Cytometry Part A最新文献

{"title":"Standardization of Suspension and Imaging Mass Cytometry Single-Cell Readouts for Clinical Decision Making.","authors":"Ruben Casanova, Shuhan Xu, Pierre Bost, Sujana Sivapatham, Andrea Jacobs, Stefanie Engler, Mitchell P Levesque, Reinhard Dummer, Bernd Bodenmiller, Stéphane Chevrier","doi":"10.1002/cyto.a.24940","DOIUrl":"https://doi.org/10.1002/cyto.a.24940","url":null,"abstract":"<p><p>Suspension and imaging mass cytometry are single-cell, proteomic-based methods used to characterize tissue composition and structure. Data assessing the consistency of these methods over an extended period of time are still sparse and are needed if mass cytometry-based methods are to be used clinically. Here, we present experimental and computational pipelines developed within the Tumor Profiler clinical study, an observational clinical trial assessing the relevance of cutting-edge technologies in guiding treatment decisions for advanced cancer patients. By using aliquots of frozen antibody panels, batch effects between independent experiments performed within a time frame of 1 year were minimized. The inclusion of well-characterized reference samples allowed us to assess and correct for batch effects. A systematic evaluation of a test tumor sample analyzed in each run showed that our batch correction approach consistently reduced signal variations. We provide an exemplary analysis of a representative patient sample including an overview of data provided to clinicians and potential treatment suggestions. This study demonstrates that standardized suspension and imaging mass cytometry measurements generate robust data that meet clinical requirements for reproducibility and provide oncologists with valuable insights on the biology of patient tumors.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":" ","pages":""},"PeriodicalIF":2.5,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144126975","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Volume 107A, Number 5, May 2025 Cover Image","authors":"","doi":"10.1002/cyto.a.24865","DOIUrl":"https://doi.org/10.1002/cyto.a.24865","url":null,"abstract":"","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"107 5","pages":""},"PeriodicalIF":2.5,"publicationDate":"2025-05-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24865","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144091660","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Workflow to Achieve Saturation of Fluorophore-Conjugated Monoclonal Antibodies for Robust Comparison of Biomarker Expression.","authors":"Natalie Smith, Helen McGuire, Barbara Fazekas de St Groth","doi":"10.1002/cyto.a.24938","DOIUrl":"https://doi.org/10.1002/cyto.a.24938","url":null,"abstract":"<p><p>Antibody titration is an important step in every cytometric workflow, with the goal being to determine antibody concentrations that ensure highly reproducible results. When aiming to compare antigen expression between samples using mean or median fluorescence intensity (MFI), reagents should be used at a saturating concentration so that unavoidable variations in staining conditions do not affect the fluorescence signal. The recommended concentrations of commercially available fluorophore-labeled monoclonal antibodies (mAbs) may not achieve plateau staining, and their saturating concentration may be too high to be experimentally useful. To address these common concerns, we present a novel method to achieve saturation of fluorophore-conjugated mAbs, by 'spiking-in' unlabelled antibody of the same clone. Here, we demonstrate the application of this workflow to human anti-CD3 (clone OKT3, mouse IgG2a) and anti-TCRαβ (clone IP26, mouse IgG1), two mAbs that do not achieve saturation at 2-fold above their commercially recommended concentrations. First, the saturating concentration of unlabelled (purified) OKT3 and IP26 was determined by detection with a fluorophore-labeled anti-mouse IgG (H + L) secondary antibody. Titration curves of unlabelled and labeled mAbs were compared for each clone to determine whether labeling had resulted in any loss in binding activity. Unlabelled antibody was then 'spiked' into the labeled antibody at varying ratios, and those that achieved saturation while maintaining an adequate fluorescence signal were identified. We demonstrate that antibody saturation can be achieved with an optimized mixture of labeled and unlabelled antibody, while maintaining a clear signal from the fluorophore. While this workflow has only been applied to OKT3 and IP26, it has potential applicability for any antibody clone for which both labeled and unlabelled preparations are available. This method has significance for robust comparison of biomarker expression when fluorophore labeled reagents do not reach saturation under standard staining conditions.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":" ","pages":""},"PeriodicalIF":2.5,"publicationDate":"2025-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143970772","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Volume 107A, Number 4, April 2025 Cover Image","authors":"","doi":"10.1002/cyto.a.24863","DOIUrl":"https://doi.org/10.1002/cyto.a.24863","url":null,"abstract":"","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"107 4","pages":""},"PeriodicalIF":2.5,"publicationDate":"2025-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24863","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143883822","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"OMIP-114: A 36-Color Spectral Flow Cytometry Panel for Detailed Analysis of T Cell Activation and Regulation in Small Human Blood Volumes.","authors":"Marie-Theres Thieme-Ehlert, Thomas Jacobs, Johannes Brandi, Maria Sophia Mackroth","doi":"10.1002/cyto.a.24937","DOIUrl":"https://doi.org/10.1002/cyto.a.24937","url":null,"abstract":"<p><p>This 36-color flow cytometry panel is designed to characterize multiple lymphocyte compartments, with a focus on T cells, their memory subpopulations, and immune checkpoints in human whole blood samples. In clinical settings, the amount of blood available from patients for scientific research is often limited. This restriction may be further exacerbated when working with samples from small children or in resource-poor settings-both scenarios commonly encountered in malaria and infectious disease research. Accordingly, this panel is designed to maximize the information that can be obtained from as little as 200 μL whole blood using flow cytometry. This panel allows a phenotypic characterization of the main subpopulations within T cells, as well as B cells and NK cells. It includes markers for the analysis of memory subpopulations, regulatory T cell subsets, and T follicular helper cells. Furthermore, surface and intracellular markers for activation and differentiation, effector functions, exhaustion, and immune checkpoints are included, allowing detailed characterization of the main lymphocyte subsets, in particular T cells. This panel was optimized for the analysis of fresh human blood samples obtained from malaria patients, but it may be adapted to the analysis of isolated PBMC or tissue samples, as well as samples from patients with other infectious or inflammatory diseases.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":" ","pages":""},"PeriodicalIF":2.5,"publicationDate":"2025-04-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143973410","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Correction to \"Potential and Challenges of Clinical High-Dimensional Flow Cytometry: A Call to Action\".","authors":"","doi":"10.1002/cyto.a.24936","DOIUrl":"https://doi.org/10.1002/cyto.a.24936","url":null,"abstract":"","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":" ","pages":""},"PeriodicalIF":2.5,"publicationDate":"2025-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143980964","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"BL-FlowSOM: Consistent and Highly Accelerated FlowSOM Based on Parallelized Batch Learning","authors":"Fumitaka Otsuka,&nbsp;Kenji Yamane,&nbsp;Koji Futamura,&nbsp;Junichiro Enoki,&nbsp;Yuji Nishimaki,&nbsp;Yoshiki Tanaka,&nbsp;Akihide Higuchi,&nbsp;Motohiro Furuki","doi":"10.1002/cyto.a.24934","DOIUrl":"10.1002/cyto.a.24934","url":null,"abstract":"<p>The recent increase in the dimensionality of cytometry data has led to the development of various computational analysis methods. FlowSOM is one of the best-performing clustering methods but has room for improvement in terms of the consistency and speed of the clustering process. Here, we introduce Batch Learning FlowSOM (BL-FlowSOM), which is a consistent and highly accelerated FlowSOM based on parallelized batch learning. The change of the learning algorithm from online learning to batch learning with principal component analysis initialization improves consistency and eliminates randomness in the clustering process. It also enables the parallelization of the learning process, leading to significant acceleration of the clustering process with clustering quality equivalent to that of FlowSOM. BL-FlowSOM is available on Sony's Spectral Flow Analysis (SFA)-Life sciences Cloud Platform (https://www.sonybiotechnology.com/us/instruments/sfa-cloud-platform/).</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"107 5","pages":"333-343"},"PeriodicalIF":2.5,"publicationDate":"2025-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24934","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143968115","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"OMIP-113: Characterization of Cytokine Producing T Cells in Swine","authors":"Riccardo Arrigucci,&nbsp;Abby Patterson,&nbsp;Chloe Brown,&nbsp;Peter Dube","doi":"10.1002/cyto.a.24935","DOIUrl":"10.1002/cyto.a.24935","url":null,"abstract":"<p>T cells are essential for preventing diseases and providing long-term protective immunity. The functional capacity of T cells and the quality of their responses to antigens can be measured by the cytokines they produce. We developed a conventional flow cytometry panel utilizing commercially available antibodies to measure antigen-specific T cell mediated immune responses in swine. The panel can simultaneously detect Th1 and Th17 cytokines (IFN-γ, TNF, and IL-17A) to characterize multifunctional αβ and γδ T cells. We also included CD40L (CD154) to identify cells that are activated upon antigen recall and that may contribute to B-cell help or activate antigen presenting cells. The assay can be applied to study T cell mediated immune responses to vaccines and diseases and can be used with cryopreserved or freshly isolated peripheral blood mononuclear cells.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"107 5","pages":"289-292"},"PeriodicalIF":2.5,"publicationDate":"2025-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24935","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143969320","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Volume 107A, Number 3, March 2025 Cover Image","authors":"","doi":"10.1002/cyto.a.24861","DOIUrl":"https://doi.org/10.1002/cyto.a.24861","url":null,"abstract":"","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"107 3","pages":""},"PeriodicalIF":2.5,"publicationDate":"2025-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24861","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143809423","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Fluorescent Cell Barcoding of Peripheral Blood Mononuclear Cells for High-Throughput Assessment of Vaccine-Induced T Cell Responses in Low-Volume Research Samples","authors":"Janna R. Shapiro,&nbsp;Nathalie Simard,&nbsp;Shelly Bolotin,&nbsp;Tania H. Watts","doi":"10.1002/cyto.a.24933","DOIUrl":"10.1002/cyto.a.24933","url":null,"abstract":"<p>T cell responses are rarely measured in large-scale human vaccine studies due to the sample volumes required, as well as the logistical, technical, and financial challenges associated with available assays. Fluorescent cell barcoding has been proposed in other contexts to allow for more high-throughput flow cytometry-based assays. Here, we aimed to expand on existing barcoding approaches to develop a reagent and sample-sparing assay for in-depth assessment of T cell responses to vaccine antigens. By using various concentrations of two fixable viability dyes in a matrix format, up to 25 samples that were pooled and acquired together could be successfully deconvoluted based on their unique fluorescent signature. This fluorescent cell barcoding approach was then combined with extracellular and intracellular staining to identify functional (i.e., producing at least one cytokine) and polyfunctional (i.e., producing multiple cytokines) T cells in response to vaccine antigen stimulation. As a proof-of-concept, we plated just 200,000 peripheral blood mononuclear cells (PBMC) per condition, and by staining and acquiring only two pooled samples, we were able to detect rare antigen-specific T cell responses in eight donors to four stimulants each. The frequencies of antigen-induced cytokine-positive cells detected in barcoded samples with 200,000 input PBMC were strongly correlated with those detected in non-barcoded samples from the same donors with 1 million input PBMC, demonstrating the validity of this approach. In conclusion, by reducing the number of PBMC needed by five-fold, and the volume of staining reagents needed by 25-fold, this assay has widespread potential applications to human vaccine studies.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"107 5","pages":"321-332"},"PeriodicalIF":2.5,"publicationDate":"2025-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24933","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143810734","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}