Cytometry Part A最新文献

{"title":"CompensAID: An Automated Detection Tool for Reference Errors","authors":"Rosan Olsman,&nbsp;Sarah Bonte,&nbsp;Mattias Hofmans,&nbsp;Malicorne Buysse,&nbsp;Katrien Van der Borght,&nbsp;Yvan Saeys,&nbsp;Vincent H. J. van der Velden,&nbsp;Sofie Van Gassen","doi":"10.1002/cyto.a.70016","DOIUrl":"10.1002/cyto.a.70016","url":null,"abstract":"<p>In flow cytometry, raw detector values do not directly represent fluorochrome abundances and must be mathematically unmixed using coefficients derived from single-stained reference controls. Inaccuracies in these controls, referred to as reference errors, can distort the estimated fluorochrome abundances, leading to skewed population distributions. However, as panel complexity and dataset sizes increase, manually inspecting all marker combinations for such artifacts becomes impractical. To address this challenge, we developed CompensAID, an open-source R-based tool that automatically flags marker combinations potentially affected by reference errors, thereby supporting quality control workflows in flow cytometry. Preprocessed data from both conventional and spectral flow cytometry were used to develop and validate the tool. CompensAID applies a density-based cutoff detection to automatically gate the negative and positive populations. The positive population is then divided into equally sized segments, after which the Secondary Stain Index (SSI) is computed for each segment. Marker combinations are flagged if the last segment yields an SSI value below −1. The tool's performance was evaluated against the consensus of five flow cytometry specialists. For the conventional dataset, 1761 out of 2240 marker combinations (79%) were unanimously classified as free of reference errors, while 24 marker combinations received a consensus score of three or higher, indicating that most flow cytometry specialists suspected reference errors. CompensAID successfully identified 23 out of these 24 marker combinations, achieving a sensitivity of 0.96. The single missed case involved a spillover spread error. In the spectral dataset, 1505 out of 1656 marker combinations (90%) were deemed free of reference errors. Of the 28 marker combinations suspected of reference errors, CompensAID flagged 21 marker combinations (sensitivity, 0.74). Five out of the seven missed marker combinations had SSI values close to the threshold. In both datasets, false positive findings were observed, often attributable to suboptimal gating or low event counts within the segments contributing to negative SSI values. CompensAID offers a robust approach for detecting marker combinations exhibiting signs of reference errors. While some false positives occur, the tool significantly reduces the burden of manual inspection. Its integration into quality control pipelines can enhance the reliability of flow cytometry data analysis. The R package is available at https://github.com/Olsman/CompensAID and will be made available through the Bioconductor platform.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"109 2","pages":"124-134"},"PeriodicalIF":2.1,"publicationDate":"2026-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.70016","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147276002","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":"Traceable Refractive Index Measurements of Liquids to Standardize Extracellular Vesicle Flow Cytometry","authors":"Martine Kuiper,&nbsp;Richard Koops,&nbsp;Rienk Nieuwland,&nbsp;Ton G. van Leeuwen,&nbsp;Edwin van der Pol","doi":"10.1002/cytoa.70015","DOIUrl":"10.1002/cytoa.70015","url":null,"abstract":"<p>Extracellular vesicles (EVs) are potential disease biomarkers released by cells into body fluids. Flow cytometers measure EV concentrations within their size detection range. These size ranges can be quantified by relating the arbitrary units of measured light scattering signals to the optical diameter of EVs with Mie theory. However, Mie theory requires input parameters, including the refractive index (RI) of the suspension liquid surrounding the measured particles, which is not accurately known. Here we measure traceable RIs of suspension liquids, including Dulbecco's phosphate buffered saline (DPBS) and human blood plasma dilutions with an uncertainty of <span></span><math>\u0000 \u0000 <semantics>\u0000 \u0000 <mrow>\u0000 \u0000 <mn>1.4</mn>\u0000 \u0000 <mo>×</mo>\u0000 \u0000 <msup>\u0000 \u0000 <mn>10</mn>\u0000 \u0000 <mrow>\u0000 \u0000 <mo>−</mo>\u0000 \u0000 <mn>5</mn>\u0000 </mrow>\u0000 </msup>\u0000 </mrow>\u0000 </semantics>\u0000 </math> for relevant wavelengths between 405 and 644 nm. We show that differences between traceably measured and currently used RIs for suspension liquids lead to differences in applied optical diameter gates and reported EV concentrations. The currently assumed RI of DPBS in Rosetta Calibration leads to a difference of 9% to 35% between reported and actual EV concentrations in diluted plasma, depending on the lower detection limit of the flow cytometer. A traceably measured RI of the suspension liquid should be used to calibrate light scattering signals with Mie theory to improve accuracy of optical diameter and concentration measurements by EV flow cytometry.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"109 2","pages":"115-123"},"PeriodicalIF":2.1,"publicationDate":"2026-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cytoa.70015","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147275969","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":"","authors":"","doi":"","DOIUrl":"","url":null,"abstract":"","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"109 3","pages":"167-179"},"PeriodicalIF":2.1,"publicationDate":"2026-03-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148072738","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":"","authors":"","doi":"","DOIUrl":"","url":null,"abstract":"","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"109 3","pages":"180-191"},"PeriodicalIF":2.1,"publicationDate":"2026-03-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148072737","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":"A Computational Workflow for Cell Line Profiling by Imaging Mass Cytometry.","authors":"Alexandre Bouzekri, Amanda Esch, Olga Ornatsky","doi":"10.1002/cytoa.70009","DOIUrl":"10.1002/cytoa.70009","url":null,"abstract":"<p><p>In single-cell spatial phenotyping biology, imaging mass cytometry (IMC) stands out as a cutting-edge, highly multiplexed technology driving discoveries across various disease areas. In vitro profiling relies on tumor-derived cancer cell lines, known for their diverse morphologies and phenotypes. A comprehensive analysis of those cell lines presents a direct challenge in predicting cancer progression and drug treatment responses. We demonstrate in our study an adaptable computational workflow we have named IMC Cell Line Profiler as a suite of versatile open-source tools for visual rendering and quantitative segmentation-based analysis of tumor-derived cell lines profiled by IMC. By leveraging our workflow with IMC resolution and multiplexing capabilities, we scrutinized the morphology, phenotypic traits, and spatial arrangement of 10 cultured cell lines from diverse tissues using custom panels of 20-30 metal-labeled antibodies. This allowed us to generate intricate high-dimensional datasets elucidating diverse cellular states with multiple markers. This computational approach was applied to a cisplatin exposure treatment in a resistant cell line to track morpho-phenotypic changes. Our IMC computational study presents new perspectives and applications to profile cell lines as new types of samples and expand the potential of IMC to new fields of discovery.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":" ","pages":"192-209"},"PeriodicalIF":2.9,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147484911","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":"The Squalene Oil-in-Water Nanoemulsion Vaccine Carrier AddaVax Potentiates TLR-Induced B-Cell Responses.","authors":"Egest J Pone, Jiin Felgner, Jenny Hernandez-Davies, D Huw Davies","doi":"10.1002/cyto.a.70018","DOIUrl":"10.1002/cyto.a.70018","url":null,"abstract":"<p><p>For vaccination, the mechanisms of action of antigens and of immunostimulatory pathogen-associated molecular pattern (PAMP) adjuvants are better understood than the roles of the depot, carrier, and other formulation-related influences. Here, we carried out cytometric studies of vaccine formulations and the effect of adjuvant formulations on B-cell responses. In particular, the AddaVax squalene oil-in-water nanoemulsion formulated with MPLA and CpG, termed IVAX-1, which is particularly effective in eliciting influenza hemagglutinin (HA)-specific mono-reactive or dual-reactive antibodies, was used as the model adjuvant to explore the requirements for generation of these B-cell antibody responses in vitro and in vivo. Using an in vitro inducible germinal center (GC) reaction, we found that AddaVax-mediated pre-assembly of B-cell stimuli for antigen, TLR agonists, CD40 ligand, and cytokines enhanced antibody class switching compared to aqueous buffer solutions. Covalent HA labeling was used to provide high-fidelity detection of mono- and dual-reactive B cells. In immunized mice, MPLA and CpG together enhanced the numbers of total and HA-specific plasma cells relative to each alone. Interestingly, titration of green fluorescent protein (GFP) and fluorescent HAs delivered in AddaVax nanoemulsion revealed that > 50% of input antigens are adsorbed onto the surface of these nanoparticles at the level of ~50-200 protein antigens per nanoparticle. Likewise, several protein antigens, including HAs, a bacterial outer membrane protein, CBU1910 from Coxiella burnetii , and GFP, were all found to associate with AddaVax emulsion nanoparticles in flow cytometry assays. Finally, the AddaVax carrier was more potent in inducing total and dual-reactive antigen-reactive B cells compared to a liposomal carrier, the Immunosome-NTA(Ni). These data cumulatively indicate that concentrated oil-in-water nanoemulsions are effective carriers for molecular adjuvants and antigen in extemporaneous formulations of research-grade protein subunit vaccines.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":" ","pages":"180-191"},"PeriodicalIF":2.9,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13552475/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147376448","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":"Gate Shape Matters.","authors":"Yolanda D Mahnke, Bartek Rajwa","doi":"10.1002/cyto.a.70023","DOIUrl":"10.1002/cyto.a.70023","url":null,"abstract":"","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":" ","pages":"165-166"},"PeriodicalIF":2.1,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147608128","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":"Superior Intracellular Detection of Cytokines, Transcription Factors, and Phosphoproteins by CyTOF Compared With Fluorescent Cytometry.","authors":"Michael J Cohen, Erika L Smith-Mahoney, Madison Bailey, Ling Wang, Lauren J Tracey, Laura Polanco, David King, Christina Loh, Amedeo J Cappione, Anna C Belkina, Jennifer E Snyder-Cappione","doi":"10.1002/cytoa.70011","DOIUrl":"10.1002/cytoa.70011","url":null,"abstract":"<p><p>Unraveling biological complexity, such as immune subset distribution in infectious disease(s), autoimmunity, or tumor heterogeneity, requires technologies capable of single-cell proteomic analysis such as flow cytometry. Surface phenotyping alone is often insufficient, as interrogating functional capacity is required to determine cellular mechanisms and effectively inform diagnostic biomarker discovery as well as development of novel therapeutics and vaccines. However, large flow panels with intracellular markers are subject to numerous challenges, including spectral overlap and background cellular autofluorescence, reducing resolving power for rare subsets or populations defined by low-abundance expression. We posited that use of mass cytometry may overcome such limitations; to address this, three small (10-12-plex) clone-matched antibody panels were evaluated by spectral flow and mass cytometry. Panels were comprised of surface and intracellular targets (phospho-epitopes, transcription factors or cytokines) and designed to minimize fluorescence spectral overlap. Overall, CyTOF technology demonstrated superior signal resolution compared with fluorescent counterparts for all three types of the intracellular targets that were compared. There was clear stimulation-specific resolution of IL-10 and IL-13 cytokine-producing cells from using mass cytometry that was not seen in the fluorescent panel and is not routinely detectable using that platform. Thus, accurate detection of immune cells with distinct functional signatures was enabled, permitting more unique and understudied populations to be quantified, including T regulatory 1 (Tr1) and cytotoxic type 2 (Tc2) T cell subsets. These results indicate that CyTOF technology has a unique ability to provide a high-resolution, comprehensive picture of the immuno-diversity present in a sample. Therefore, we posit that a new focus on use of mass cytometry for intracellular readouts could catalyze seminal discoveries in functional immune profiling, driving therapeutic design and diagnostics.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":" ","pages":"167-179"},"PeriodicalIF":2.9,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147389749","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":"A Mass Cytometry-Based Blood Cell Phenotyping Workflow Enabling Inclusion of Resource-Limited and Rural Sites in Immune System Studies.","authors":"Natalie J Smith, Michael Cohen, Lauren Tracey, Julie Alipaz, Christina Loh, David King, Neha Pulyani, Rebecca Auzins, Elin Gray, Sandra Taylor, Rajat Rai, Steven Kao, Barbara Fazekas de St Groth, Helen M McGuire","doi":"10.1002/cyto.a.70026","DOIUrl":"10.1002/cyto.a.70026","url":null,"abstract":"<p><p>Insight into disease detection and treatment through comprehensive immune phenotyping relies on the generation of high-quality data. However, the execution of robust immune monitoring in clinical trials by flow cytometry is complex due to logistical challenges in sample preparation and reagent stability. These challenges are exacerbated when considering remote and rural communities in Australia, which are burdened by both an increased prevalence and a worse prognosis of many chronic and infectious pathologies. This is not unique to the Australian context as globally remote and rural communities are underrepresented in biomedical research studies, contributing to persistent health inequities. To address these challenges, we have harnessed the unique benefits of mass cytometry to develop and test a workflow that enables inclusion of remote communities in high-parameter immune phenotyping studies. The stability of heavy-metal conjugated antibodies and the lack of any signal derived from cellular autofluorescence allows samples stained for mass cytometry to be cryopreserved and stored long-term. In this approach, whole blood (WB) samples are collected and stained fresh with dry-format antibodies specific for 38 surface targets, then cryopreserved without washing. By removing the need for centrifugation, we enable staining and preservation of fresh samples in resource-limited settings without access to specialized equipment. Samples are delivered to the central site, where neutrophils are removed and samples are barcoded with a 6-channel cocktail, pooled and stained for an additional 8 intracellular targets, achieving 52-plex coverage. In a pilot to demonstrate workflow feasibility, we characterized blood immune and myeloid cell populations from cancer patients recruited at 3 sites across Australia, including 26 patients recruited from Dubbo Base Hospital which is located approximately 400 km from the state capital city, Sydney. We find consistency in immune phenotype between WB samples prepared with our simplified WB approach and matched PBMC samples prepared in parallel. This is the first time, to our knowledge, that a multi-centre high-parameter immune phenotyping study has included cancer patients from regional Australia. In summary, we show here that the preparation of samples for high-dimensional mass cytometry can be simplified to be compatible with resource-limited environments while conserving data quality.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":" ","pages":"210-229"},"PeriodicalIF":2.9,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147638243","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 109A, Number 1, January 2026 Cover Image","authors":"","doi":"10.1002/cytoa.70014","DOIUrl":"https://doi.org/10.1002/cytoa.70014","url":null,"abstract":"","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"109 1","pages":""},"PeriodicalIF":2.1,"publicationDate":"2026-02-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cytoa.70014","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146154664","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}