Cytometry Part A最新文献

{"title":"Single-Cell Morphometrics From Hematoxylin and Eosin-Stained Images Reveals Subtype-Specific Features in Chronic Lymphocytic Leukemia","authors":"María Cecilia Cabral Lorenzo,&nbsp;Juan Quarroz Braghini,&nbsp;Gregorio Cordini,&nbsp;Tomás Lombardo,&nbsp;Laura Kornblihtt,&nbsp;Guillermo Blanco","doi":"10.1002/cytoa.70010","DOIUrl":"10.1002/cytoa.70010","url":null,"abstract":"<div>\u0000 \u0000 <p>Chronic lymphocytic leukemia (CLL) is a highly heterogeneous B-cell malignancy, spanning a spectrum from indolent conventional forms (C-CLL) to Richter transformation. Accelerated CLL (A-CLL) represents an intermediate subtype, histologically and clinically more aggressive than C-CLL, yet its diagnosis relies on subjective histological criteria. We hypothesized that high-dimensional single-cell morphometrics from routine hematoxylin and eosin-stained lymph node biopsies could provide complementary metrics for positioning cases along the disease continuum. We implemented a novel, open-source methodology using CellProfiler and Seurat to segment cells and measure hundreds of parameters from two indolent and two aggressive cases. Datasets were integrated to mitigate technical batch effects. Our analysis combined an unsupervised approach with trajectory analysis and a pathologist-guided supervised machine learning model. Both unsupervised and supervised approaches successfully and independently distinguished aggressive from indolent cases. Aggressive cases were enriched in morphometric clusters corresponding to paraimmunoblasts, while indolent cases were enriched in those corresponding to small lymphocytes. Pseudo-bulk analysis using a subset of key parameters also successfully classified patients. In this highly exploratory, proof-of-concept study utilizing a small, well-characterized cohort (<i>n</i> = 4 CLL patients plus 2 RT reference cases), we present a robust and traceable single-cell morphometric pipeline. By providing objective metrics of cellular and subcellular morphology, our method reveals strong segregation patterns reflecting the CLL continuum. Our findings warrant validation in larger cohorts, but offer novel, quantifiable insights that could potentially complement diagnostic criteria and aid in patient stratification.</p>\u0000 </div>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"109 1","pages":"62-79"},"PeriodicalIF":2.1,"publicationDate":"2026-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146009227","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":"TimeFlow 2: An Unsupervised Cell Lineage Detection Method for Flow Cytometry Data","authors":"Margarita Liarou,&nbsp;Thomas Matthes,&nbsp;Stéphane Marchand-Maillet","doi":"10.1002/cytoa.70006","DOIUrl":"10.1002/cytoa.70006","url":null,"abstract":"<p>Cell lineage detection refers to the inference of differentiation pathways from immature cells to distinct mature cell types. We developed TimeFlow 2, a new method for lineage inference in large flow cytometry datasets. It uses a single static snapshot of unordered cells and does not require prior knowledge of the number of pathways, cell types or temporal labels. TimeFlow 2 uses the cell orderings from TimeFlow and defines coarse cell states along pseudotime segments. By connecting these states, it constructs paths at the cell state level. To approximate the trajectory structure, it further groups the paths based on an optimal transport-based cost function. We used TimeFlow 2 on three healthy bone marrow samples and accurately assigned monocytes, neutrophils, erythrocytes and B-cells of different maturation stages to four distinct pathways. Marker dynamics across the inferred pathways showed highly correlated patterns for the corresponding lineages in all three patients. We compared the performance of TimeFlow 2 and three other established methods using standard classification and correlation metrics. TimeFlow 2 outperformed the others on flow cytometry datasets and remained competitive on the challenging mass cytometry datasets. Overall, TimeFlow 2 detects biologically informative pathways, allowing bioinformaticians to model and compare marker dynamics across cell lineages in a data-driven way. Source code in Python and tutorials are available at https://github.com/MargaritaLiarou1/TimeFlow2.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"109 1","pages":"42-61"},"PeriodicalIF":2.1,"publicationDate":"2026-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cytoa.70006","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145931533","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":"Matrix Recovery Algorithm for Reconstructing Mixing Matrices From Raw Observations and Ordinary Least Squares Unmixed Abundance Values","authors":"Ryan Kmet,&nbsp;David Novo","doi":"10.1002/cytoa.70005","DOIUrl":"10.1002/cytoa.70005","url":null,"abstract":"&lt;div&gt;\u0000 \u0000 &lt;p&gt;Flow cytometric analysis requires accurate spectral unmixing using a mixing matrix (&lt;span&gt;&lt;/span&gt;&lt;math&gt;\u0000 &lt;semantics&gt;\u0000 &lt;mrow&gt;\u0000 &lt;mi&gt;M&lt;/mi&gt;\u0000 &lt;/mrow&gt;\u0000 &lt;/semantics&gt;&lt;/math&gt;) to deconvolve overlapping fluorescent signals into individual fluorochrome abundances. However, current Flow Cytometry Standard (FCS) data formats inadequately support the archival storage of &lt;span&gt;&lt;/span&gt;&lt;math&gt;\u0000 &lt;semantics&gt;\u0000 &lt;mrow&gt;\u0000 &lt;mi&gt;M&lt;/mi&gt;\u0000 &lt;/mrow&gt;\u0000 &lt;/semantics&gt;&lt;/math&gt; alongside experimental data, significantly compromising analytical reproducibility and method transparency. We present a novel matrix recovery (MR) algorithm that computationally reconstructs the original &lt;span&gt;&lt;/span&gt;&lt;math&gt;\u0000 &lt;semantics&gt;\u0000 &lt;mrow&gt;\u0000 &lt;mi&gt;M&lt;/mi&gt;\u0000 &lt;/mrow&gt;\u0000 &lt;/semantics&gt;&lt;/math&gt; from archived raw detector observations and previously calculated unmixed abundance values. For ordinary least squares (OLS) unmixing methodologies, our algorithm achieves mathematically exact recovery with numerical errors below &lt;span&gt;&lt;/span&gt;&lt;math&gt;\u0000 &lt;semantics&gt;\u0000 &lt;mrow&gt;\u0000 &lt;msup&gt;\u0000 &lt;mn&gt;10&lt;/mn&gt;\u0000 &lt;mrow&gt;\u0000 &lt;mo&gt;−&lt;/mo&gt;\u0000 &lt;mn&gt;4&lt;/mn&gt;\u0000 &lt;/mrow&gt;\u0000 &lt;/msup&gt;\u0000 &lt;/mrow&gt;\u0000 &lt;/semantics&gt;&lt;/math&gt; using the closed-form solution &lt;span&gt;&lt;/span&gt;&lt;math&gt;\u0000 &lt;semantics&gt;\u0000 &lt;mrow&gt;\u0000 &lt;mi&gt;M&lt;/mi&gt;\u0000 &lt;mo&gt;=&lt;/mo&gt;\u0000 &lt;msup&gt;\u0000 &lt;mfenced&gt;\u0000 &lt;mrow&gt;\u0000 &lt;mi&gt;A&lt;/mi&gt;\u0000 &lt;mo&gt;⋅&lt;/mo&gt;\u0000 &lt;msup&gt;\u0000 &lt;mi&gt;O&lt;/mi&gt;\u0000 &lt;mo&gt;+&lt;/mo&gt;\u0000 &lt;/msup&gt;\u0000 &lt;/mrow&gt;\u0000 &lt;/mfenced&gt;\u0000 &lt;mo&gt;+&lt;/mo&gt;\u0000 &lt;/msup&gt;\u0000 &lt;/mrow&gt;\u0000 &lt;/semantics&gt;&lt;/math&gt;, where matrix &lt;span&gt;&lt;/span&gt;&lt;math&gt;\u0000 &lt;semantics&gt;\u0000 &lt;mrow&gt;\u0000 &lt;mi&gt;A&lt;/mi&gt;\u0000 &lt;/mrow&gt;\u0000 &lt;/semantics&gt;&lt;/math&gt; contains unmixed abundances and matrix &lt;span&gt;&lt;/span&gt;&lt;math&gt;\u0000 &lt;semantics&gt;\u0000 &lt;mrow&gt;\u0000 &lt;mi&gt;O&lt;/mi&gt;\u0000 &lt;/mrow&gt;\u0000 &lt;/semantics&gt;&lt;/math&gt; contains raw observations. Comprehensive validation across six commercial cytometric platforms, encompassing detector arrays ranging from 55 to 182 channels and endmember panels from 10 to 47 fluorochromes, confirmed algorithmic accuracy for OLS-based compensation systems. While weighted least squares (WLS) recovery remains theoretically feasible, computational complexity renders current implementations intractable for practical applications. This matrix recovery approach pr","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"109 1","pages":"80-84"},"PeriodicalIF":2.1,"publicationDate":"2026-01-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145899212","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":"Cyt-Geist: Current and Future Challenges in Cytometry: Reports of the CYTO 2025 Conference Workshops","authors":"Paul K. Wallace,&nbsp;Evan R. Jellison,&nbsp;Sherry Thornton,&nbsp;Kathleen Kluepfel,&nbsp;Jessica Back,&nbsp;Thomas C. Beadnell,&nbsp;Attila Bebes,&nbsp;Jochen Behrends,&nbsp;Anna C. Belkina,&nbsp;Michele Black,&nbsp;Goce Bogdanoski,&nbsp;Mariela Bollati-Fogolín,&nbsp;Sarah Bonte,&nbsp;Katrien Van der Borght,&nbsp;Ryan R. Brinkman,&nbsp;Kathleen Brundage,&nbsp;Tim Bushnell,&nbsp;Daniel T. Chiu,&nbsp;Norman Chow,&nbsp;Christopher O. Ciccolella,&nbsp;Matthew Cochran,&nbsp;Kamila Czechowska,&nbsp;Kleopatra Dagla,&nbsp;Benjamin Daniel,&nbsp;Gelo de la Cruz,&nbsp;Julie Van Duyse,&nbsp;Laura Ferrer Font,&nbsp;Òscar Fornas,&nbsp;Sara Garcia-Garcia,&nbsp;Rui Gardner,&nbsp;Sofie Van Gassen,&nbsp;Daniel Gimenes,&nbsp;Richard Grenfell,&nbsp;Madeline J. Grider-Hayes,&nbsp;Randall Grose,&nbsp;Christopher Hall,&nbsp;Kathryn E. Hally,&nbsp;Marjolijn Hameetman,&nbsp;Karen Hogg,&nbsp;Jessica Houston,&nbsp;Jonathan M. Irish,&nbsp;Gert Van Isterdael,&nbsp;Maria Jaimes,&nbsp;Sylvia Janetzki,&nbsp;Cheryl Kim,&nbsp;Abhishek Koladiya,&nbsp;Jochen Lamote,&nbsp;Joanne Lannigan,&nbsp;Julien Leconte,&nbsp;Virginia Litwin,&nbsp;Ana Longhini,&nbsp;Nicolas Loof,&nbsp;Estefanía Lozano-Andrés,&nbsp;Kelly Lundsten,&nbsp;Peter Mage,&nbsp;Florian Mair,&nbsp;Catarina Gregório Martins,&nbsp;Megan McCausland,&nbsp;Helen M. McGuire,&nbsp;Justin Meskas,&nbsp;William Murphy,&nbsp;John Nolan,&nbsp;Barbara Oliveira,&nbsp;Diana Ordoñez-Rueda,&nbsp;Eva Orlowski-Oliver,&nbsp;Charlotte Christie Petersen,&nbsp;Nicole J. Poulton,&nbsp;Givanna Putri,&nbsp;Karen J. Quadrini,&nbsp;Beata Ramasz,&nbsp;Donald Ruhrmund,&nbsp;Vikas V. Singh,&nbsp;Sam J. Small,&nbsp;Natalie J. Smith,&nbsp;Josef Spidlen,&nbsp;Camille Stegen,&nbsp;Tamar Tak,&nbsp;Sam Thompson,&nbsp;Michael Thomson,&nbsp;Daniel Vocelle,&nbsp;Rachael V. Walker,&nbsp;Robin E. Walsh,&nbsp;Lili Wang,&nbsp;Yu-Fen Wang,&nbsp;Andrea,&nbsp;Meredith Weglarz,&nbsp;Moritz Winker,&nbsp;James C. S. Wood,&nbsp;Stacie Woolard,&nbsp;Nai-Yu Yeh,&nbsp;Raif Yuecel,&nbsp;Bartek Rajwa","doi":"10.1002/cytoa.70002","DOIUrl":"10.1002/cytoa.70002","url":null,"abstract":"&lt;p&gt;Organized by the International Society for Advancement of Cytometry (ISAC), the CYTO conference is one of the most important events for everyone interested in cytometry and quantitative single-cell analysis. It is an annual gathering of cytometry experts and novices in the field, all with a passion for cytometry. This meeting is an opportunity to learn exciting, cutting-edge research directly from the source by attending the Frontiers and State-of-the-Art lectures, research and technology sessions, and Scientific Tutorials.&lt;/p&gt;&lt;p&gt;CYTO conferences are also the premier live forum for discussing current and emerging challenges in cytometry and associated sciences through an interactive workshop format. Workshops attract considerable attention within and outside the cytometry community as they address where we stand in specific fields of interest to cytometrists, how current issues with the evolving technologies within the field can be addressed and solved, and what future areas for development exist. Due to the limited time frame allocated to this format, however, workshops run in parallel, preventing attendees from participating in all sessions of interest. Moreover, those unable to attend the CYTO conference miss the content entirely. Therefore, we continue the tradition of summarizing all workshops to disseminate the information provided and to capture the state-of-the-art view of the community. The primary purpose of creating such a combined summary is to preserve the record of live discussions and main conclusions, propose potential guidelines, and make them available to the broader public.&lt;/p&gt;&lt;p&gt;The publication of workshop summaries and their availability to a global audience not only captures live discussions but also undoubtedly seeds new initiatives to support emerging topics or trends. This “Cyt-Geist,” a play on the German &lt;i&gt;Zeitgeist&lt;/i&gt;, meaning “spirit of the age”, aims to document the defining spirit and collective consciousness of the cytometry field at this moment in time. Just as &lt;i&gt;Zeitgeist&lt;/i&gt; captures the prevailing ideas and beliefs of an era, &lt;i&gt;Cyt-Geist&lt;/i&gt; captures the current challenges, innovations, and forward momentum that characterize our community. The joint effort begun in 2018 and continued in 2019 by Kamila Czechowska proved to be a valuable reference [&lt;span&gt;1, 2&lt;/span&gt;]. Building on that spirit, we present summaries from CYTO 2025, held in Denver, Colorado, from May 31 to June 4.&lt;/p&gt;&lt;p&gt;This manuscript serves as a summary report of 15 workshops held at CYTO 2025. We present, in concise form, the current and future challenges in cytometry identified by workshop organizers and participants. The manuscript is organized into three thematic sections: Building the Cytometry Infrastructure of the Future (Standardization, Sharing, and Sustainability in Cytometric Practice); Applied Innovation Across Modalities (Expanding Possibilities: From Fluorescence to Imaging and Automated Annotation); and the People Behind the P","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"109 1","pages":"5-41"},"PeriodicalIF":2.1,"publicationDate":"2025-12-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cytoa.70002","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145862299","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-119: A 36-Color Full-Spectrum Flow Cytometry Panel for Deep Immunophenotyping of Peripheral Blood and Ex Vivo Expanded Human T Cells.","authors":"Robert J Harris, Benjamin Arkle, Elizabeth Evans, Charles H V Smith, Rebecca Teevan, Natalie Bath","doi":"10.1002/cytoa.70001","DOIUrl":"10.1002/cytoa.70001","url":null,"abstract":"<p><p>The increasing uptake of adoptive CAR-T and TCR-T cell therapies into clinical practice has intensified the need for robust and detailed immunophenotyping of ex vivo expanded T cells. We have developed and optimized an extended 36-color full-spectrum flow cytometry panel suitable for in-depth immunophenotyping of both peripheral blood and ex vivo expanded human T cells. Our panel allows for analysis of CD4⁺ and CD8⁺ memory subpopulations (Tn/Tscm, Tcm, Tem, and Temra), Treg subsets (CD45RA⁺ and CD39⁺), and helper T cell subsets (Tfh, Th1, Th2, and Th17). Intracellular staining facilitates the evaluation of functional markers granzyme B (cytotoxicity) and Ki-67 (proliferation). Our panel allows detailed investigation of T cell activation using markers carrying a range of expression kinetics. Exhaustion and senescence features, known to correlate with resistance to immunotherapies, can also be thoroughly evaluated. TCR staining with pHLA-dextramer or anti-TCR antibody is also included to allow evaluation of TCR specificity and/or transduction efficiency. A PD-1 receptor occupancy component enables quantification of T cells bound to antibodies from immune checkpoint inhibitor (ICI) therapies. Overall, we have generated a fully optimized and fit-for-purpose extended T cell profiling panel with particular relevance to the field of immunotherapies including ICIs and adoptive T cell therapies.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":" ","pages":"787-792"},"PeriodicalIF":2.9,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145741422","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":"Detection and Validation of Influenza Virus Hemagglutinin Specific, Cross-Reactive, and Heteroclitic B Cells Induced by a Combination Vaccine Adjuvant.","authors":"Egest J Pone, Jiin Felgner, Jenny Hernandez-Davies, Johannes S Gach, Tyler Albin, D Huw Davies","doi":"10.1002/cytoa.70000","DOIUrl":"10.1002/cytoa.70000","url":null,"abstract":"<p><p>Inherent cross-reactivity for shared epitopes is thought to be critical for vaccines, since this is likely the only way in which a vaccine can be anticipatory, similar to immune responses induced by natural infections. However, measuring antigen-specific and cross-reactive B cells remains technically challenging due to low abundance of these cells and background binding. Here, we have assessed different antigen-labeling methods to optimize the measurement of B cell specificity, cross-reactivity, heterocliticity, and average affinity for antigen by flow cytometry. Antigens covalently labeled with NHS ester amine-reactive dyes gave reliable results compared to other antigen labeling methods: namely conjugation of His-tagged antigen on fluorescent streptavidin nanoparticles, or on soluble streptavidin tetramers, bridged via the biomolecular adapter biotin-trisNTA(Ni). Blocking with unlabeled H1 or H7 clearly outcompeted H1- and/or H7-specific cells, indicating that cross-reactive antibodies possess lower affinities compared to the mono-reactive or the rare heteroclitic antibodies. Overall, these studies suggest that evaluating the antigen specificity and breadth of candidate vaccines, including multi-season or universal vaccines, may be best accomplished using flow cytometric quantification of clonally distributed B lineage cells using antigens covalently derivatized with fluorescent dyes, which is also analogous with the standard flow cytometry approach of quantifying antigen biomarkers on cells using fluorescent monoclonal antibodies (mAbs).</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":" ","pages":"817-836"},"PeriodicalIF":2.9,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13435353/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145755443","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":"Lost in Translation: Harmonizing Terminology and Defining Mathematical Tools for Panel Optimization.","authors":"Bartek Rajwa, Mario Roederer","doi":"10.1002/cyto.a.24972","DOIUrl":"10.1002/cyto.a.24972","url":null,"abstract":"<p><p>Spectral flow cytometry has evolved from a contentious idea into a mainstay of high-parameter single-cell analysis, yet its vocabulary (and the statistical reasoning behind it) remains a patchwork of overlapping, sometimes contradictory terms. This position paper highlights the terminological fragmentation and translation gap, and aims to harmonize the field's lexicon while providing a cohesive information-theoretic framework for panel optimization. We expose the limits of popular ad hoc heuristics, matrix condition numbers, and pairwise cosine similarities, and promote stronger surrogates: effective rank and information efficiency, which together capture spectral independence and numerical stability, and the Cramér-Rao lower bound (CRLB) matrix, which directly predicts fluorochrome-specific spreading error. Building on this foundation, we revive the optimal-design criteria: D-optimality maximizes total information; A-optimality minimizes the average parameter variance; E-optimality constrains worst-direction inflation. By aligning precise definitions with actionable design rules, we provide a roadmap for consistent terminology, next-generation panel construction, and objective instrument benchmarking.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":" ","pages":"793-816"},"PeriodicalIF":2.9,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145803269","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":"An Opportunity for ISAC and ICCS to Lead on Universal Cytometry Standards.","authors":"Marc Sorigue, Jordi Petriz","doi":"10.1002/cytoa.70003","DOIUrl":"10.1002/cytoa.70003","url":null,"abstract":"<p><p>Flow cytometry, a complex and important methodology, is exacerbating the systemic reproducibility crisis in biological research. Our systematic review of 100 published manuscripts on PD-1 flow cytometry data found systemic deficiencies in the clarity and completeness of methodological documentation across top-tier journals. We urgently request that the International Society for Advancement of Cytometry (ISAC) and dedicated journal editors from the scientific publishing community lead a global initiative mandating a unique common standard for all submissions (MIFlowCyt). This adoption is critical for enforcing transparency, enabling rigorous peer review, and fundamentally strengthening the scientific integrity of published cytometry data.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":" ","pages":"785-786"},"PeriodicalIF":2.9,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145803244","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":"Optimization of a 40-Color Spectral Flow Cytometry Panel for Human Blood Immunophenotyping: Impact of Blood Volume, Sample Preservation, and Overnight Staining.","authors":"Friedrich Neubauer, Andreas Spittler, Angelika Berger, Lukas Wisgrill","doi":"10.1002/cytoa.70004","DOIUrl":"10.1002/cytoa.70004","url":null,"abstract":"<p><p>Spectral flow cytometry and high-parameter panels provide a powerful tool to meet the growing demands for deep immunophenotyping. This study aimed to adapt a published 40-color spectral flow cytometry panel for whole blood analysis, evaluate the impact of cryopreservation, and explore the potential benefits of overnight staining on data quality and cost-effectiveness. The optimization process revealed several artifacts, including loss of IgG signal and spectral profile changes in PerCP-eFluor710, which were addressed through protocol and reagent modifications. Cryopreservation resulted in significant alterations to granulocyte morphology and viability, limiting direct comparisons with fresh blood samples. Preservation also led to altered distributions in 30% of gated populations, particularly affecting T cell, dendritic cell, and monocyte subsets. Overnight staining with reduced antibody concentrations enhanced resolution for key markers while allowing for 5- to 20-fold reductions in antibody usage, substantially lowering panel costs. Contrary, extended incubation times also decreased the detection of markers critical for identification of NKT-like and regulatory T cell subsets, including CD2 and CD39. This underscores the importance of fluorochrome interference, preservation effects, and extended incubation times when optimizing high-dimensional panels, suggesting opportunities to improve staining protocols in clinical and epidemiological research, particularly where resolution or cost-effectiveness are primary concerns.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":" ","pages":"837-853"},"PeriodicalIF":2.9,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145843529","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 11, November 2025 Cover Image","authors":"","doi":"10.1002/cyto.a.24877","DOIUrl":"https://doi.org/10.1002/cyto.a.24877","url":null,"abstract":"","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"107 11","pages":""},"PeriodicalIF":2.1,"publicationDate":"2025-11-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24877","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145619252","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}