Cytometry Part A最新文献

{"title":"OMIP-109: 45-color full spectrum flow cytometry panel for deep immunophenotyping of the major lineages present in human peripheral blood mononuclear cells with emphasis on the T cell memory compartment","authors":"Lily M. Park,&nbsp;Joanne Lannigan,&nbsp;Quentin Low,&nbsp;Maria C. Jaimes,&nbsp;Diana L. Bonilla","doi":"10.1002/cyto.a.24900","DOIUrl":"10.1002/cyto.a.24900","url":null,"abstract":"<p>The need for more in-depth exploration of the human immune system has moved the flow cytometry field forward with advances in instrumentation, reagent development and availability, and user-friendly implementation of data analysis methods. We developed a high-quality human 45-color panel, for comprehensive characterization of major cell lineages present in circulation including T cells, γδ T cells, NKT-like cells, B cells, NK cells, monocytes, basophils, dendritic cells, and ILCs. Assay optimization steps are described in detail to ensure that each marker in the panel was optimally resolved. In addition, we highlight the outstanding discernment of cell activation, exhaustion, memory, and differentiation states of CD4+ and CD8+ T cells using this 45-color panel. The panel enabled an in-depth description of very distinct phenotypes associated with the complexity of the T cell memory response. Furthermore, we present how this panel can be effectively used for cell sorting on instruments with a similar optical layout to achieve the same level of resolution. Functional evaluation of sorted specific rare cell subsets demonstrated significantly different patterns of immunological responses to stimulation, supporting functional and phenotypic differences within the T cell memory subsets. In summary, the combination of full spectrum profiling technology and careful assay design and optimization results in a high resolution multiparametric 45-color assay. This panel offers the opportunity to fully characterize immunological profiles present in peripheral blood in the context of infectious diseases, autoimmunity, neurodegeneration, immunotherapy, and biomarker discovery.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"105 11","pages":"807-815"},"PeriodicalIF":2.5,"publicationDate":"2024-10-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24900","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142521288","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":"Overcoming fixation and permeabilization challenges in flow cytometry by optical barcoding and multi-pass acquisition","authors":"Marissa D. Fahlberg,&nbsp;Sarah Forward,&nbsp;Emane Rose Assita,&nbsp;Michael Mazzola,&nbsp;Anna Kiem,&nbsp;Maris Handley,&nbsp;Seok-Hyun Yun,&nbsp;Sheldon J. J. Kwok","doi":"10.1002/cyto.a.24904","DOIUrl":"10.1002/cyto.a.24904","url":null,"abstract":"<p>The fixation and permeabilization of cells are essential for labeling intracellular biomarkers in flow cytometry. However, these chemical treatments often alter fragile targets, such as cell surface and fluorescent proteins (FPs), and can destroy chemically-sensitive fluorescent labels. This reduces measurement accuracy and introduces compromises into sample workflows, leading to losses in data quality. Here, we demonstrate a novel multi-pass flow cytometry approach to address this long-standing problem. Our technique utilizes individual cell barcoding with laser particles, enabling sequential analysis of the same cells with single-cell resolution maintained. Chemically-fragile protein markers and their fluorochrome conjugates are measured prior to destructive sample processing and adjoined to subsequent measurements of intracellular markers after fixation and permeabilization. We demonstrate the effectiveness of our technique in accurately measuring intracellular FPs and methanol-sensitive antigens and fluorophores, along with various surface and intracellular markers. This approach significantly enhances assay flexibility, enabling accurate and comprehensive cellular analysis without the constraints of conventional one-time measurement flow cytometry. This innovation paves new avenues in flow cytometry for a wide range of applications in immuno-oncology, stem cell research, and cell biology.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"105 11","pages":"838-848"},"PeriodicalIF":2.5,"publicationDate":"2024-10-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142521299","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":"Potential and challenges of clinical high-dimensional flow cytometry: A call to action","authors":"Thomas Liechti,&nbsp;Iva Lelios,&nbsp;Aaron Schroeder,&nbsp;Vilma Decman,&nbsp;Christele Gonneau,&nbsp;Christopher Groves,&nbsp;Cherie Green,&nbsp;Enrique Gomez Alcaide","doi":"10.1002/cyto.a.24902","DOIUrl":"10.1002/cyto.a.24902","url":null,"abstract":"<p>Clinical biomarker strategies increasingly integrate translational research to gain new insights into disease mechanisms or to define better biomarkers in clinical trials. High-dimensional flow cytometry (HDFCM) holds the promise to enhance the exploratory potential beyond traditional, targeted biomarker strategies. However, the increased complexity of HDFCM poses several challenges, which need to be addressed in order to fully leverage its potential and to align with current regulatory requirements in clinical flow cytometry. These challenges include among others extended timelines for assay development and validation, the necessity for extensive knowledge in HDFCM, and sophisticated data analysis strategies. However, no guidelines exist on how to manage such challenges in adopting clinical HDFCM. Our CYTO 2024 workshop “Potential and challenges of clinical high-dimensional flow cytometry” aimed to find consensus across the pharmaceutical industry and broader scientific community on the overall benefits and most urgent challenges of HDFCM in clinical trials. Here, we summarize the insights we gained from our workshop. While this report does not provide a blueprint, it is a first step in defining and summarizing the most pressing challenges in implementing HDFCM in clinical trials. Furthermore, we compile current efforts with the goal to overcome some of these challenges. As such we bring the scientific community and health authorities together to build solutions, which will accelerate and simplify the full adoption of HDFCM in clinical trials.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"105 11","pages":"829-837"},"PeriodicalIF":2.5,"publicationDate":"2024-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142496717","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 105A, Number 10, October 2024 Cover Image","authors":"","doi":"10.1002/cyto.a.24760","DOIUrl":"https://doi.org/10.1002/cyto.a.24760","url":null,"abstract":"","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"105 10","pages":""},"PeriodicalIF":2.5,"publicationDate":"2024-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24760","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142447592","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":"SCIP: A scalable, reproducible and open-source pipeline for morphological profiling of image cytometry and microscopy data","authors":"Maxim Lippeveld,&nbsp;Daniel Peralta,&nbsp;Andrew Filby,&nbsp;Yvan Saeys","doi":"10.1002/cyto.a.24896","DOIUrl":"10.1002/cyto.a.24896","url":null,"abstract":"<p>Imaging flow cytometry (IFC) provides single-cell imaging data at a high acquisition rate. It is increasingly used in image-based profiling experiments consisting of hundreds of thousands of multi-channel images of cells. Currently available software solutions for processing microscopy data can provide good results in downstream analysis, but are limited in efficiency and scalability, and often ill-adapted to IFC data. In this work, we propose Scalable Cytometry Image Processing (SCIP), a Python software that efficiently processes images from IFC <i>and</i> standard microscopy datasets. We also propose a file format for efficiently storing IFC data. We showcase our contributions on two large-scale microscopy and one IFC datasets, all of which are publicly available. Our results show that SCIP can extract the same kind of information as other tools, in a much shorter time and in a more scalable manner.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"105 11","pages":"816-828"},"PeriodicalIF":2.5,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24896","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142343358","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-108: 22-color flow cytometry panel for detection and monitoring of chimerism and immune reconstitution in porcine-to-baboon models of operational xenotransplant tolerance studies","authors":"M. Esad Gunes,&nbsp;Daniel H. Wolbrom,&nbsp;Emilie Ditlev Nygaard,&nbsp;Elin Manell,&nbsp;Philip Jordache,&nbsp;Susan Qudus,&nbsp;Alexander Cadelina,&nbsp;Joshua Weiner,&nbsp;Greg Nowak","doi":"10.1002/cyto.a.24899","DOIUrl":"10.1002/cyto.a.24899","url":null,"abstract":"","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"105 11","pages":"800-806"},"PeriodicalIF":2.5,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142249533","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 105A, Number 9, September 2024 Cover Image","authors":"","doi":"10.1002/cyto.a.24758","DOIUrl":"https://doi.org/10.1002/cyto.a.24758","url":null,"abstract":"","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"105 9","pages":""},"PeriodicalIF":2.5,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24758","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142233210","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-069 version 2: Update to the 40-color full Spectrum flow cytometry panel for deep immunophenotyping of major cell subsets in human peripheral blood","authors":"Lily M. Park,&nbsp;Joanne Lannigan,&nbsp;Quentin Low,&nbsp;Maria C. Jaimes,&nbsp;Diana L. Bonilla","doi":"10.1002/cyto.a.24898","DOIUrl":"10.1002/cyto.a.24898","url":null,"abstract":"&lt;p&gt;Optimized Multicolor Immunofluorescence Panel (OMIP)-069 was the first optimized flow cytometry panel demonstrating that 40 different fluorochromes can be effectively used in combination, without compromising the resolution of each individual marker [&lt;span&gt;1&lt;/span&gt;]. Since its publication, the panel has been adopted in some laboratories (personal communications and [&lt;span&gt;2, 3&lt;/span&gt;]). Throughout its adoption, challenges that limited the ability to use the full panel have been reported.&lt;/p&gt;&lt;p&gt;The challenges can be grouped into two main categories: reagent availability and reagent performance. Concerning reagent availability, CD20 Pacific Orange™ (Thermo Fisher Cat. MHCD2030), CD25 Phycoerythrin (PE) Alexa Fluor™ 700 (Thermo Fisher Cat. MHCD2524), CD24 PE-Alexa Fluor™ 610 (Thermo Fisher Cat. MHCD2422), and CD127 Allophycocyanin (APC) R700 (BD Biosciences Cat. 565185) have been reported to be often on back order. Regarding reagent performance, HLA-DR PE/Fire™ 810 (Biolegend Cat. 307683) and TCRγδ Peridinin-Chlorophyll-Protein (PerCP) eFluor® 710 (Thermo Fisher Cat. 46–9959-42), have shown technical issues, due to tandem degradation or unexpected spectrum signatures, respectively. Finally, it was also documented that spread between fluorescein isothiocyanate (FITC) and Brilliant™ Blue (BB) BB515, the most challenging fluorochrome combination in the panel (similarity index 0.98; spillover spread value of BB515 into FITC 29), was not always consistent with the one reported in the publication, making it difficult to use that fluorochrome pair.&lt;/p&gt;&lt;p&gt;These issues were investigated, and solutions were identified to enable the use of the full 40-color panel without compromises in performance. First, regarding the PE/Fire 810 tandem stability, the manufacturer stated the fluorochrome meets specifications and the authors did not observe tandem degradation when the reagent was used before its expiration date. To better understand the issues reported by the readership, a stability test was performed with exposure to fixation and light. No stability issues were observed with fixation with paraformaldehyde (1% or 4% in Phosphate buffered saline (PBS)). However, changes in the PE/Fire 810 spectrum and higher spillover values into PE were observed starting at 2 h of light exposure. Tandem breakdown can have serious impacts on the data, in this case specifically with any PE conjugates. For comparison, the PE/Cyanine 7 (Cy7) reagent included in the panel was tested in parallel and exhibited a more stable behavior &lt;b&gt;(&lt;/b&gt;Figure 1A&lt;b&gt;).&lt;/b&gt; The data suggests that PE/Fire 810 might need to be handled with even more care than other tandems. Next, multiple spectra were observed when using TCRγδ PerCP-eFluor710, without consistency across lots &lt;b&gt;(&lt;/b&gt;Figure 1B&lt;b&gt;).&lt;/b&gt; Data unmixing and TCRγδ population resolution are highly impacted by the presence of multiple spectra &lt;b&gt;(&lt;/b&gt;Figure 1C&lt;b&gt;).&lt;/b&gt; Finally, we observed that the use of BB515 and FITC in combination lea","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"105 11","pages":"791-799"},"PeriodicalIF":2.5,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24898","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142249534","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-107: 8-color whole blood immunophenotyping panel for the characterization and quantification of lymphocyte subsets and monocytes in swine","authors":"Riccardo Arrigucci,&nbsp;Abby Patterson,&nbsp;Peter Dube","doi":"10.1002/cyto.a.24897","DOIUrl":"10.1002/cyto.a.24897","url":null,"abstract":"<p>We developed this whole blood immunophenotyping panel with the aim to monitor and quantify major lymphocyte subsets (CD4⁺, CD8⁺, CD4⁺CD8⁺ αβ T cells, γδ-T cells, B and NK cells) and monocytes in pigs. The panel involved the use of commercially available reagents, avoiding secondary antibody staining or in-house antibody conjugations, with the aim to make the assay accessible and reproducible across laboratories. The assay is accurate, robust and represents a useful tool for immune monitoring of swine in the pharmacology and toxicology fields, or to monitor the immune status in response to vaccination and diseases.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"105 10","pages":"737-740"},"PeriodicalIF":2.5,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24897","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142249535","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":"An AI-based imaging flow cytometry approach to study erythrophagocytosis","authors":"S. Neri,&nbsp;E. T. Brandsma,&nbsp;F. P. J. Mul,&nbsp;T. W. Kuijpers,&nbsp;H. L. Matlung,&nbsp;R. van Bruggen","doi":"10.1002/cyto.a.24894","DOIUrl":"10.1002/cyto.a.24894","url":null,"abstract":"<p>Erythrophagocytosis is a process consisting of recognition, engulfment and digestion by phagocytes of antibody-coated or damaged erythrocytes. Understanding the dynamics that are behind erythrophagocytosis is fundamental to comprehend this cellular process under specific circumstances. Several techniques have been used to study phagocytosis. Among these, an interesting approach is the use of Imaging Flow Cytometry (IFC) to distinguish internalization and binding of cells or particles. However, this method requires laborious analysis. Here, we introduce a novel approach to analyze the phagocytosis process by combining Artificial Intelligence (AI) with IFC. Our study demonstrates that this approach is highly suitable to study erythrophagocytosis, categorizing internalized, bound and non-bound erythrocytes. Validation experiments showed that our pipeline performs with high accuracy and reproducibility.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"105 10","pages":"763-771"},"PeriodicalIF":2.5,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142153395","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}