Cytometry Part A最新文献

{"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}

{"title":"Size and fluorescence calibrated imaging flow cytometry: From arbitrary to standard units","authors":"Wouter W. Woud,&nbsp;Haley R. Pugsley,&nbsp;Britta A. Bettin,&nbsp;Zoltán Varga,&nbsp;Edwin van der Pol","doi":"10.1002/cyto.a.24895","DOIUrl":"10.1002/cyto.a.24895","url":null,"abstract":"<p>Imaging flow cytometry (IFCM) is a technique that can detect, size, and phenotype extracellular vesicles (EVs) at high throughput (thousands/minute) in complex biofluids without prior EV isolation. However, the generated signals are expressed in arbitrary units, which hinders data interpretation and comparison of measurement results between instruments and institutes. While fluorescence calibration can be readily achieved, calibration of side scatter (SSC) signals presents an ongoing challenge for IFCM. Here, we present an approach to relate the SSC signals to particle size for IFCM, and perform a comparability study between three different IFCMs using a plasma EV test sample (PEVTES). SSC signals for different sizes of polystyrene (PS) and hollow organosilica beads (HOBs) were acquired with a 405 nm 120 mW laser without a notch filter before detection. Mie theory was applied to relate scatter signals to particle size. Fluorescence calibration was accomplished with 2 μm phycoerythrin (PE) and allophycocyanin (APC) MESF beads. Size and fluorescence calibration was performed for three IFCMs in two laboratories. CD235a-PE and CD61-APC stained PEVTES were used as EV-containing samples. EV concentrations were compared between instruments within a size range of 100–1000 nm and a fluorescence intensity range of 3–10,000 MESF. 81 nm PS beads could be readily discerned from background based on their SSC signals. Fitting of the obtained PS bead SSC signals with Mie theory resulted in a coefficient of determination &gt;0.99 between theory and data for all three IFCMs. 216 nm HOBs were detected with all instruments, and confirmed the sensitivity to detect EVs by SSC. The lower limit of detection regarding EV-size for this study was determined to be ~100 nm for all instruments. Size and fluorescence calibration of IFCM data increased cross-instrument data comparability with the coefficient of variation decreasing from 33% to 21%. Here we demonstrate – for the first time – scatter calibration of an IFCM using the 405 nm laser. The quality of the scatter-to-diameter relation and scatter sensitivity of the IFCMs are similar to the most sensitive commercially available flow cytometers. This development will support the reliability of EV research with IFCM by providing robust standardization and reproducibility, which are pre-requisites for understanding the biological significance of EVs.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"105 10","pages":"752-762"},"PeriodicalIF":2.5,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24895","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142139573","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-106: A 30-color panel for analysis of check-point inhibitory networks in the bone marrow of acute myeloid leukemia patients","authors":"Jan Musil,&nbsp;Antonin Ptacek,&nbsp;Sarka Vanikova","doi":"10.1002/cyto.a.24892","DOIUrl":"10.1002/cyto.a.24892","url":null,"abstract":"<p>Acute myeloid leukemia (AML) is the most common form of acute leukemia diagnosed in adults. Despite advances in medical care, the treatment of AML still faces many challenges, such as treatment-related toxicities, that limit the use of high-intensity chemotherapy, especially in elderly patients. Currently, various immunotherapeutic approaches, that is, CAR-T cells, BiTEs, and immune checkpoint inhibitors, are being tested in clinical trials to prolong remission and improve the overall survival of AML patients. However, early reports show only limited benefits of these interventions and only in a subset of patients, showing the need for better patient stratification based on immunological markers. We have therefore developed and optimized a 30-color panel for evaluation of effector immune cell (NK cells, γδ T cells, NKT-like T cells, and classical T cells) infiltration into the bone marrow and analysis of their phenotype with regard to their differentiation, expression of inhibitory (PD-1, TIGIT, Tim3, NKG2A) and activating receptors (DNAM-1, NKG2D). We also evaluate the immune evasive phenotype of CD33⁺ myeloid cells, CD34⁺CD38⁻, and CD34⁺CD38⁺ hematopoietic stem and progenitor cells by analyzing the expression of inhibitory ligands such as PD-L1, CD112, CD155, and CD200. Our panel can be a valuable tool for patient stratification in clinical trials and can also be used to broaden our understanding of check-point inhibitory networks in AML.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"105 10","pages":"729-736"},"PeriodicalIF":2.5,"publicationDate":"2024-08-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24892","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142079625","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}