Cytometry Part A最新文献

{"title":"The Diamond Age of Cytometry: From Fulwyler to Spectral Multiplexing","authors":"J. Paul Robinson","doi":"10.1002/cyto.a.24970","DOIUrl":"10.1002/cyto.a.24970","url":null,"abstract":"<p>Over the past 6 decades, cytometry has evolved from a niche experimental method into a cornerstone of modern biomedical research. The development of the first cell sorter by Mack Fulwyler laid the groundwork for technologies that now define single-cell analysis. From the early challenges of instrument operation and laser alignment to today's era of automated, high-parameter systems, the field has undergone continual transformation. We now enter what may be termed the “diamond age” of cytometry, an era of exceptional sensitivity, resolution, and analytical depth, driven by innovations such as spectral flow cytometry. Here, we reflect on the historical milestones that shaped the discipline, the cultural and educational shifts within the community, and the future challenges of standardization and quantitative rigor.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"107 11","pages":"713-715"},"PeriodicalIF":2.1,"publicationDate":"2025-11-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24970","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145458156","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 Quantitative Microfluidic Flow Cytometer Based on Spaced Uniform Optical Field","authors":"Chiyuan Gao,&nbsp;Mingze Sun,&nbsp;Long Fan,&nbsp;Junbo Wang,&nbsp;Xiaoye Huo,&nbsp;Xiaosu Zhao,&nbsp;Yi Zhang,&nbsp;Jian Chen","doi":"10.1002/cyto.a.24971","DOIUrl":"10.1002/cyto.a.24971","url":null,"abstract":"<div>\u0000 \u0000 <p>Rapid, quantitative analysis of single-cell proteins with arbitrary spatial distributions carries pivotal significance across fundamental biological research and clinical applications. However, as the golden method, flow cytometry can only quantify cell surface proteins due to non-uniform excitation illumination and the lack of calibration microbeads with known numbers of internal fluorescent molecules. This study reported a quantitative microfluidic flow cytometer utilizing aligned microfabricated metal apertures to selectively extract the central portions of Gaussian beams, forming a spaced uniform optical field. Based on this spaced uniform optical field, multicolor fluorescence signals originating from antibody-binding events with arbitrary distributions on single microbeads were converted into the number of proteins. The geometries of the microfluidic cuvette and the uniformity of fluorescence intensities across the excitation fields were quantitatively validated. Using this methodology, calibration curves with high linearities for all nine fluorescence channels were established, and a mixture of rainbow calibration microbeads was analyzed, where five types of fluorescent probes were quantitatively measured, enabling the classification of the rainbow mixture into five distinct subpopulations. The presented flow cytometer enables quantitative analysis of single-cell proteins with arbitrary spatial distributions and thus demonstrates strong potential for fundamental biological research and clinical applications at the single-cell level.</p>\u0000 </div>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"107 11","pages":"770-778"},"PeriodicalIF":2.1,"publicationDate":"2025-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145444278","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 10, October 2025 Cover Image","authors":"","doi":"10.1002/cyto.a.24875","DOIUrl":"https://doi.org/10.1002/cyto.a.24875","url":null,"abstract":"","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"107 10","pages":""},"PeriodicalIF":2.1,"publicationDate":"2025-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24875","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145384929","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":"Metabolic Changes in Living Human Lymphoma Cells Intervening NAD\u0000 + Metabolism as Revealed by NAD(P)H-Fluorescence Lifetime Imaging and Para-Hydrogen-Induced Polarization NMR","authors":"Lea Marie Jeude,&nbsp;Ruth Leben,&nbsp;Wiebke Beninga,&nbsp;Yonghong Ding,&nbsp;Gabriele Stevanato,&nbsp;Stefan Glöggler,&nbsp;Raluca A. Niesner,&nbsp;Dieter Kube","doi":"10.1002/cyto.a.24946","DOIUrl":"10.1002/cyto.a.24946","url":null,"abstract":"<p>Proliferating cells have a sustained high demand for regeneration of electron acceptors as nicotinamide dinucleotide (phosphate) (NAD(P)⁺/NAD(P)H) is involved in a number of critical redox reactions within cells. However, their analysis in living cells is still challenging. We propose that combining label-free NADH and NADPH fluorescence lifetime imaging (NAD(P)H-FLIM) and signal-enhanced nuclear magnetic resonance (NMR) spectroscopy allows new, deeper insights into changes in specific metabolic pathways in living cells. For proof of principle, NAD⁺-metabolism was perturbed by specific inhibition of the rate-limiting enzyme of the NAD⁺ “Salvage pathway” Nicotinamide phosphoribosyltransferase (NAMPT) by FK866 in RAMOS human lymphoma cells. FK866 treatment leads to NAD(H) reduction, followed by reduced RAMOS cell proliferation. The NAD(P)H-FLIM analysis revealed increased general NAD(P)H-dependent metabolic activity indicated by increased ratios of enzyme-bound to total NAD(P)H concentration upon NAMPT inhibition. More importantly, a marked reduction in lactate dehydrogenase (LDH) activity accompanied by NADPH oxidase activity increase is observed. Using signal-enhanced NMR spectroscopy a reduced flux of pyruvate to lactate catalyzed by LDH is detectable in real time in living cells. This strongly supports NAD(P)H-FLIM analysis and demonstrates that intervening in the NAD⁺ “Salvage pathway” can have specific and global consequences for cells. Our principle study shows how spatially-resolved metabolic imaging techniques, that is, NAD(P)H-FLIM, are complemented by real-time NMR, paving the way toward a comprehensive spatiotemporal understanding of metabolic pathways in living cells.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"107 11","pages":"750-762"},"PeriodicalIF":2.1,"publicationDate":"2025-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24946","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145307144","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":"Best Practices for Optimization of Phytoplankton Analysis in Natural Waters Using CytoSense Flow Cytometers","authors":"Clémentine Gallot,&nbsp;Zéline Hubert,&nbsp;Lumi Haraguchi,&nbsp;Hedy Aardema,&nbsp;Luis Felipe Artigas,&nbsp;Amel Bellaaj Zouari,&nbsp;Arnaud Cauvin,&nbsp;Raffaella Casotti,&nbsp;Véronique Créach,&nbsp;Georges Dubelaar,&nbsp;Alexandre Epinoux,&nbsp;Gérald Grégori,&nbsp;Oliver Grosso,&nbsp;Joanna Kolasinki,&nbsp;Harrie Kools,&nbsp;Rob Lievaart,&nbsp;Arnaud P. Louchart,&nbsp;Glaucia Moreira Fragoso,&nbsp;Maialen Palazot,&nbsp;Machteld Rijkeboer,&nbsp;Kévin Robache,&nbsp;Joseph Rolland,&nbsp;Thomas Rutten,&nbsp;Melilotus Thyssen","doi":"10.1002/cyto.a.24964","DOIUrl":"10.1002/cyto.a.24964","url":null,"abstract":"<p>The use of flow cytometry to investigate phytoplankton functional groups is rapidly expanding worldwide, using lab- or ship-based instruments or autonomous environmental monitoring platforms. Automation, coupled with greater autonomy, allows for higher spatial and temporal resolution of phytoplankton groups, enhancing understanding of their dynamics and patterns, generating large datasets. The level of resolution is determined by both instrumental capabilities and optimization of its acquisition settings. Sharing these datasets with the scientific community, whether to improve global phytoplankton distribution resolution or facilitate the intercomparison of environmental indicators among monitoring laboratories, strongly relies on quality-controlled instruments and standardized data acquisition and analysis. This article focuses on CytoSense-type (CytoBuoy, NL) flow cytometers, which operate by recording the optical pulse shapes of particles as they pass through a laser beam. Different configurations such as laser wavelength and power, sheath fluid management, sample inlet design, and dataset output format were not considered, in order to focus on optimization and protocol standardization to resolve the whole phytoplankton size spectrum, from the smallest autofluorescing prokaryotes to colonies and chain-forming species. In this study, coincidence, PMT voltage, trigger threshold optimization, and regular quality control procedures are considered and discussed, using datasets from three types of instruments and two contrasted marine coastal waters as case studies. The primary goal of this study is to establish a framework to guide and support the exploration and application of this type of flow cytometer, ultimately achieving a reliable and optimal resolution for sample acquisition of natural waters.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"107 11","pages":"730-744"},"PeriodicalIF":2.1,"publicationDate":"2025-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24964","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145299056","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":"Transparency and Reporting in the Evaluation of PD1 by Flow Cytometry","authors":"Marc Sorigue,&nbsp;Roser Salvia,&nbsp;Jordi Petriz","doi":"10.1002/cyto.a.24969","DOIUrl":"10.1002/cyto.a.24969","url":null,"abstract":"<div>\u0000 \u0000 <p>Transparent and reproducible reporting is essential in flow cytometry, particularly in cancer research where it is used to evaluate biomarkers like PD1. However, inadequate reporting remains a significant concern. In this study, we conducted a methodological review of 100 recent manuscripts from various journals that assessed PD1 expression by flow cytometry in cancer. We evaluated reporting across six domains: methods, dotplot availability, gating strategy, robustness of analysis, figure presentation, and data availability. We found that compliance with basic standards was suboptimal, with only 35%–70% of manuscripts meeting these criteria across different domains. While reagent and device reporting was strong (~90%), transparency regarding key data, such as representative PD1 dotplots and complete gating sequences, was lacking. Contrary to our initial expectations, journal impact factor and whether a journal was focused on cytometry did not correlate consistently with reporting quality. Our findings highlight an urgent need for authors to improve their reporting practices, for reviewers to increase their vigilance, and for journal editors to enforce minimal reporting standards.</p>\u0000 </div>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"107 11","pages":"745-749"},"PeriodicalIF":2.1,"publicationDate":"2025-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145299037","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":"Pluronic Acid Prevents Sorter-Induced Cell Stress in Myeloid Cells From Mouse Peritoneal Cavity","authors":"Johann Mar Gudbergsson,&nbsp;Ida Holten-Møller,&nbsp;Morgane Moulin,&nbsp;Sune Jonathan Keidser-Nilsson,&nbsp;Thomas Wittenborn,&nbsp;Anja Bille Bohn,&nbsp;Charlotte Christie Petersen,&nbsp;Anders Etzerodt","doi":"10.1002/cyto.a.24968","DOIUrl":"10.1002/cyto.a.24968","url":null,"abstract":"<div>\u0000 \u0000 <p>Sorter-induced cell stress (SICS) is a recognized challenge within the fluorescence-activated cell sorting (FACS) community; however, it remains significantly underreported in the scientific literature, with limited studies proposing effective mitigation strategies. In the present study, we observed that myeloid cells isolated from the peritoneal cavity exhibited heightened sensitivity to FACS-induced stress relative to other immune cell populations. Notably, we identified that the addition of pluronic acid to cell suspensions prior to sorting substantially improved post-sort cell viability. These findings suggest that pluronic acid may represent a broadly applicable approach to preserving the viability of sensitive cell types, potentially extending to cells derived from other tissues.</p>\u0000 </div>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"107 11","pages":"763-769"},"PeriodicalIF":2.1,"publicationDate":"2025-10-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145285737","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":"Validation of an Optimized DuraClone Phenotyping Kit Workflow for TBNK Subset Quantification","authors":"Carlos Agustin Villegas-Valverde,&nbsp;Imilla Casado Hernandez,&nbsp;Yaima Zuñiga Rosales,&nbsp;Fatma Abdou,&nbsp;Shadi Sharif Shamat,&nbsp;Antonio Alfonso Bencomo-Hernandez,&nbsp;Yendry Ventura-Carmenate","doi":"10.1002/cyto.a.24967","DOIUrl":"10.1002/cyto.a.24967","url":null,"abstract":"<p>Quantification of T-cells, B-cells, and NK-cells assay is crucial for diagnosing and monitoring immune diseases and evaluating lymphodepleting therapies. To standardize and validate an optimized workflow of the Beckman Coulter DuraClone IM Phenotyping Basic Kit for quantification of TBNK subsets in peripheral blood. Procedural changes included the use of an alternative lysis buffer, the addition of counting beads, the elimination of centrifugation steps, and an increase in acquisition volume. Validation followed CLSI H42-A2 and H62 guidelines, assessing accuracy, precision, linearity, and limit of quantification (LLOQ). Accuracy was evaluated by comparison with Immuno-Trol controls and by Bland–Altman analysis against standard immunophenotyping methods. External proficiency was assessed through participation in the College of American Pathologists (CAP) TBNK program in 2024. Procedural steps were reduced by 50%, and processing time by 38.6%. The modified method demonstrated high accuracy (−3 &lt; bias &lt; 35; cells/μL), a low bias based on Immuno-Trol targets, and strong agreement in the Bland–Altman analysis. The method successfully passed three CAP external proficiency tests in 2024, confirming interlaboratory reliability. Coefficients of variation for precision were below 10% for all subsets. Linearity exceeded <i>R</i>\u0000 ² &gt; 0.99 across clinically relevant ranges. Most subsets demonstrated an LLOQ below 10–50 cells/μL, which is suitable for clinical applications. The proposed modifications to the DuraClone IM kit protocol improved workflow efficiency and analytical performance without compromising accuracy or reproducibility. The validated method provides a standardized, reliable, and time-efficient alternative for lymphocyte subset quantification.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"107 10","pages":"695-708"},"PeriodicalIF":2.1,"publicationDate":"2025-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24967","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145250212","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":"Cleanet: Robust Doublet Detection in Cytometry Data Based on Protein Expression Patterns","authors":"Matei Ionita,&nbsp;Michelle L. McKeague,&nbsp;Mark M. Painter,&nbsp;Divij Mathew,&nbsp;Ajinkya Pattekar,&nbsp;Ayman Rezk,&nbsp; Shwetank,&nbsp;Damian Maseda,&nbsp;E. John Wherry,&nbsp;Allison R. Greenplate","doi":"10.1002/cyto.a.24961","DOIUrl":"10.1002/cyto.a.24961","url":null,"abstract":"<p>Flow and mass cytometry experiments are essential for profiling immune cells at single-cell resolution. Better understanding of human immunology increasingly involves analyzing studies at the scale of hundreds or thousands of samples, with data analysis a significant bottleneck. This trend increases the demand for automated analysis methods. In particular, a common preprocessing step in cytometry data analysis is distinguishing single cells from doublets (or multiplets), events in which two (or more) cells pass simultaneously through the detector. Typically, doublets are identified on two-dimensional density plots, using their high measured values for DNA intercalators (mass cytometry) or scattering channels (flow cytometry). Despite its popularity, this bivariate gating method is sometimes imprecise: for example, we show that bivariate gating of mass cytometry data can mistake single eosinophils for doublets, due to their high DNA content. Taking inspiration from methods already used in single-cell transcriptomics, but not in the cytometry community, we propose an alternative approach. Our method, called Cleanet, first simulates doublet events, then identifies true events with protein expression similar to the simulated doublets. This simple method is completely automated and detects both homotypic and heterotypic doublets. We validate it in datasets acquired with mass and flow cytometry; moreover, we verify with imaging flow cytometry data from ImageStream and Discover A8 instruments that most events predicted to be doublets truly consist of multiple cells. Cleanet can also classify doublets based on their component cell types, which potentially enables the study of cell–cell interactions, mining extra information out of doublet events that would otherwise be discarded. As a proof of concept, we demonstrate that Cleanet can detect a treatment-specific increase in interactions between two cell lines. By automating doublet detection and classification, we aim to streamline the data analysis in large cytometry studies and provide a more accurate picture of both immune cell populations and cell–cell interactions.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"107 11","pages":"716-729"},"PeriodicalIF":2.1,"publicationDate":"2025-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24961","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145250198","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-118: A 38-Marker Spectral Flow Cytometry Panel to Assess Human Regulatory T Cell Phenotype and Lineage Plasticity","authors":"Nilika Bhattacharya,&nbsp;Collin Jugler,&nbsp;Jessica C. Hill,&nbsp;Kade R. Copple,&nbsp;Sameena Nikhat,&nbsp;Mohsen Khosravi-Maharlooei,&nbsp;Casey R. Ager","doi":"10.1002/cyto.a.24966","DOIUrl":"10.1002/cyto.a.24966","url":null,"abstract":"<p>We report the optimization of a 38-parameter spectral flow cytometry panel to identify, phenotype, and assess lineage plasticity of human regulatory T cells (Tregs). Tregs are an indispensable T cell lineage with pleiotropic tolerogenic functions whose activities contribute critically to numerous disease settings including cancer, autoimmunity, and infectious disease, among others. Phenotypic and functional heterogeneity within the Treg lineage has been appreciated, but the etiology and impact of Treg plasticity across disease states remain incompletely understood. Better tools are thus needed to deeply characterize human Treg phenotypic heterogeneity at the single cell level to advance discovery of Treg-based biomarkers in disease and to assess the effects of Treg-directed therapeutics. To this end, our 38-parameter panel consists of a 13-marker PBMC backbone module to broadly phenotype PBMCs while specifically discriminating Tregs, and a 25-marker Treg phenotyping module to determine Treg differentiation state, activation profile, and lineage subtype. In contrast to many high-parameter OMIPs that rely on surface staining only, we incorporated several intracellular targets in this panel. This afforded the opportunity to thoroughly evaluate binding characteristics of all antibodies in both pre-fixation and post-fixation and permeabilization settings; we identify several antibodies eligible for overnight post-fixation staining that require substantially reduced titers as compared to traditional pre-fixation staining, resulting in significant cost savings. Dimensionality reduction and semi-supervised clustering on healthy donor PBMC-derived Tregs profiled by our panel reveal up to 14 discrete Treg phenotypes. In sum, this panel enables deep phenotypic characterization of human Treg heterogeneity in peripheral blood specimens by spectral flow cytometry.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"107 10","pages":"649-657"},"PeriodicalIF":2.1,"publicationDate":"2025-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24966","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145231701","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}