Poster #1:
Title: Development of high dimensional spectral flow cytometry panel for Immunophenotypic Characterization of fixed whole blood samples
Author: Vaishnavi Parthasarathy, Allen Institute for Immunology
Abstract: Cryopreserved peripheral blood mononuclear cells (PBMCs) are important for deep immune cell characterization in clinical trials, but standard PBMC isolation practices present logistical challenges, especially in underserved communities with limited access to research facilities. To overcome this, we utilized a whole blood fixation protocol that stabilizes surface protein antigens/markers rapidly and thereby enabling us to perform deep immunophenotyping. Here, we designed and optimized two 35 marker spectral flow cytometry panels to assess the distribution of backbone lineage cells, naïve and memory T cell populations, B cells and NK cells subsets, monocyte, dendritic cells and granulocytes. We address the challenges in panel design for fixed whole blood, the choice of antibody clones, strategies to overcome non-specific binding, and expression of certain chemokine markers. We compared the performance of these panels on fresh and fixed whole blood on all major markers such as neutrophils with a CV of 0.6%, Myeloid cells with a CV of 6.5%, NK cells with a CV of 5.5%, T cells with 4% and B cells with 3%. To further evaluate the stability of the fixed whole blood sample, data from a frozen healthy donor was analyzed over the course of a year and were able to get consistent WBC frequencies with a CV of 1%. Results from our experiment further supports the utility of this method for longitudinal immune monitoring, making it a powerful tool for deep immunophenotyping in clinical trials and resource-limited settings.
Poster #2:
Title: Enabling rapid and convenient human immune profiling in fresh and long term stabilized whole blood samples with CyTOF flow cytometry
Author: Anita Kant, Standard Biotools
Abstract: Immunotherapy is an essential part of treatment strategies for aggressive cancers. Deep immune profiling of peripheral blood leukocytes reveals immune signatures that inform immunotherapy selection and patient outcomes. Whole blood (WB) is best processed for analysis within 24 hours of collection to capture clinically relevant immune signatures. But WB collection and cytometric analysis are often performed at different sites, which can lead to significant delays. Fixation of WB with stabilization reagents can overcome these challenges. However, not all antibody panels are compatible with these reagents.
Methods: The Human Broad Immune Profiling CyTOF ® Panel was created to be compatible with commercial WB stabilizers and enables fast and convenient human immune profiling. CyTOF flow cytometry uses metal-tagged antibodies and has multiple advantages over fluorescence-based cytometry. Compensation and spectral unmixing are not required since CyTOF flow cytometry has low signal spillover and no autofluorescence. To demonstrate the flexibility of the panel with different WB staining and stabilization workflows, samples from three healthy donors were assessed using two stabilization workflows with Proteomic Stabilizer PROT1 and Cytodelics Whole Blood Cell Stabiliser. The 20 liquid antibodies from the panel were pooled together and frozen at –80 °C as single-use aliquots to reduce technical variability from staining throughout the course of the study. In addition, samples were barcoded and acquired as a single tube to reduce variability from sample acquisition.
Results: The panel successfully identified more than 30 immune cell populations in fresh and stabilized multiplexed WB samples. The panel provided sufficient resolution and did not show increased background in fixed samples. For customization, there are more than 30 open channels to easily drop in and analyze markers of interest. Moreover, the frozen antibody cocktails yielded equivalent signal intensities (CV: 13.1%) without changes in bimodal distribution of events one, seven and 14 days after freezing.
Conclusions: The Human Broad Immune Profiling CyTOF Panel overcomes challenges caused by logistical delays as WB samples can be frozen before staining, after staining and before acquisition. Furthermore, freezing antibody cocktails is a unique feature of CyTOF flow cytometry, ensuring staining consistency. Panels with more than 50 markers can be rapidly designed and conveniently stained and acquired in a single tube. Together, CyTOF workflows enable fast and convenient cytometry of WB samples and can facilitate the study of immunotherapy responses in cancer patients.
Poster #3:
Title: High-parameter Immuno-oncology profiling of immune checkpoints and cytokines in cancer patient T Cells using CyTOF flow cytometry
Author: Anita Kant, Standard Biotools
Abstract: Accurate phenotyping of immune cells from cancer patients is critical for understanding mechanism of action and disease prognoses and monitoring clinical efficacy of immunotherapies. Functional characterization of immune checkpoint markers and cytokines produced by single cells helps decipher immune evasion and antitumor response, potentially identifying mechanistic predictors of response and elucidating new therapeutic targets, overcoming immunotherapy resistance. By using novel metal-tagged antibodies to interrogate 50-plus cellular marker simultaneously, CyTOF® technology offers exceptional resolution, simple panel design and optimization with no reference controls required. Compensation and spectral unmixing are not required since CyTOF has low signal spillover and no autofluorescence. Moreover, antibody cocktails and stained samples can be frozen for later use and acquisition, enabling a standardized, streamlined, and flexible workflow in clinical research.
Methods: Two high-parameter panels were used to characterize (panel 1, 34 markers) and functionally measure (panel 2, 40 markers) the immune competency of cancer patient T cells. Both panels consist of five to seven subpanels. The modular nature of these panels allows users to interrogate the cytokine proficle of immune cells by simply swapping out two subpanels from panel 1. Furthermore, the 11–17 open channels provide diverse options for users to build the customized panel using “drop-in” antibodies for various research purposes. PBMCs isolated from prostate cancer and melanoma patients were stimulated with cancer antigen- specific peptides or immunodominant viral peptides followed by surface staining (panel 1) or a combination of surface and intracellular staining (panel 2). Healthy donor PBMCs were used as controls. Samples were barcoded and frozen before acquisition on a CyTOF XT™ instrument in automated batch acquisition mode. T cell phenotype and functionality were assessed.
Results: In cancer patient PBMC, these high-parameter panels identified over 50 immune cell subsets, enabling comprehensive profiling of T cells with immune checkpoints. Antigen-specific T cell responses were detected in cancer patient PBMC as indicated by functional markers mediating T cell activation or exhaustion as well as intracellular cytokine production. The modular design of these panels allowed for simultaneous detection of both key immune checkpoints and functional cytokines in cancer patients and healthy donors.
Conclusion: These high-parameter CyTOF panels empower comprehensive immuno-oncology profiling, providing crucial insights into the functionality of cancer patient T cells. Combined with the convenient workflow, high flexibility, and customizability of CyTOF flow cytometry, these panels are powerful tools to facilitate mechanism of action studies in cancer research to accelerate therapeutic development.
Poster #4:
Title: A 45-color panel for comprehensive immunophenotyping of human leukocytes with the Agilent NovoCyte Opteon spectral flow cytometer
Author: Dr. HaiGuang Zhang, Agilent
Abstract: Agilent has developed the NovoCyte Opteon spectral flow cytometer, which consists of up to five lasers (349nm, 405nm, 488nm, 561nm, and 637nm) and 73 detectors (70 fluorescence detection channels, FSC, BSSC, and VSSC). Taking an OMIP-069[1] panel as a backbone, a 45-color immunophenotyping panel was designed and optimized for the Agilent NovoCyte Opteon spectral flow cytometer. Five additional biomarkers were added for broader research applications. These biomarkers are CD31, CD45RO, CD69, CD33 and LAG-3. The addition of CD31 and CD45RO enables the identification of recent thymic emigrants (RTEs) within the CD4+ T cells. CD69 is an early activation marker that is upregulated on T cells upon stimulation. LAG-3 enables the analysis of exhausted T cells together with PD-1. CD33 allows the phenotyping of DCs and monocytes.
Poster #5:
Title: A 45-color panel for comprehensive immunophenotyping of human leukocytes with the Agilent NovoCyte Opteon spectral flow cytometer
Author: Alex Sutton, Cytek Biosciences
Abstract: Cell Painting is a method for staining cell organelles to establish a standard phenotype profile for each cell type and can be used to identify normal cells from those responding to drug treatments. This approach is advantageous in that it uses a standard “cell painting” protocol without the need to develop novel assays for each new drug being tested. Fluorescent dyes are applied to ‘paint’ various cellular components, including, but not limited to: DNA, mitochondria, the cytoskeleton, and the endoplasmic reticulum and Golgi apparatus. Cells can be treated with chemical or genetic perturbagens and images analyzed for morphological changes, allowing for the collection of many morphological measurements on a single cell. The current method for acquiring data for the Cell Painting assay is generally through High-Content Screening (HCS); however, this method is typically applied to adherent cells in a plate-based assay. The Cell Painting method can be adopted for cells in suspension using the Cytek® Amnis® ImageStream®X Mk II system.
The Cytek® Amnis® ImageStream®X Mk II imaging flow cytometer combines the high throughput performance of a flow cytometer with the imagery and functional insights of microscopy and is useful to characterize the behavior of adherent and suspension cells. In this study, we treated cells in suspension with varying doses of the biguanide phenformin, a drug known to induce lactic acidosis through inhibition of mitochondrial complex I, to determine if cellular morphological changes could be detected and quantified. We performed the Cell Painting assay using SYTO™ 14 (nucleoli/RNA), MitoTracker™ Orange (mitochondria), concanavalin A (endoplasmic reticulum), wheat germ agglutinin (Golgi plasma membrane), phalloidin (actin filaments), and Hoechst (DNA) dyes to identify changes to cellular components. The cell phenotype profiles were established with image analysis software, and the features that had the greatest response to phenformin and exhibited any morphological changes were quantified with the machine learning module. Cell profiles were used to determine if cells change their morphology in response to various treatments. Our data demonstrates that the Cell Painting assay and the Cytek® Amnis® ImageStream®X Mk II imaging flow cytometer can detect changes to organelles, such as aggregates and granularity caused by phenformin treatment, and thus, help uncover targeted therapeutic effects of countless compounds using cells in suspension.
Poster #6:
Title: Separation of Blood-Derived Extracellular Vesicles Using Droplet-Based Sorting
Author: Maria Garcia Mendoza, Cytek Biosciences
Abstract: Extracellular vesicles (EVs) are cell-derived, membrane-bound small particles less than a micron in size that demonstrate potential as valuable biomarkers and therapeutic agents in many disease environments. Due to their small size and heterogeneity, EVs can be difficult to characterize; however, single vesicle flow cytometry is a proven method to detect and analyze EVs. There is growing interest in using high-speed, droplet-based sorting to isolate specific EV populations for further characterization in both functional assays and to explore their use in therapeutics. The work described herein outlines a proof-of-concept assay that demonstrates the ability to effectively purify specific EV populations using a droplet-based cell sorter.
A mixture of platelet- and red blood cell-derived EVs were analyzed using the Cellarcus Single Vesicle Flow Cytometry (vFC™) assay, which includes instrument qualification, fluorescent channel calibration, sizing, and surface cargo measurements, and follows the MIFlowCyt-EV guidelines. A Cytek Aurora™ CS system with Enhanced Small Particle™ (ESP™) Detection Option was set up with a 70 μm nozzle. Outfitted with the ESP Detection Option, this sorter has increased violet side scatter resolution that resolves 70 nm polystyrene beads and provides the same high-sensitivity fluorescence detection as Cytek Full Spectrum Profiling™ (FSP™) analyzers. The sheath pressure was set at 30 psi to improve the resolution of small particles by slowing the sheath velocity in the flow cell. The threshold was set to membrane dye (vFRed™) positive EVs. Final sort gates were drawn to isolate CD41-positive platelet-derived EVs and CD235ab-positive red blood cell-derived EVs.
The two populations were sorted using the multiway sort mode at an average sort rate of 500 events per second. The results of three independent experiments were similar, with an average sort efficiency of 98% and an average purity of 98%. The average time to sort 1 million EVs was 25 minutes.
Here, we describe a feasibility study demonstrating the purification of two EV populations using fluorescent, cell-of-origin surface markers and a high-sensitivity cell sorter. The droplet-based sorting of EVs may help address the challenges of EV analysis and opens novel research avenues for advancements in therapy and diagnostic development. This endeavor presents a starting point for the technique; further work is needed to determine desired downstream workflows.
Poster #7:
Title: Implementing Dynamic Automated Flow Cytometry Analysis for Rapid Cell Therapy Manufacturing
Author: Jacob van Vloten, Accellix
Abstract: Cell subset phenotyping and quantification are used for process development and decision-making during the manufacturing and release of cell therapy products. Consistent and logical threshold placement during cell subset identification is crucial for setting quantitative acceptance criteria for QC testing and reproducible manufacturing. Ambiguity in threshold placement due to noisy or poorly defined borders leads to subjectivity and uncertainty. Automated flow cytometry analysis allows technicians to accurately phenotype cells in real time without the need to place thresholds and with minimal training. The Accellix spectral flow cytometer offers automated sample preparation, data acquisition, and assay-specific autoclassification. Assay results are generated immediately after data acquisition and are reported on-screen for timely decision-making. Here, we verify the performance of assay-specific autoclassification algorithms for Accellix off-the-shelf T Cell, TBNK-16, and Stem Cell assays, as well as three custom transduction efficiency assays developed for CAR T cell manufacturing. Data was collected from multiple samples matching the specific assay use case and collected on several Accellix instruments. Assay-autoclassification results were compared to manual reporting by an expert analyst. We report strong agreement between the autoclassification and expert manual analysis across the off-the-shelf and custom Accellix assays, including for cell subsets with frequencies lower than 2%. The Accellix assay-specific autoclassification algorithms can dynamically identify and quantify predefined cell subsets reducing operator requirements, and simplifying cell therapy process development, manufacturing, and quality management.
Poster #8:
Title: Artificial Intelligence for Large Spectral Flow Cytometry Panel Design
Author: Duy Mai, Fred Hutchinson Cancer Center
Abstract: We used an Artificial Intelligence software by FluoroFinder to design large (fifteen plus color) flow cytometry panels. We evaluated this tool compared to other software requiring more user input, such as BD Research Cloud and Cytek Cloud. Our goal is to generate more complex panels while streamlining the development process. The evaluation is ongoing, but our current data is very promising.
Poster #9:
Title: High-Resolution Sorting of Microparticles Using BD FACS S8 Reveals Mitochondria-Containing Extracellular Vesicles as Key Mediators of Heart Failure Inflammation
Author: Denise Tong, UW Medicine Cardiology/MMC
Abstract: Introduction/Hypothesis: Inflammation is a critical driver of heart failure (HF) progression, yet therapeutic development remains limited by the lack of biological targets and an incomplete understanding of the underlying mechanisms. Mitochondria-containing extracellular vesicles (MitoEVs), due to their pro-inflammatory properties, may serve as key mediators of HF-associated sterile inflammation.
Methods and Results: Plasma samples from 12 Stage-D HF patients and 12 healthy controls were analyzed. MitoEVs were first identified and quantified using the BD FACSymphony A3 via volumetric flow analysis with mitochondrial (MTG) and cytoplasmic (CFSE) markers. HF plasma exhibited a 2.5-fold increase in MitoEVs (MTG+/CFSE+), which accounted for approximately 60% of total microparticles. MitoEVs isolated by size-exclusion chromatography were significantly more potent than soluble protein fractions in inducing cytokine production in macrophages, implicating MitoEVs as a primary source of HF plasma immunogenicity. Surface marker analysis by flow identified CD14+ monocytes as a major source of HF plasma MitoEVs.
To further characterize microparticle subpopulations, we used the BD FACS S8 to successfully sort microparticles (~200 nm) into four distinct groups: MitoEVs (MTG+/CFSE+), naked mitochondria (MTG+/CFSE-), EVs lacking mitochondria (MTG-/CFSE+), and non-specific particles (MTG-/CFSE-), with sorting efficiency confirmed by Western blot. Among these, MitoEVs exhibited the strongest immunogenic potential, reinforcing their role in HF-associated inflammation.
Conclusion: Circulating MitoEVs, released by monocytes, are pivotal mediators of sterile inflammation in HF. This study demonstrates that the BD Symphony A3 enables high-throughput MitoEV quantification, while the BD FACS S8 effectively sorts microparticles as small as ~200 nm. These findings highlight the potential of flow cytometry-based approaches for advancing our understanding of microparticle biology in HF and beyond.
Poster #10:
Title: Label-free Technology to Assess Protein Loadings on the Surface of Biological Sample
Author: Evan Davis, Beckman Coulter Life Sciences
Abstract: Interest in identifying and characterizing nano-sized particles like extracellular vesicles (EVs), viruses, and liposomes is growing due to their wide applications in biomedicine. Flow cytometry offers a powerful, high throughput method for single-particle detection, and this is particularly exemplified by the CytoFLEX nano flow cytometer, which is designed for nanoparticle characterization. This instrument features two violet side scatter channels with a large dynamic range and additional channels for blue, yellow, and red scatter signals. High scatter sensitivity of CytoFLEX nano enables detection of small changes of medium scatter intensities caused by varying amount of protein loaded on the surface of biological sample. In this study, we demonstrate capabilities of the instrument to detect and analyze the change of refractive index caused by different number of antibodies bound to the surface of Murine Leukemia Virus (MLV).
Poster #11:
Title: Label-free Characterization of Biological Nanoparticles
Author: Evan Davis, Beckman Coulter Life Sciences
Abstract: Flow cytometry is a powerful method to analyze heterogeneous particle populations based on single particle detection and is widely used for cellular analysis. However, the sensitivity of most current conventional cytometers has limitations to detect nanoparticles with sizes below 200 nm, creating a challenge for extracellular vesicle analysis. This work aims to demonstrate the performance of CytoFLEX nano flow cytometer as a sensitive label-free tool to characterize nanoparticles by leveraging its capability to collect and analyze scatter parameters at multiple wavelengths.
Poster #12:
Title: A 37-Marker Spectral Flow Cytometric Triage Panel for the Efficient Diagnosis of Lymphoid Neoplasms in Blood, Bone Marrow, Tissues, and Fluids
Author: Patty Davis, ARUP Laboratories
Abstract: While spectral flow cytometry has gained traction in research in recent years, it has yet to be implemented widely in clinical laboratories. Spectral flow has significant potential advantages in analysis of specimens, allowing for more complex and informative multicolor analyses as well as maximizing information gathered from limited specimens. Limitations of reagent availability for the additional spectral channels and limited instrument software options for efficient high-volume workflow have until recently hampered efforts for clinical utilization. Here we report that advances in new classes of fluorochromes with distinct spectral signatures and improvements of instrument software have enabled development of a clinical triage panel using a 5-laser Cytek® Aurora™ spectral cytometer for characterization of hematolymphoid neoplasms in a high-volume reference lab. The panel consists of a 25-color backbone used on all specimen types and an 11-color panel extension for bone marrow, a 6-color panel extension for peripheral blood, and a 3-color panel extension for tissues and fluids. Single-stained cells and Cytek® FSP™ CompBeads were used as reference controls for spectral unmixing. The configuration of the backbone panel and specimen-specific panel extensions, as well as gating strategies and normal and neoplastic case studies, with comparisons to our current 10-color triage panel, will be presented. This backbone panel with sample-specific panel extensions can resolve most leukemias and lymphomas without the need for add-on studies; a significant benefit in streamlining workflows and maximizing recovery from limited specimens.
Poster #13:
Title: Use of spectral flow cytometry to identify adaptive and innate immune responses to Mycobacterium tuberculosis among young children
Author: Zhen Gu, OHSU
Abstract: Mycobacterium tuberculosis (Mtb) is thought to infect nearly one-quarter of the world’s population and can lead to symptomatic Tuberculosis disease (TB). Young children are uniquely susceptible to progress to TB following Mtb-infection, and the immunologic features associated with protection versus vulnerability to disease remain unknown. Here, using cryopreserved peripheral blood mononuclear cells isolated from children under 5 years old who were exposed to TB in their homes, we will compare the phenotypes and cytokine-response profiles of adaptive and innate immune effectors between children who developed TB versus those who remained healthy for 1 year following their TB exposure, using a longitudinal study design. A 24-color spectral flow panel that includes a resting and four different stimulation conditions has been developed. Challenges in spectral flow panel development and validation, and the solutions identified to optimize panel performance with a focus on approaches towards spectral unmixing, will be described.
Poster #14:
Title: Development of a T-cell activation panel using image cytometry for research applications
Author: Blessing Musgrove, Allen Institute for Immunology
Abstract: Phytohemagglutinin (PHA) P is a lectin that in T-cells triggers the signaling pathways leading to nuclear factor of activated T-cell (NFAT) activation. In this work, an Activation Induced Marker (AIM) assay was developed and demonstrated a multiplex detection method to monitor T-cell activation in a panel consisting of CD3, CD69, CD25, and CD279 (PD-1) surface markers using the Cellaca® PLX Image Cytometer (Revvity Health Sciences, Inc., Lawrence, MA). First, a dosage titration of PHA-P was performed on PBMCs to stimulate T-cell activation. Once stimulated, CD3 positive T-cells were evaluated for the activation markers, CD69 and CD25, and the checkpoint/exhaustion molecule CD279 for disease specific compatibility. After 24- and 48- hours, the level of surface marker expression (median fluorescent intensity (MFI) values) were used to determine optimal PHA-P dosage and image cytometric parameters. Additionally, an orthogonal cytokine detection assay was performed using electrochemiluminescence via MSD technology to confirm activation of T-cells using interleukin 2 (IL-2) as a reference. Results showed concentration dependent increases in the expression levels of CD69, CD25, and CD279 on T-cells. Based on the CD25 titration curve compared to an unstimulated control sample, the calculated half maximal effective concentration (EC50) value of PHA-P at 24-hours for the PLX was 15.75 µg/mL and on a traditional cytometer was 16.16 µg/mL. At 48 hours, the PLX EC50 value was 13.73 µg/mL and for traditional flow it was 19.69 µg/mL. IL-2 cytokine release results also showed a concentration dependent increase in expression, further confirming activation. The AIM assay showed to be reproducible and has the potential to be automation compatible. The proposed image cytometric AIM assay may prove to be an efficient tool to rapidly screen clinical patient samples for T-cell activation in the setting of disease treatment or progression.
Poster #15:
Title: Human metastatic lymph nodes are no longer functional hubs of immune control
Author: Eva Domenjo, Fred Hutchinson Cancer Center
Abstract: There is emerging evidence that tumor-draining lymph nodes (tdLN) are a target of current immunotherapies and renewed interest in understanding how tdLNs orchestrate antitumor immune responses. Most studies assess the tumor microenvironment for immune correlates to predict the response to immunotherapies, but immune responses in tdLN and their alterations during tdLN metastasis in human cancer patients are still poorly characterized. Human head and neck squamous cell carcinomas (HNSCC) provide a clinically unique setting because surgical resection is typically the first line of treatment and lymph nodes are removed to assess metastasis. Here we analyzed patient-matched tumor tissue, normal and metastatic tdLNs using high parameter flow cytometry, multi-modal single-cell sequencing (transcriptomics, proteomics, and T cell receptor), and spatial transcriptomics. Protein expression analyses revealed that antigen-presenting cells (APCs) in metastatic tdLNs have an immature phenotype and exhibit more immunosuppressive properties resembling those of tumor-infiltrating APCs when compared to normal tdLNs. Additionally, metastatic tdLNs had an accumulation of activated regulatory T cell (Tregs) that was associated with clonal expansion. Likewise, Treg clones that expanded the most also expressed costimulatory markers at levels comparable to tumor Tregs. By spatial transcriptomics, metastatic tdLNs had changes in the architecture supporting the notion that tdLN metastases alter the lymphatic cellular niches and interfere with proper immune cell functioning. Future work is aimed at dissecting APC – T cell interactions that occur in normal tdLN and may be altered in the context of the metastatic tdLN and the tumor microenvironment.
Poster #16:
Title: Assay transfer of two 12-color panels for CAR detection and assessment of T-cell function using a BD FACSLyric™ Flow Cytometer
Author: Chris Sequeira, BD Biosciences
Abstract: We compared assay transfer results between instruments for two 12-color immunophenotyping panels applied to identify CAR T-cells (anti-CD19 CAR/NFAT Jurkat cell line) and differentiate them from monocytes, B-, T-, and NK-cells along with assessing markers of T-cell function. Both panels share nine fluorescent conjugates: a set of seven backbone markers to identify the main cell subsets (CD45-BV786, CD3-BV510, CD4-BV711, CD8-APC-H7, CD19-R718, CD16/56-RB780, and CD14-BV605), a viability dye (7-AAD), and a CAR detection reagent (CD19Ag-PE). Panels differed on three markers applied to characterize Effector and Memory (E&M) T-cells (CD45RA-BB515, CCR7-APC, and CD95- BV421) or to characterize exhaustion states (TIM3-BB515, LAG3-AF647, and PD-1-BV421). Fluorochrome assignment was optimized to ensure clear resolutionof target cell populations on a BD FACSLyric™ Flow Cytometer. Cells from healthy donor blood and CAR T cells were stained and processed with each panel, then mixed before sample acquisition. For the assessment of exhaustion markers, cells were stimulated in cultures for 72h prior to staining. Settings were optimized and samples acquired on a BD FACSLyric™ Flow Cytometer then successfully reacquired on two different BD FACSLyric™ Flow Cytometer, after transferring the settings using the BD FACSuite™ Application assay export and transfer feature. To compare across three instruments, CV% in MFI of T, B, NK, and CAR-T cells ranged 0.7%-6.2% for E&M panel, 1.7%-5.7% for T-exhaustion panel; while CV% in %Parent ranged 0.2%-3.0% for E&M panel, 0.2%-1.0% for Texhaustion panel. Results of assay transfer for the two 12-color immunophenotyping panels were satisfactory.
Poster #17:
Title: CellEngine’s automatic gate adjustment tool quickly and accurately recapitulates manual gating: assessment in a COVID longitudinal clinical study
Author: Zach Bjornson, CellCarta
Abstract: Gating is the backbone of cytometry analysis. Intersample variability can require adjusting gate position and size on a file-by-file basis. This process can be time-consuming, particularly when multiple gates need to be adjusted in large datasets. Various algorithms have been developed for supervised gating, but they are challenging to use, slow, and/or perform poorly, especially on rare populations. CellCarta developed a machine-learning algorithm that uses a manually gated subset of samples to quickly and consistently gate the remaining dataset, following the user’s defined hierarchy, and incorporated it into the flow cytometry analysis software CellEngine.
In order to explore the quality and time savings of using the automatic gate tailoring, we examined two markers with high variability in a large clinical study of COVID patients. We found that the algorithm saved a significant amount of time and produced populations highly similar to the populations gated manually.
Poster #18:
Title: Utilizing high-parameter spectral flow cytometry to dissect T cell function in inflamed mucosal tissues.
Author: Nicole Potchen, Fred Hutchinson Cancer Center
Abstract: T cells play a key role in both driving and resolving tissue inflammation. At barrier sites like the oral mucosa, T cells are critical to maintain both protection against infections and tolerance to innocuous antigens. Currently, T cell phenotype and function during inflammation is poorly defined in human tissues. To enable a holistic assessment of the T cell compartment, our lab has developed a novel 37-color spectral flow cytometry panel to assess T cells in human tissue. Using this panel, we analyzed 52 gingival samples from donors undergoing routine periodontic procedures, ranging from clinically healthy and mild gingivitis cases to severely inflamed Stage III-IV periodontitis. We assess cell phenotype directly ex vivo and function following activation through a 6-hour stimulation with PMA and ionomycin. Our spectral panel allows us to phenotype T cells from gingival samples and describe cellular functions, including activation, inhibition, proliferation, tissue residency, memory, and effector molecules like cytokines. Additionally, we are evaluating how cellular autofluorescence affects the resolution of multiple markers on the Discover S8. This high-parameter panel is critical for analysis of tissues of limited availability or for samples with limited cell numbers. By understanding which T cell subsets are present, along with the functional markers that contribute to various inflammatory states, we can identify targets for downstream therapeutics. Unexpectedly, upon activation, both CD4 and CD8 T cells from severely inflamed tissue maintain their ability to express amphiregulin (AREG), a key player in tissue healing. Continued studies will focus on seemingly paradoxical subsets, such as those that express both pro-inflammatory and tissue repair or homeostatic markers.
Poster #19:
Title: Adapting a broad 30-color spectral flow cytometry panel into a targeted 12-color panel for routine assessment of T-cell biology using conventional flow cytometry
Author: Kent Smith & Michelle Underwood-Ditto, BD Biosciences
Abstract: Large flow cytometry panels comprising numerous markers and fluorochromes are applied for investigation and discovery. These panels require expertise on panel design (more fluorochromes), sample preparation (more pipetting), instrument setup (more parameters) and data analysis (more dimensions). Adapting large panels suitable for discovery, into smaller panels suitable for routine assessments might be beneficial when data collection needs scaling and reproduction. In this study, we illustrate the transformation of a comprehensive 30-color immunophenotyping panel originally designed for profiling T cells, B cells, dendritic cells (DCs), and natural killer (NK) cells using spectral flow cytometry (BD FACSymphony™ A5 SE Cell Analyzer) into a targeted panel suitable for routine use on a conventional flow cytometer (BD FACSLyric™ Clinical Cell Analyzer). The goal was to streamline the panel selecting 12 relevant markers for T cell characterization. Following the selection of markers based on their biological relevance, antibodies were reassigned to fluorochromes compatible with the BD FACSLyric™ System instrument configuration. Fluorochrome assignment was optimized to ensure clear resolution of target cell populations while minimizing spillover. The consistency in the frequencies of different cell subsets across both platforms confirmed the reliability of the adapted panel. Our study exemplifies the successful transformation of a large, complex, discovery panel into a smaller, targeted panel suitable for routine assessments. This adjusted approach not only simplifies complexity and accelerates sample processing time but also preserves the high resolution essential for effective cell characterization.
Poster #20:
Title: Measuring the Efficiency of in-situ Reverse Transcription in Bacteria via Flow Cytometry and Fluorescence Microscopy
Author: Karl Gaisser, Institute for Systems Biology
Abstract: Microbial single-cell RNA sequencing by split-pool barcoding (MicroSPLiT) is a single cell transcriptomics method developed for bacteria. During this process, cells are grown, fixed in formaldehyde, and permeabilized. After, cells are split into 96 different wells, each containing a different barcode primer, where they undergo reverse transcription. Following reverse transcription, the cells are then pooled and redistributed into another 96 well plate containing a different set of barcodes, which are then ligated. This split-pooling process is then repeated an additional time, which leads to each cell containing a unique combination of the three barcodes. All of these reverse transcription and ligation steps take place within the cell itself, which acts as an in-situ reactor.
The efficiency of this process is dependent on how well the cells are permeabilized. This poses a challenge to using each cell as an in-situ reactor, as bacteria have a diverse range of cell wall and membrane compositions, leading to varying levels of strength and resistance to permeabilization. If cells are not permeabilized enough, the reverse transcription enzyme and other reagents will not be able to enter the cell. If the treatment is too harsh, the cell will break apart and lose intracellular contents. For optimal results, the permeabilization method needs to be tailored to each bacterial species.
To test the efficiency of different permeabilization methods, we present two methods for measuring the in-situ efficiency of reverse transcription in bacterial cells using flow cytometry and fluorescence microscopy. The first method uses an acridine orange fluorescent dye solution to stain the cells before and after reverse transcription. Acridine orange is a well characterized metachromatic dye that differentially stains RNA and DNA. By comparing fluorescence intensity in cells before and after reverse transcription, the relative efficiency can be measured. The second method uses ChromaTide Alexa Fluor 488-5-UTP, which is a fluorescently labeled deoxyuridine triphosphate. This reagent is added to the dNTP mix during reverse transcription. The fluorescence intensity is correlated with cDNA generated, and indicates how effectively reverse transcription reagents are able to enter the cell. For both methods, these fluorescent cells are examined via both flow cytometry and fluorescence microscopy. Flow cytometry provides a high throughput measurement of individual cells, while the cell structure and intracellular fluorescence are examined with fluorescence microscopy.