requiring more than 29 markers. Recently, however, with the introduction of full spectrum flow cytometry (FSFC), it has become possible to break the fluorescence barrier and go beyond 29 fluorescent parameters. In this study, in collaboration with the Stanford Human Immune Monitoring Center (HIMC), we compared five patient samples using an established immune panel developed by the HIMC using their MC platform. Using split samples and the same antibody panel, we were able to demonstrate highly comparable results between the two technologies using multiple data analysis approaches. We report here a direct comparison of two technology platforms (MC and FSFC) using a 32-marker flow cytometric immune monitoring panel that can identify all the previously described and anticipated immune subpopulations defined by this panel.

OMIP-084: 28-color full spectrum flow cytometry panel for the comprehensive analysis of human γδ T cells.

Using full spectrum flow cytometry, we designed a 28-color panel for the analysis of markers known to be associated with the γδ T cell immune response. This panel allows the classification of γδ T cell subsets via specific V gene usage (Vγ9, Vδ1, Vδ2, and Vδ3) of their T cell receptor (TCR) and according to their functional differentiation. Phenotypical surface receptors to distinguish different stages of cell maturation included CD45RA, CD27, CD28, CD127, CD57, and CD16; chemokine receptors CXCR6, CCR5, CCR6, and CX3CR1; NK-associated markers NKG2A, NKG2D, CD56, and CD161, checkpoint-inhibitor PD-1, and activating receptors CD38 and CD25. T cell lineage markers for the analysis of αß T cells (CD4 and CD8) and MAIT cells (Vα7.2) were also included. This optimized multicolor panel allows a comprehensive immune-profiling of all main human γδ T cell subsets and is suitable for longitudinal or exploratory analysis of γδ T cell development and γδ T cell dynamics in clinical cohorts.

A comprehensive 26-color immunophenotyping panel to study the role of the gut-liver axis in chronic liver diseases.

The gut-liver axis includes the bidirectional communication between the gut and the liver, and thus covers signals from liver-to-gut and from gut-to-liver. Disruptions of the gut-liver axis have been associated with the progression of chronic liver diseases, including alcohol-related and metabolic dysfunction-associated steatotic liver disease and cholangiopathies. Immune cells and their expression of pattern recognition receptors, activation markers or immune checkpoints might play an active role in the communication between gut and liver. Here, we present a 26-color full spectrum flow cytometry panel for human cells to decipher the role of circulating immune cells in gut-liver communication during the progression of chronic liver diseases in a non-invasive manner, which has been optimized to be used on patient-derived whole blood samples, the most abundantly available clinical material. Our panel focuses on changes in pattern recognition receptors, including toll-like receptors (TLRs) or Dectin-1, and also includes other immunomodulatory molecules such as bile acid receptors and checkpoint molecules. Moreover, this panel can be utilized to follow the progression of chronic liver diseases and could be used as a tool to evaluate the efficiency of therapeutic targets directed against microbial mediators or modulating immune cell activation.

Immunophenotyping challenging tissue types using high-dimensional full spectrum flow cytometry.

Technological advancements in fluorescence flow cytometry and an ever-expanding understanding of the complexity of the immune system, have led to the development of large flow cytometry panels, reaching up to 40 markers at the single-cell level. Full spectrum flow cytometry, that measures the full emission range of all the fluorophores present in the panel instead of only the emission peaks is now routinely used in many laboratories internationally, and the demand for this technology is rapidly increasing. With the capacity to use larger and more complex staining panels, optimized protocols are required for the best panel design, panel validation and high-dimensional data analysis outcomes. In addition, for ex vivo experiments, tissue preparation methods for single-cell analysis should also be optimized to ensure that samples are of the highest quality and are truly representative of tissues in situ. Here we provide optimized step-by-step protocols for full spectrum flow cytometry panel design, tissue digestion and panel optimization to facilitate the analysis of challenging tissue types.

Panel Design and Optimization for Full Spectrum Flow Cytometry.

Technological advancements in fluorescence flow cytometry and an ever-expanding understanding of the complexity of the immune system have led to the development of large flow cytometry panels, reaching up to 40 markers at the single-cell level. Full spectrum flow cytometry, which measures the full emission range of all the fluorophores present in the panel instead of only the emission peaks, is now routinely used in laboratories around the world, and the demand for this technology is rapidly increasing. With the ability to use larger and more complex staining panels, optimized protocols are vital for achieving the best panel design, panel optimization, and high-dimensional data analysis outcomes. In addition, a better understanding of how to fully characterize the autofluorescence of the sample, coupled with an intelligent panel design approach, allows improved marker resolution on highly autofluorescent tissues or cells. Here, we provide optimized step-by-step protocols for full spectrum flow cytometry, covering panel design and optimization, autofluorescence evaluation and strategy selection, and methods for performing longitudinal studies.

Resolving 31 colors on a standard 3-laser full spectrum flow cytometer for immune monitoring of human blood samples.

Immune monitoring of patients on a single-cell level is becoming increasingly important in various diseases. Due to the often very limited availability of human specimens and our increased understanding of the immune systems there is an increasing demand to analyze as many markers as possible simultaneously in one panel. Full spectrum flow cytometry is emerging as a powerful tool for immune monitoring since 5-laser instruments enable characterization of 40 parameters or more in a single sample. Nevertheless, even if only machines with fewer lasers are available, development of novel fluorophore families enables increasing panel sizes. Here, we demonstrate that careful panel design enables the use of 31-color panels on a 3-laser Cytek® Aurora cytometer for analyzing human peripheral blood leukocytes, without the need for custom configuration and using only commercially available fluorochromes. The panel presented here should serve as an example of a 31-fluorochrome combination that can be resolved on a 3-laser full spectrum cytometer and that can be adapted to comprise other (and possibly more) markers of interest depending on the research focus.

Ensuring Full Spectrum Flow Cytometry Data Quality for High-Dimensional Data Analysis.

Full spectrum flow cytometry (FSFC) allows for the analysis of more than 40 parameters at the single-cell level. Compared to the practice of manual gating, high-dimensional data analysis can be used to fully explore single-cell datasets and reduce analysis time. As panel size and complexity increases so too does the detail and time required to prepare and validate the quality of the resulting data for use in downstream high-dimensional data analyses. To ensure data analysis algorithms can be used efficiently and to avoid artifacts, some important steps should be considered. These include data cleaning (such as eliminating variable signal change over time, removing cell doublets, and antibody aggregates), proper unmixing of full spectrum data, ensuring correct scale transformation, and correcting for batch effects. We have developed a methodical step-by-step protocol to prepare full spectrum high-dimensional data for use with high-dimensional data analyses, with a focus on visualizing the impact of each step of data preparation using dimensionality reduction algorithms. Application of our workflow will aid FSFC users in their efforts to apply quality control methods to their datasets for use in high-dimensional analysis, and help them to obtain valid and reproducible results. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Data cleaning Basic Protocol 2: Validating the quality of unmixing Basic Protocol 3: Data scaling Basic Protocol 4: Batch-to-batch normalization.

Twenty-four-color full spectrum flow cytometry panel for minimal residual disease detection in acute myeloid leukemia.

Full spectrum flow cytometry brings a breakthrough for minimal residual disease (MRD) detection in acute myeloid leukemia (AML). We aimed to explore the role of a new panel in MRD detection. We established a 24-color full-spectrum flow cytometry panel. A tube of 24-color antibodies included CD45, CD117, CD34, HLA-DR, CD15, CD64, CD14, CD11c, CD11b, CD13, CD33, CD371, CD7, CD56, CD19, CD4, CD2, CD123, CD200, CD38, CD96, CD71, CD36, and CD9. We discovered that when a tube meets 26 parameters (24 colors), these markers were not only limited to the observation of MRD in AML, but also could be used for fine clustering of bone marrow cells. Mast cells, basophils, myeloid dendritic cells, and plasmacoid dendritic cells were more clearly observed. In addition, immune checkpoint CD96 had the higher expression in CD117+ myeloid naive cells and CD56dimNK cells, while had the lower expression in CD56briNK cells in AML-MRD samples than in normal bone marrow samples. CD200 expression was remarkably enhanced in CD117+ myeloid naive cells, CD4+ T cells, T cells, activated T cells, CD56dimNK cells, and CD56briNK cells in AML-MRD samples. Our results can be used as important basis for auxiliary diagnosis, prognosis judgment, treatment guidance, and immune regulation in AML.

Using Full-Spectrum Flow Cytometry to Phenotype Memory T and NKT Cell Subsets with Optimized Tissue-Specific Preparation Protocols.

Full-spectrum flow cytometry is now routinely used in many laboratories internationally, and the demand for this technology is rapidly increasing. With capacity to use larger and more complex staining panels, standardized protocols are required for optimal panel design and analysis. Importantly, for ex vivo analysis, tissue preparation methods also need to be optimized to ensure samples are truly representative of tissues in situ. This is particularly relevant given the recent interest in adaptive immune cells that form residency in specific organs. Here we provide optimized protocols for tissue processing and phenotyping of memory T cells and natural killer T (NKT) cell subsets from liver, lung, spleen, and lymph node using full-spectrum flow cytometry. We provide a 21-color antibody panel for identification of different memory subsets, including tissue-resident memory T (TRM ) cells, which are increasingly regarded as important effectors in adaptive immunity. We show that processing procedures can affect outcomes, with liver TRM cells particularly sensitive to heat, such that accurate evaluation requires fast processing at defined temperatures. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Processing mouse liver for flow cytometric analysis of memory T and NKT cell subsets Basic Protocol 2: Processing mouse spleen for flow cytometric analysis of memory T and NKT cell subsets Basic Protocol 3: Processing mouse lungs for flow cytometric analysis of memory T and NKT cell subsets Basic Protocol 4: Processing mouse lymph nodes for flow cytometric analysis of memory T and NKT cell subsets Basic Protocol 5: Staining and flow cytometric analysis of samples for memory T and NKT cell subsets Support Protocol: Obtaining cell counts from flow cytometry data.

Panel Optimization for High-Dimensional Immunophenotyping Assays Using Full-Spectrum Flow Cytometry.

Technological advancements in fluorescence flow cytometry and an ever-expanding understanding of the complexity of the immune system have led to the development of large flow cytometry panels reaching up to 43 colors at the single-cell level. However, as panel size and complexity increase, so too does the detail involved in designing and optimizing successful high-quality panels fit for downstream high-dimensional data analysis. In contrast to conventional flow cytometers, full-spectrum flow cytometers measure the entire emission spectrum of each fluorophore across all lasers. This allows for fluorophores with very similar emission maxima but unique overall spectral fingerprints to be used in conjunction, enabling relatively straightforward design of larger panels. Although a protocol for best practices in full-spectrum flow cytometry panel design has been published, there is still a knowledge gap in going from the theoretically designed panel to the necessary steps required for panel optimization. Here, we aim to guide users through the theory of optimizing a high-dimensional full-spectrum flow cytometry panel for immunophenotyping using comprehensive step-by-step protocols. These protocols can also be used to troubleshoot panels when issues arise. A practical application of this approach is exemplified with a 24-color panel designed for identification of conventional T-cell subsets in human peripheral blood. © 2021 Malaghan Institute of Medical Research, Cytek Biosciences. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation and evaluation of optimal spectral reference controls Support Protocol 1: Antibody titration Support Protocol 2: Changing instrument settings Basic Protocol 2: Unmixing evaluation of fully stained sample Basic Protocol 3: Evaluation of marker resolution Support Protocol 3: Managing heterogeneous autofluorescence Basic Protocol 4: Assessment of data quality using expert gating and dimensionality reduction algorithms.