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  • Deep Learning‐Based Single‐Cell Optical Image Studies
    Cytom. Part A (IF 3.433) Pub Date : 2020-01-25
    Jing Sun; Attila Tárnok; Xuantao Su

    Optical imaging technology that has the advantages of high sensitivity and cost‐effectiveness greatly promotes the progress of nondestructive single‐cell studies. Complex cellular image analysis tasks such as three‐dimensional reconstruction call for machine‐learning technology in cell optical image research. With the rapid developments of high‐throughput imaging flow cytometry, big data cell optical images are always obtained that may require machine learning for data analysis. In recent years, deep learning has been prevalent in the field of machine learning for large‐scale image processing and analysis, which brings a new dawn for single‐cell optical image studies with an explosive growth of data availability. Popular deep learning techniques offer new ideas for multimodal and multitask single‐cell optical image research. This article provides an overview of the basic knowledge of deep learning and its applications in single‐cell optical image studies. We explore the feasibility of applying deep learning techniques to single‐cell optical image analysis, where popular techniques such as transfer learning, multimodal learning, multitask learning, and end‐to‐end learning have been reviewed. Image preprocessing and deep learning model training methods are then summarized. Applications based on deep learning techniques in the field of single‐cell optical image studies are reviewed, which include image segmentation, super‐resolution image reconstruction, cell tracking, cell counting, cross‐modal image reconstruction, and design and control of cell imaging systems. In addition, deep learning in popular single‐cell optical imaging techniques such as label‐free cell optical imaging, high‐content screening, and high‐throughput optical imaging cytometry are also mentioned. Finally, the perspectives of deep learning technology for single‐cell optical image analysis are discussed. © 2020 International Society for Advancement of Cytometry

    更新日期:2020-01-26
  • 更新日期:2020-01-17
  • An Evaluation of T‐Cell Functionality After Flow Cytometry Sorting Revealed p38 MAPK Activation
    Cytom. Part A (IF 3.433) Pub Date : 2020-01-16
    Immanuel Andrä; Hanna Ulrich; Susi Dürr; Dominik Soll; Lynette Henkel; Corinne Angerpointner; Julia Ritter; Sabine Przibilla; Herbert Stadler; Manuel Effenberger; Dirk H. Busch; Matthias Schiemann

    Cell alterations during isolation and preparation for flow cytometry cell sorting by antibodies, temperature, homogenization, buffer composition and mitogens are well known. In contrast, little is known about cell alteration caused by the instrument or the sorting process itself. We systematically evaluated cellular responses to different sorter‐induced physical forces. In summary, flow cytometry cell‐sorting induced forces can affect cellular signaling cascades, especially the MAPK p38. Functional assays, related to the p38 MAPK pathway, of human primary T cells after flow cytometry sorting did lead to minor physiological modulation but no functional impairments. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

    更新日期:2020-01-16
  • Cell Cycle Analysis and Relevance for Single‐Cell Gating in Mass Cytometry
    Cytom. Part A (IF 3.433) Pub Date : 2020-01-14
    Idun D. Rein; Heidi Ø. Notø; Monica Bostad; Kanutte Huse; Trond Stokke
    更新日期:2020-01-15
  • Volume 97A, Number 1, January 2020 Cover Image
    Cytom. Part A (IF 3.433) Pub Date : 2020-01-09

    The overview of somatic mutations in a single Circulating Tumor Cell. See article by Xu et al. https://doi.org/10.1002/cyto.a.23902. In this Special Issue on Prevalent Cancers in Asia. Cover design by Bärbel Beran [www.beran‐design.de].

    更新日期:2020-01-10
  • Advances of In Vivo Flow Cytometry on Cancer Studies
    Cytom. Part A (IF 3.433) Pub Date : 2019-07-05
    Yuanzhen Suo; Zhenqin Gu; Xunbin Wei

    Cancer is a big threat to human life. Asia has about 60% of the global population and accounts for half of global cancer incidence and mortality. Circulating tumor cells (CTCs) have been a good biomarker for cancer diagnosis, staging, and prognosis. Conventional detection methods of CTCs require drawing blood. It may disturb the biological environment and limited real‐time monitoring. in vivo flow cytometry (IVFC) is a burgeoning technique that allows noninvasive detection of CTCs in vivo. Here, we review the technical development of IVFC based on various contrast principles, including fluorescence IVFC, photoacoustic IVFC, imaging IVFC, and label‐free IVFC. This powerful tool has been applied widely in many areas of cancer‐related studies, especially the CTC studies. We review applications of IVFC in preclinical studies on prevalent cancers in Asia, including liver cancer, blood cancer, and so forth. Other cancer‐related studies in breast cancer, prostate cancer, cancer‐related stem cell research and drug studies are also reviewed. © 2019 International Society for Advancement of Cytometry

    更新日期:2020-01-10
  • Two‐Dimensional Light Scattering Anisotropy Cytometry for Label‐Free Classification of Ovarian Cancer Cells via Machine Learning
    Cytom. Part A (IF 3.433) Pub Date : 2019-07-16
    Xuantao Su; Tao Yuan; Zhiwen Wang; Kun Song; Rongrong Li; Cunzhong Yuan; Beihua Kong

    We develop a single‐mode fiber‐based cytometer for the obtaining of two‐dimensional (2D) light scattering patterns from static single cells. Anisotropy of the 2D light scattering patterns of single cells from ovarian cancer and normal cell lines is investigated by histograms of oriented gradients (HOG) method. By analyzing the HOG descriptors with support vector machine, an accuracy rate of 92.84% is achieved for the automatic classification of these two kinds of label‐free cells. The 2D light scattering anisotropy cytometry combined with machine learning may provide a label‐free, automatic method for screening of ovarian cancer cells, and other types of cells. © 2019 International Society for Advancement of Cytometry

    更新日期:2020-01-10
  • Classification and Segmentation of Hyperspectral Data of Hepatocellular Carcinoma Samples Using 1‐D Convolutional Neural Network
    Cytom. Part A (IF 3.433) Pub Date : 2019-08-12
    Rendong Wang; Yida He; Cuiping Yao; Sijia Wang; Yuan Xue; Zhenxi Zhang; Jing Wang; Xiaolong Liu
    更新日期:2020-01-10
  • Single‐Cell Detection and Photostimulation on a Microfluidic Chip Aided with Gold Nanorods
    Cytom. Part A (IF 3.433) Pub Date : 2019-07-07
    Yujie Zhu; Hui Xu; Xunbin Wei; Hao He

    Gold nanorods (GNRs) can be easily designed and synthesized to respond to photons in the near infrared (NIR) band. The photostimulation by laser irradiation can be mediated and enhanced by GNRs to introduce localized damage to cells for photodynamic/photothermal therapy (PDT or PTT). In this study, we show that cells stained with GNRs can be detected and stimulated simultaneously by short flashes of femtosecond‐laser irradiation on a microfluidic system effectively. In the relatively high‐throughput cell flow, the two‐photon luminescence from GNRs can be excited and detected. The GNRs also mediate and enhance the transient photostimulation of the cells. After photostimulation, cells can remain alive, go to apoptosis, or necrosis, respectively. The stimulation effect is strongly dependent on the photon density and stimulation duration. We found the cells remain alive, go to apoptosis or necrosis, dependent on the GNR staining, the laser illumination pattern and duration. Hence, our system provides a simple and effective method for high‐throughput cell stimulation and analysis on chip. © 2019 International Society for Advancement of Cytometry

    更新日期:2020-01-10
  • An Integrated Microfluidic Chip and Its Clinical Application for Circulating Tumor Cell Isolation and Single‐Cell Analysis
    Cytom. Part A (IF 3.433) Pub Date : 2019-10-09
    Mingxin Xu; Hui Zhao; Jun Chen; Wenwen Liu; Encheng Li; Qi Wang; Lichuan Zhang
    更新日期:2020-01-10
  • 更新日期:2020-01-10
  • Myeloid Neoplasms with Elevated Plasmacytoid Dendritic Cell Differentiation Reflect the Maturation Process of Dendritic Cells
    Cytom. Part A (IF 3.433) Pub Date : 2019-12-26
    Yumei Huang; Yazhe Wang; Yan Chang; Xiaoying Yuan; Le Hao; Hongxia Shi; Yueyun Lai; Xiaojun Huang; Yanrong Liu

    To date, the research on dendritic cells (DCs) and their correlated neoplasms has not been clear. Blastic plasmacytoid dendritic cell neoplasm (BPDCN) and mature plasmacytoid dendritic cell proliferation (MPDCP) are two types of malignancies originating from plasmacytoid dendritic cells (pDCs). Some evidence has indicated the existence of other pDC neoplasms. In addition, cases of myeloid neoplasms (MNs), acute myeloblastic leukemia (AML), and myelodysplastic syndrome (MDS) with increased pDCs (AML/MDS‐pDCs) seem to have immature DCs according to the vaguely consistent expression of markers among MNs and pDCs, which appear to fit the developmental pattern of normal DCs. We analyzed 14 AML/MDS‐pDC cases mainly for their immunophenotype by flow cytometry and inferred their CD expression pattern. The patients' clinical information and other laboratory data were collected and reviewed. AML/MDS‐pDCs show a different pattern of markers from BPDCN and MPDCP. Three maturation‐involved stages were found in these AML/MDS‐pDCs patients. Stage I was the most immature stage and displayed an expression profile of CD34+/st+ CD117+/st+ BDCA2− BDCA4− CD123+ HLA‐DR+/st+ CD4− CD45dim+; Stage II was the more immature stage displayed a phenotype of CD34dim+ CD117dim+ BDCA2−/dim+ BDCA4−/dim+ CD123st+ HLA‐DR+/st+ CD4− CD45+; and Stage III was the mature stage showed CD34− CD117− BDCA2+/BDCA4+ CD123st+ HLA‐DR+/st+ CD4+ CD45+/st+. Three maturation‐involved stages overlapped well with the phenotypes of normal DC progenitors in a continuously developmental process: granulocyte, monocyte, and DC progenitors (GMDPs) and/or monocyte and DC progenitors (MDPs), common DC progenitors (CDPs), pDCs, and/or pre‐DCs. In this study, we considered AML/MDS‐pDCs as entities that were distinct from BPDCN and MPDCP and correlated the components of this tumor with the normal DC differentiation pathway, which provides new evidence for understanding DC neoplasms. © 2019 International Society for Advancement of Cytometry

    更新日期:2020-01-10
  • Yiqi Chutan Tang Reduces Gefitinib‐Induced Drug Resistance in Non‐Small‐Cell Lung Cancer by Targeting Apoptosis and Autophagy
    Cytom. Part A (IF 3.433) Pub Date : 2019-08-14
    Jue Zhang; Lingling Sun; Jian Cui; Jing Wang; Xiaomin Liu; Thazin Nwe Aung; Zhipeng Qu; Zhuangzhong Chen; David L. Adelson; Lizhu Lin

    High incidence and mortality rates for non‐small‐cell lung cancer (NSCLC) lead to low survival rates. Epidermal growth factor receptor‐tyrosine kinase inhibitors (EGFR‐TKI) are commonly first prescribed for NSCLC patients with EGFR mutations. However, most patients with sensitizing EGFR mutations become resistant to EGFR‐TKI after 9–13 months treatment. Yiqi Chutan Tang (YQCT) has been prescribed as a treatment to this issue for over 20 years. In this report, high‐performance liquid chromatography (HPLC) analysis, flow cytometry, western blot analysis, and functional annotation analysis were applied to uncover the molecular mechanisms of YQCT. Our results show the application of YQCT reduces gefitinib‐induced drug resistance, induces slight cell cycle arrest, enhances gefitinib‐induced apoptosis, and activates the autophagy. These results indicate that at the molecular level YQCT can reduce drug resistance and improve anti‐cancer effects when associated with gefitinib, which could be a result of enhancement of apoptosis and autophagy in the EGFR‐TKI resistant cells of NSCLC. This research provides a new treatment strategy for patients with EGFR‐TKI resistance in NSCLC. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

    更新日期:2020-01-10
  • A Design of Microfluidic Chip with Quasi‐Bessel Beam Waveguide for Scattering Detection of Label‐Free Cancer Cells
    Cytom. Part A (IF 3.433) Pub Date : 2019-12-25
    Ning Lv; Lu Zhang; Lili Jiang; Amir Muhammad; Huijun Wang; Li Yuan

    Light scattering detection in microfluidic chips provides an important tool to identify cancer cells without any label processes. However, forward small‐angle scattering signals of cells, which are related to their sizes and morphologies, are hard to be detected accurately when scattering angle is less than 11° in microfluidic chips by traditional lighting design due to the influence of incident beam. Therefore, cell's size and morphology being the golden standard for clinical detection may lose their efficacy in recognizing cancer cells from healthy ones. In this article, a novel lighting design in microfluidic chips is put forward in which traditional incident Gaussian beam can be modulated into quasi‐Bessel beam by a microprism and waveguide. The quasi‐Bessel beam's advantages of nondiffraction theoretically make forward scattering (FS) detection less than 11° possibly. Our experimental results for peripheral blood lymphocytes of human beings and cultured HeLa cells show that the detection rates increase by 47.87% and 46.79%, respectively, by the novel designed microfluidic chip compared to traditional Gaussian lighting method in microfluidic chips. © 2019 International Society for Advancement of Cytometry

    更新日期:2020-01-10
  • Discriminating Bacterial Phenotypes at the Population and Single‐Cell Level: A Comparison of Flow Cytometry and Raman Spectroscopy Fingerprinting
    Cytom. Part A (IF 3.433) Pub Date : 2019-12-30
    Cristina García‐Timermans; Peter Rubbens; Jasmine Heyse; Frederiek‐Maarten Kerckhof; Ruben Props; Andre G. Skirtach; Willem Waegeman; Nico Boon
    更新日期:2019-12-31
  • Intravital Imaging Techniques for Biomedical and Clinical Research
    Cytom. Part A (IF 3.433) Pub Date : 2019-12-30
    Anouchka Coste; Maja H. Oktay; John S. Condeelis; David Entenberg

    Intravital imaging, the direct visualization of cells and tissues within a living animal, is a technique that has been employed for the better part of a century. The advent of confocal and multiphoton microscopy has dramatically improved the power of intravital imaging, making it possible to obtain optical sections of tissues non‐destructively. This review discusses the various techniques used for intravital imaging, describes how intravital imaging provides information about cellular and tissue dynamics not possible to be garnered by other techniques, and details several ways in which intravital imaging is making a direct impact on the clinical care of patients. © 2019 International Society for Advancement of Cytometry

    更新日期:2019-12-31
  • A “No‐Touch” Antibody‐Staining Method of Adherent Cells for High‐Throughput Flow Cytometry in 384‐Well Microplate Format for Cell‐Based Drug Library Screening
    Cytom. Part A (IF 3.433) Pub Date : 2019-12-26
    Annelisa M. Cornel; Celina L. Szanto; Niek P. van Til; Jeroen F. van Velzen; Jaap J. Boelens; Stefan Nierkens

    In the last decade, screening compound libraries on live cells has become an important step in drug discovery. The abundance of compounds in these libraries requires effective high‐throughput (HT) analyzing methods. Although current cell‐based assay protocols are suitable for HT analyses, the analysis itself is often restrained to simple, singular outcomes. Incorporation of HT samplers on flow cytometers has provided an interesting approach to increase the number of measurable parameters and increase the sensitivity and specificity of analyses. Nonetheless, to date, the labor intensive and time‐consuming strategies to detach and stain adherent cells before flow cytometric analysis has restricted use of HT flow cytometry (HTFC) to suspension cells. We have developed a universal “no‐touch” HTFC antibody staining protocol in 384‐well microplates to bypass washing and centrifuging steps of conventional flow cytometry protocols. Optimizing culture conditions, cell‐detachment and staining strategies in 384‐well microplates resulted in an HTFC protocol with an optimal stain index with minimal background staining. The method has been validated using six adherent cell lines and simultaneous staining of four parameters. This HT screening protocol allows for effective monitoring of multiple cellular markers simultaneously, thereby increasing informativity and cost‐effectiveness of drug screening. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

    更新日期:2019-12-27
  • PySpacell: A Python Package for Spatial Analysis of Cell Images
    Cytom. Part A (IF 3.433) Pub Date : 2019-12-24
    France Rose; Luca Rappez; Sergio H. Triana; Theodore Alexandrov; Auguste Genovesio

    Technologies such as microscopy, sequential hybridization, and mass spectrometry enable quantitative single‐cell phenotypic and molecular measurements in situ. Deciphering spatial phenotypic and molecular effects on the single‐cell level is one of the grand challenges and a key to understanding the effects of cell–cell interactions and microenvironment. However, spatial information is usually overlooked by downstream data analyses, which usually consider single‐cell read‐out values as independent measurements for further averaging or clustering, thus disregarding spatial locations. With this work, we attempt to fill this gap. We developed a toolbox that allows one to test for the presence of a spatial effect in microscopy images of adherent cells and estimate the spatial scale of this effect. The proposed Python module can be used for any light microscopy images of cells as well as other types of single‐cell data such as in situ transcriptomics or metabolomics. The input format of our package matches standard output formats from image analysis tools such as CellProfiler, Fiji, or Icy and thus makes our toolbox easy and straightforward to use, yet offering a powerful statistical approach for a wide range of applications. © 2019 International Society for Advancement of Cytometry

    更新日期:2019-12-25
  • Effects of Flow‐Induced Microfluidic Chip Wall Deformation on Imaging Flow Cytometry
    Cytom. Part A (IF 3.433) Pub Date : 2019-12-19
    Yaxiaer Yalikun; Nobutoshi Ota; Baoshan Guo; Tao Tang; Yuqi Zhou; Cheng Lei; Hirofumi Kobayashi; Yoichiroh Hosokawa; Ming Li; Hector Enrique Muñoz; Dino Di Carlo; Keisuke Goda; Yo Tanaka

    Imaging flow cytometry is a powerful tool by virtue of its capability for high‐throughput cell analysis. The advent of high‐speed optical imaging methods on a microfluidic platform has significantly improved cell throughput and brought many degrees of freedom to instrumentation and applications over the last decade, but it also poses a predicament on microfluidic chips. Specifically, as the throughput increases, the flow speed also increases (currently reaching 10 m/s): consequently, the increased hydrodynamic pressure on the microfluidic chip deforms the wall of the microchannel and produces detrimental effects lead to defocused and blur image. Here, we present a comprehensive study of the effects of flow‐induced microfluidic chip wall deformation on imaging flow cytometry. We fabricated three types of microfluidic chips with the same geometry and different degrees of stiffness made of polydimethylsiloxane (PDMS) and glass to investigate material influence on image quality. First, we found the maximum deformation of a PDMS microchannel was >60 μm at a pressure of 0.6 MPa, while no appreciable deformation was identified in a glass microchannel at the same pressure. Second, we found the deviation of lag time that indicating velocity difference of migrating microbeads due to the deformation of the microchannel was 29.3 ms in a PDMS microchannel and 14.9 ms in a glass microchannel. Third, the glass microchannel focused cells into a slightly narrower stream in the X‐Y plane and a significantly narrower stream in the Z‐axis direction (focusing percentages were increased 30%, 32%, and 5.7% in the glass channel at flow velocities of 0.5, 1.5, and 3 m/s, respectively), and the glass microchannel showed stabler equilibrium positions of focused cells regardless of flow velocity. Finally, we achieved the world's fastest imaging flow cytometry by combining a glass microfluidic device with an optofluidic time‐stretch microscopy imaging technique at a flow velocity of 25 m/s. © 2019 International Society for Advancement of Cytometry

    更新日期:2019-12-20
  • Volume 95A, Number 12, December 2019 Cover Image
    Cytom. Part A (IF 3.433) Pub Date : 2019-12-13

    Multitude colors or parameters in high‐content single cell analysis by cytometry technologies illustrate the colorfulness of cytometry in diagnosis and research and bring end‐of‐the year emotions. With that I wish you all a great end‐of‐2019 and a good start of the new year. Cover design by Bärbel Beran [www.beran-design.de].

    更新日期:2019-12-13
  • OMIP‐061: 20‐Color Flow Cytometry Panel for High‐Dimensional Characterization of Murine Antigen‐Presenting Cells
    Cytom. Part A (IF 3.433) Pub Date : 2019-08-19
    Anthony T. DiPiazza; Juliane P. Hill; Barney S. Graham; Tracy J. Ruckwardt

    This 20‐color flow cytometry panel was designed to resolve the cellular heterogeneity of antigen‐presenting cells and was optimized for lymph node tissue. Reagents were carefully selected and optimized for identification of B cells (B220), neutrophils (Ly6G), monocytes and macrophages (Ly6C, CD169, F4/80), and dendritic cells (XCR1, CD172a, CD11c, I‐A/I‐E, CD24, CD64, pDCA‐1, CD103, CD11b). Inclusion of additional functional markers involved in cell migration (CCR7), co‐stimulation (CD80), and adhesion (ICAM‐1) enabled further phenotypic characterization. Finally, this panel has been tested and is compatible with fluorescently labeled antigens such as Alexa Fluor 488 (Ax488) for the study of antigen‐bearing cells in vivo. Published 2019. This article is a U.S. Government work and is in the public domain in the USA.

    更新日期:2019-12-13
  • Proposed Dynamics of CDB3 Activation in Human Erythrocytes by Nifedipine Studied with Scanning Flow Cytometry
    Cytom. Part A (IF 3.433) Pub Date : 2019-11-21
    Ekaterina S. Yastrebova; Anastasiya I. Konokhova; Dmitry I. Strokotov; Andrei A. Karpenko; Valeri P. Maltsev; Andrei V. Chernyshev

    Nifedipine is calcium channels and pumps blocker widely used in medicine. However, mechanisms of nifedipine action in blood are not clear. In particular, the influence of nifedipine on erythrocytes is far from completely understood. In this work, applying scanning flow cytometry, we observed experimentally for the first time the dynamics behind a significant increase of HCO3−/Cl− transmembrane exchange rate of CDB3 (main anion exchanger, AE1, Band 3, SLC4A1) of human erythrocytes in the presence of nifedipine in blood. It was found that the rate of CDB3 activation is not limited by the rate of nifedipine binding and/or Ca2+ transport. In order to explain the experimental data, we suggested a kinetic model assuming that the rate of CDB3 activation is limited by the dynamics of the balance between two intracellular processes (1) the activation of CDB3 limited by its interaction with intracellular Ca2+, and (2) the spontaneous deactivation of CDB3. Thus the use of scanning flow cytometry allowed to clarify quantitatively the molecular kinetic mechanism of nifedipine action on human erythrocytes. In particular, the efficiency (~30) and rates of activation (~0.3 min−1) and deactivation (~10−3 min−1) of CDB3 in human erythrocytes was evaluated for two donors. © 2019 International Society for Advancement of Cytometry

    更新日期:2019-12-13
  • Chromosome Microdissection on Semi‐Archived Material
    Cytom. Part A (IF 3.433) Pub Date : 2019-09-18
    Ahmed B. Hamid Al‐Rikabi; Marcelo de Bello Cioffi; Thomas Liehr

    Glass needle‐based chromosome microdissection (midi) is a standard approach developed in the 1980s and remains more frequently applied in testing than the comparable technique using laser‐based platforms. As the amount of DNA extracted by this technique is minimal and often in the range of picograms, the isolated DNA must be further amplified prior to use; the isolated amplified product can be readily utilized in multiple molecular research and diagnostic investigation. DNA libraries created by midi are either chromosome‐ or chromosome‐region‐specific. However, a critical component to this process is the need for timely chromosome preparation via the air‐drying method not to exceed a ~2–3 h before midi is performed. Failure of this time‐sensitive step often results in the chromosomes drying out after dropping, and upon initiation of the midi technique, the dissected material can jump away while touching by the needle, and collection of a suitable sample is inhibited. Herein, we demonstrate with a simple adaptation of the standard procedure, midi can be performed on semi‐archived material stored for longer periods at −20°C. Thus, the critical step to obtain well‐spread chromosome preparations can be completed under established conditions, for example, in the primary laboratory, stored at −20°C, and sent directly to specialized reference laboratories offering midi. In our study, we were able to obtain high‐quality DNA libraries, as verified by gel electrophoreses and reverse fluorescence in situ hybridization, via midi extracted chromosome spreads derived from human, fish, snake, lampbrush, and insect stored for up to 6 months. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

    更新日期:2019-12-13
  • Optimization in Detection of Antigen‐Specific T Cells Through Differentially Labeled MHC Multimers
    Cytom. Part A (IF 3.433) Pub Date : 2019-12-06
    Natasja Wulff Pedersen, Karoline Laske, Dominik Maurer, Marij Welters, Steffen Walter, Cécile Gouttefangeas, Sine Reker Hadrup

    A large variety of fluorescent molecules are used on a regular basis to tag major histocompatibility complex (MHC) multimers for detection of antigen‐specific T cells. We have evaluated the way in which the choice of fluorescent label can impact the detection of MHC multimer binding T cells in an exploratory proficiency panel where detection of MHC multimer binding T cells was assessed across 16 different laboratories. We found that the staining index (SI) of the multimer reagent provided the best direct correlation with the value of a given fluorochrome for T cell detection studies. The SI is dependent on flow cytometer settings and chosen antibody panel; hence, the optimal fluorochrome selection may differ from lab to lab. Consequently, we describe a strategy to evaluate performance of the detection channels and optimize the SI for selected fluorescent molecules. This approach can easily be used to test and optimize fluorescence detection in relation to MHC multimer staining and in general, for antibody‐based identification of rare cell populations. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

    更新日期:2019-12-06
  • Precise Quantitative Analysis of Cell Targeting by Particle‐Based Agents Using Imaging Flow Cytometry and Convolutional Neural Network
    Cytom. Part A (IF 3.433) Pub Date : 2019-12-06
    Elizaveta N. Mochalova, Ivan A. Kotov, Julian M. Rozenberg, Maxim P. Nikitin
    更新日期:2019-12-06
  • From the Cradle to the Grave of an Infection: Host‐Pathogen Interaction Visualized by Intravital Microscopy
    Cytom. Part A (IF 3.433) Pub Date : 2019-11-27
    Juliane Handschuh, Jonas Amore, Andreas J. Müller

    During infections, interactions between host immune cells and the pathogen occur in distinct anatomical locations and along defined time scales. This can best be assessed in the physiological context of an infection in the living tissue. Consequently, intravital imaging has enabled us to dissect the critical phases and events throughout an infection in real time in living tissues. Specifically, advances in visualizing specific cell types and individual pathogens permitted tracking the early events of tissue invasion of the pathogen, cellular interactions involved in the induction of the immune response as well the events implicated in clearance of the infection. In this respect, two vantage points have evolved since the initial employment of this technique in the field of infection biology. On the one hand, strategies acquired by the pathogen to establish within the host and circumvent or evade the immune defenses have been elucidated. On the other hand, analyzing infections from the immune system's perspective has led to insights into the dynamic cellular interactions that are involved in the initial recognition of the pathogen, immune induction as well as effector function delivery and immunopathology. Furthermore, an increasing interest in probing functional parameters in vivo has emerged, such as the analysis of pathogen reactivity to stress conditions imposed by the host organism in order to mediate clearance upon pathogen encounter. Here, we give an overview on recent intravital microscopy findings of host‐pathogen interactions along the course of an infection, from both the immune system's and pathogen's perspectives. We also discuss recent developments and future perspectives in extracting intravital information beyond the localization of pathogens and their interaction with immune cells. Such reporter systems on the pathogen's physiological state and immune cell functions may prove useful in dissecting the functional dynamics of host‐pathogen interactions. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

    更新日期:2019-11-29
  • Image‐Based Analysis of Protein Stability
    Cytom. Part A (IF 3.433) Pub Date : 2019-11-27
    K. Ashley Hickman, Santosh Hariharan, Jason De Melo, Jarkko Ylanko, Lindsay C. Lustig, Linda Z. Penn, David W. Andrews

    Short half‐life proteins regulate many essential processes, including cell cycle, transcription, and apoptosis. However, few well‐characterized protein‐turnover pathways have been identified because traditional methods to measure protein half‐life are time and labor intensive. To overcome this barrier, we developed a protein stability probe and high‐content screening pipeline for novel regulators of short half‐life proteins using automated image analysis. Our pilot probe consists of the short half‐life protein c‐MYC (MYC) fused to Venus fluorescent protein (MYC‐Venus). This probe enables protein half‐life to be scored as a function of fluorescence intensity and distribution. Rapid turnover prevents maximal fluorescence of the probe due to the relatively longer maturation time of the fluorescent protein. Cells expressing the MYC‐Venus probe were analyzed using a pipeline in which automated confocal microscopy and image analyses were used to score MYC‐Venus stability by two strategies: assaying the percentage of cells with Venus fluorescence above background, and phenotypic comparative analysis. To evaluate this high‐content screening pipeline and our probe, a kinase inhibitor library was screened by confocal microscopy to identify known and novel kinases that regulate MYC stability. Compounds identified were shown to increase the half‐life of both MYC‐Venus and endogenous MYC, validating the probe and pipeline. Fusion of another short half‐life protein, myeloid cell leukemia 1 (MCL1), with Venus also demonstrated an increase in percent Venus‐positive cells after treatment with inhibitors known to stabilize MCL1. Together, the results validate the use of our automated microscopy and image analysis pipeline of stability probe‐expressing cells to rapidly and quantitatively identify regulators of short half‐life proteins. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

    更新日期:2019-11-28
  • Real‐Time Monitoring of Bacteria Clearance From Blood in a Murine Model
    Cytom. Part A (IF 3.433) Pub Date : 2019-11-26
    Walter N. Harrington, Jacqueline Nolan, Dmitry A. Nedosekin, Mark S. Smeltzer, Vladimir P. Zharov

    Bloodstream infections, especially those that are antibiotic resistant, pose a significant challenge to health care leading to increased hospitalization time and patient mortality. There are different facets to this problem that make these diseases difficult to treat, such as the difficulty to detect bacteria in the blood and the poorly understood mechanism of bacterial invasion into and out of the circulatory system. However, little progress has been made in developing techniques to study bacteria dynamics in the bloodstream. Here, we present a new approach using an in vivo flow cytometry platform for real‐time, noninvasive, label‐free, and quantitative monitoring of the lifespan of green fluorescent protein‐expressing Staphylococcus aureus and Pseudomonas aeruginosa in a murine model. We report a relatively fast average rate of clearance for S. aureus (k = 0.37 ± 0.09 min−1, half‐life ~1.9 min) and a slower rate for P. aeruginosa (k = 0.07 ± 0.02 min−1, half‐life ~9.6 min). We also observed what appears to be two stages of clearance for S. aureus, while P. aeruginosa appeared only to have a single stage of clearance. Our results demonstrate that an advanced research tool can be used for studying the dynamics of bacteria cells directly in the bloodstream, providing insight into the progression of infectious diseases in circulation. © 2019 International Society for Advancement of Cytometry

    更新日期:2019-11-27
  • MiSet RFC Standards: Defining a Universal Minimum Set of Standards Required for Reproducibility and Rigor in Research Flow Cytometry Experiments
    Cytom. Part A (IF 3.433) Pub Date : 2019-11-26
    Fabienne Lucas, Jesus Gil‐Pulido, John LaMacchia, Fred Preffer, Paul K. Wallace, Peter Lopez

    Poor adherence to best practices, insufficient training, and pressure to produce data quickly may lead to publications of suboptimal biomedical research flow cytometry data, which contributes to the body of irreproducible research findings. In addition, documentation of compliance with best flow cytometry practices for submission, visualization, and publication of flow cytometry data is currently endorsed by very few scientific journals, which is particularly concerning as numerous peer‐reviewed flow cytometry publications emphasize instrumentation, experimental design, and data analysis as important sources of variability. Guidelines and resources for adequate reporting, annotation and deposition of flow cytometry experiments are provided by MIFlowCyt and the FlowRepository database, and comprehensive expert recommendations covering principles and techniques of field‐specific flow cytometry applications have been published. To facilitate the integration of quality‐defining parameters into manuscript and grant submission and publication requirements across biomedical fields that rely on the use of flow‐cytometry‐based techniques, a single comprehensive yet easily and universally applicable document is needed. To produce such a list of gold‐standard parameters that assess whether a research flow cytometry experiment has been planned, conducted, interpreted, and reported at the highest standard, a new initiative defining the minimum set of standards a robust and rigorous research flow experiment must fulfill (MiSet RFC Standards) was proposed at CYTO 2019. MiSet RFC Standards will integrate and simplify existing resources to provide a universal benchmark a flow cytometry experiment can easily be measured against. The goal of MiSET RFC Standards is its integration into peer‐review and publication procedures through partnership with stakeholders, journals and publishers in biomedical and translational research. This article introduces the aims and anticipated timeline and discusses strategies for interdisciplinary consensus and implementation. A single‐resource broadly applicable guideline will harmonize standards across different fields of biomedical research and lead to publication of more robust research findings. © 2019 International Society for Advancement of Cytometry

    更新日期:2019-11-27
  • Preparing a Single Cell Suspension from Zebrafish Retinal Tissue for Flow Cytometric Cell Sorting of Müller Glia
    Cytom. Part A (IF 3.433) Pub Date : 2019-11-25
    Kristin Allan, Rose DiCicco, Michael Ramos, Kewal Asosingh, Alex Yuan

    Preparation of a single cell suspension from solid tissue is vital for a successful flow cytometry experiment. We report a detailed and reproducible method to produce a quality cell suspension from the zebrafish retina. Zebrafish retinas, especially their Müller glia cells, are of particular interest for their inherent regenerative capacity, making them a useful model for regenerative medicine and cell therapy research. Here, we detail a papain‐based dissociation that is gentle enough to keep cells intact, but strong enough to disrupt cell‐cell and cell‐matrix interactions to yield a cell suspension that produces clean and reliable flow cytometric cell sorting results. This procedure consistently results in over 90% viability and three populations of cells based on GFP expression. The dissociation procedure described herein has been optimized for the collection of Müller glia from Tg(apoe:gfp) zebrafish retinas; however, the overall process may be applicable to other cell types in the fish retina, additional flow cytometric techniques, or preparing cell suspensions from similar tissues. © 2019 International Society for Advancement of Cytometry

    更新日期:2019-11-27
  • Intravital Multiphoton Imaging of the Bone and Bone Marrow Environment
    Cytom. Part A (IF 3.433) Pub Date : 2019-11-23
    JungMo Kim, Maria Gabriele Bixel

    Over the last two decades, numerous advances in our understanding of bone cell biology and bone mineral homeostasis have been achieved. As a dynamic connective and supportive tissue, bone is constantly sensing and responding to both external mechanical forces and internal systemic and local signals. A variety of intravital imaging approaches have been investigated to identify molecular and cellular processes and to decipher signaling pathways involved in the cellular communication between different types of bone cells that form bone multicellular units. Furthermore, bone multicellular units interact with cells of the immune and hematopoietic system to maintain bone homeostasis. Bone‐forming osteoblasts and bone‐degrading osteoclasts are situated on the endosteal surface of bone influencing the dynamic remodeling and the regeneration of bone tissue. Osteocytes are found at very unique locations in the bone, closely surrounded by bone matrix, forming a cellular network through their interconnected dendritic processes. Bone marrow cells fill the numerous large cavities inside the bones with various blood cell lineages arising from hematopoietic stem and progenitor cells. A highly complex and interconnected network of arterial vessels and sinusoidal capillaries span through the bone marrow spaces forming an interface between the blood circulation and the bone marrow which allows cell trafficking between both compartments. Live imaging of animals using multiphoton microscopy represents a powerful approach to address the cellular behaviors of bone and bone marrow cells over time and space in their natural tissue microenvironment. The in vivo environment is crucial, because the dynamic behavior of cells is critically influenced by many tissue factors including extracellular components, cytokine and growth factor gradients, and fluid forces, such as blood flow. The review article focuses upon recent advances in multiphoton imaging technologies as well as novel experimental approaches in the understanding of the dynamic molecular and cellular mechanisms underlying bone tissue homeostasis, remodeling, and regeneration under physiological and pathological conditions. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

    更新日期:2019-11-26
  • Volume 95A, Number 11, November 2019 Cover Image
    Cytom. Part A (IF 3.433) Pub Date : 2019-11-19

    Automatically controlled microfluidic system for continuous separation of rare bacteria from blood. See article by Yoon et al. https://onlinelibrary.wiley.com/doi/full/10.1002/cyto.a.23909

    更新日期:2019-11-20
  • OMIP‐060: 30‐Parameter Flow Cytometry Panel to Assess T Cell Effector Functions and Regulatory T Cells
    Cytom. Part A (IF 3.433) Pub Date : 2019-07-23
    Thomas Liechti, Mario Roederer

    We developed this comprehensive 28‐color flow cytometry panel with the aim to measure a variety of T cell effector functions in combination with T cell differentiation markers (CCR7, CD27, CD28, CD45RO, CD95) in γδ T cells and CD4+ and CD8+ αβ T cells (Table 1). The effector functions measured in this panel include activation and co‐stimulatory molecules (CD69, CD137, and CD154), cytokines (IL‐2, IL‐13, IL‐17A, IL‐21, IL‐22, TNF, and IFNγ), the chemokine IL‐8, cytotoxic molecules (perforin and granzyme B), and the degranulation marker CD107a. In addition, Ki67 enables the identification and analysis of recently activated T cells. To characterize regulatory T cells (Tregs), we included CD25, CD39, and the canonical Tregs transcription factor FoxP3. We developed and optimized this panel for cryopreserved human peripheral blood mononuclear cells (PBMC) and stimulation with phorbol 12‐myristate 13‐acetate (PMA) and ionomycin. However, we successfully tested other types of stimulation such as staphylococcus enterotoxin B (SEB) or a mix of immunodominant peptides (CEF peptide pool) from cytomegalovirus (CMV), Epstein–Barr virus (EBV) and influenza. Published 2019. This article is a U.S. Government work and is in the public domain in the USA.

    更新日期:2019-11-20
  • Automatically Controlled Microfluidic System for Continuous Separation of Rare Bacteria from Blood
    Cytom. Part A (IF 3.433) Pub Date : 2019-10-22
    Taehee Yoon, Hui‐Sung Moon, Jae‐Woo Song, Kyung‐A Hyun, Hyo‐Il Jung

    Bloodstream infection by microorganisms is a major public health concern worldwide. Millions of people per year suffer from microbial infections, and current blood culture‐based diagnostic methods are time‐consuming because of the low concentration of infectious microorganisms in the bloodstream. In this study, we introduce an efficient automated microfluidic system for the continuous isolation of rare infectious bacteria (Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa) from blood. Bacteria received a balanced force between a fluidic drag force and a periodically controlled dielectrophoretic (DEP) force from tilted electrodes to minimize cell adhesion to the electrodes, which prevented the loss of rare infectious bacteria. Target bacteria were efficiently segregated from the undesired blood cells to ensure that only the bacteria received the DEP force under the hypotonic condition, while the blood cells received no DEP force and exited the channel via a laminar flow. Thus, the bacteria were successfully extracted from the blood with a high recovery yield of 91.3%, and the limit of the bacteria concentration for isolation was 100 cfu/ml. We also developed an automated system that performed every step from blood‐sample loading to application of electricity to the microfluidic chip for bacteria separation. It reduced the standard deviation of the bacteria recovery yield from 6.16 to 2.77 compared with the conventional batch process, providing stable bacteria‐extraction performance and minimizing errors and bacteria loss caused by user mistakes. © 2019 International Society for Advancement of Cytometry

    更新日期:2019-11-20
  • Morpho‐Rheological Fingerprinting of Rod Photoreceptors Using Real‐Time Deformability Cytometry
    Cytom. Part A (IF 3.433) Pub Date : 2019-05-20
    Tiago Santos‐Ferreira, Maik Herbig, Oliver Otto, Madalena Carido, Mike O. Karl, Stylianos Michalakis, Jochen Guck, Marius Ader

    Distinct cell‐types within the retina are mainly specified by morphological and molecular parameters, however, physical properties are increasingly recognized as a valuable tool to characterize and distinguish cells in diverse tissues. High‐throughput analysis of morpho‐rheological features has recently been introduced using real‐time deformability cytometry (RT‐DC) providing new insights into the properties of different cell‐types. Rod photoreceptors represent the main light sensing cells in the mouse retina that during development forms apically the densely packed outer nuclear layer. Currently, enrichment and isolation of photoreceptors from retinal primary tissue or pluripotent stem cell‐derived organoids for analysis, molecular profiling, or transplantation is achieved using flow cytometry or magnetic activated cell sorting approaches. However, such purification methods require genetic modification or identification of cell surface binding antibody panels. Using primary retina and embryonic stem cell‐derived retinal organoids, we characterized the inherent morpho‐mechanical properties of mouse rod photoreceptors during development based on RT‐DC. We demonstrate that rods become smaller and more compliant throughout development and that these features are suitable to distinguish rods within heterogenous retinal tissues. Hence, physical properties should be considered as additional factors that might affect photoreceptor differentiation and retinal development besides representing potential parameters for label‐free sorting of photoreceptors. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

    更新日期:2019-11-20
  • 更新日期:2019-11-20
  • Flow Cytometric Analysis of Hematopoietic Populations in Rat Bone Marrow. Impact of Trauma and Hemorrhagic Shock
    Cytom. Part A (IF 3.433) Pub Date : 2019-10-09
    Wendy R. Francis, Rachel E. Ireland, Abigail M. Spear, Dominic Jenner, Sarah A. Watts, Emrys Kirkman, Ian Pallister

    Severe injury and hemorrhagic shock (HS) result in multiple changes to hematopoietic differentiation, which contribute to the development of immunosuppression and multiple organ failure (MOF). Understanding the changes that take place during the acute injury phase may help predict which patients will develop MOF and provide potential targets for therapy. Obtaining bone marrow from humans during the acute injury phase is difficult so published data are largely derived from peripheral blood samples, which infer bone marrow changes that reflect the sustained inflammatory response. This preliminary and opportunistic study investigated leucopoietic changes in rat bone marrow 6 h following traumatic injury and HS. Terminally anesthetized male Porton Wistar rats were allocated randomly to receive a sham operation (cannulation with no injury) or femoral fracture and HS. Bone marrow cells were flushed from rat femurs and immunophenotypically stained with specific antibody panels for lymphoid (CD45R, CD127, CD90, and IgM) or myeloid (CD11b, CD45, and RP‐1) lineages. Subsequently, cell populations were fluorescence‐activated cell sorted for morphological assessment. Stage‐specific cell populations were identified using a limited number of antibodies, and leucopoietic changes were determined 6 h following trauma and HS. Myeloid subpopulations could be identified by varying levels CD11b expression, CD45, and RP‐1. Trauma and HS resulted in a significant reduction in total CD11b + myeloid cells including both immature (RP‐1(−)) and mature (RP‐1+) granulocytes. Multiple B‐cell lymphoid subsets were identified. The total percentage of CD90+ subsets remained unchanged following trauma and HS, but there was a reduction in the numbers of maturing CD90(−) cells suggesting movement into the periphery. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

    更新日期:2019-11-20
  • Challenges in the Multivariate Analysis of Mass Cytometry Data: The Effect of Randomization
    Cytom. Part A (IF 3.433) Pub Date : 2019-11-06
    Georgios Papoutsoglou, Vincenzo Lagani, Angelika Schmidt, Konstantinos Tsirlis, David‐Gómez Cabrero, Jesper Tegnér, Ioannis Tsamardinos

    Cytometry by time‐of‐flight (CyTOF) has emerged as a high‐throughput single cell technology able to provide large samples of protein readouts. Already, there exists a large pool of advanced high‐dimensional analysis algorithms that explore the observed heterogeneous distributions making intriguing biological inferences. A fact largely overlooked by these methods, however, is the effect of the established data preprocessing pipeline to the distributions of the measured quantities. In this article, we focus on randomization, a transformation used for improving data visualization, which can negatively affect multivariate data analysis methods such as dimensionality reduction, clustering, and network reconstruction algorithms. Our results indicate that randomization should be used only for visualization purposes, but not in conjunction with high‐dimensional analytical tools. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

    更新日期:2019-11-20
  • An R‐Derived FlowSOM Process to Analyze Unsupervised Clustering of Normal and Malignant Human Bone Marrow Classical Flow Cytometry Data
    Cytom. Part A (IF 3.433) Pub Date : 2019-10-02
    Francis Lacombe, Nicolas Lechevalier, Jean Philippe Vial, Marie C. Béné
    更新日期:2019-11-20
  • Generic Isolated Cell Image Generator
    Cytom. Part A (IF 3.433) Pub Date : 2019-10-08
    Marin Scalbert, Florent Couzinie‐Devy, Riadh Fezzani
    更新日期:2019-11-20
  • Live and Let Dye: Visualizing the Cellular Compartments of the Malaria Parasite Plasmodium falciparum
    Cytom. Part A (IF 3.433) Pub Date : 2019-11-18
    Marleen Linzke, Sun Liu Rei Yan, Attila Tárnok, Henning Ulrich, Matthew R. Groves, Carsten Wrenger

    Malaria remains one of the deadliest diseases worldwide and it is caused by the protozoan parasite Plasmodium spp. Parasite visualization is an important tool for the correct detection of malarial cases but also to understand its biology. Advances in visualization techniques promote new insights into the complex life cycle and biology of Plasmodium parasites. Live cell imaging by fluorescence microscopy or flow cytometry are the foundation of the visualization technique for malaria research. In this review, we present an overview of possibilities in live cell imaging of the malaria parasite. We discuss some of the state‐of‐the‐art techniques to visualize organelles and processes of the parasite and discuss limitation and advantages of each technique. © 2019 International Society for Advancement of Cytometry

    更新日期:2019-11-18
  • Automated Data Cleanup for Mass Cytometry
    Cytom. Part A (IF 3.433) Pub Date : 2019-11-18
    Charles Bruce Bagwell, Margaret Inokuma, Benjamin Hunsberger, Donald Herbert, Christopher Bray, Beth Hill, Gregory Stelzer, Stephen Li, Avinash Kollipara, Olga Ornatsky, Vladimir Baranov

    Mass cytometry is an emerging technology capable of 40 or more correlated measurements on a single cell. The complexity and volume of data generated by this platform have accelerated the creation of novel methods for high‐dimensional data analysis and visualization. A key step in any high‐level data analysis is the removal of unwanted events, a process often referred to as data cleanup. Data cleanup as applied to mass cytometry typically focuses on elimination of dead cells, debris, normalization beads, true aggregates, and coincident ion clouds from raw data. We describe a probability state modeling (PSM) method that automatically identifies and removes these elements, resulting in FCS files that contain mostly live and intact events. This approach not only leverages QC measurements such as DNA, live/dead, and event length but also four additional pulse‐processing parameters that are available on Fluidigm Helios™ and CyTOF® (Fluidigm, Markham, Canada) 2 instruments with software versions of 6.3 or higher. These extra Gaussian‐derived parameters are valuable for detecting well‐formed pulses and eliminating coincident positive ion clouds. The automated nature of this new routine avoids the subjectivity of other gating methods and results in unbiased elimination of unwanted events. © 2019 International Society for Advancement of Cytometry

    更新日期:2019-11-18
  • Computational Analysis of Multiparametric Flow Cytometric Data to Dissect B Cell Subsets in Vaccine Studies
    Cytom. Part A (IF 3.433) Pub Date : 2019-11-11
    Simone Lucchesi, Emanuele Nolfi, Elena Pettini, Gabiria Pastore, Fabio Fiorino, Gianni Pozzi, Donata Medaglini, Annalisa Ciabattini

    The generation of the B cell response upon vaccination is characterized by the induction of different functional and phenotypic subpopulations and is strongly dependent on the vaccine formulation, including the adjuvant used. Here, we have profiled the different B cell subsets elicited upon vaccination, using machine learning methods for interpreting high‐dimensional flow cytometry data sets. The B cell response elicited by an adjuvanted vaccine formulation, compared to the antigen alone, was characterized using two automated methods based on clustering (FlowSOM) and dimensional reduction (t‐SNE) approaches. The clustering method identified, based on multiple marker expression, different B cell populations, including plasmablasts, plasma cells, germinal center B cells and their subsets, while this profiling was more difficult with t‐SNE analysis. When undefined phenotypes were detected, their characterization could be improved by integrating the t‐SNE spatial visualization of cells with the FlowSOM clusters. The frequency of some cellular subsets, in particular plasma cells, was significantly higher in lymph nodes of mice primed with the adjuvanted formulation compared to antigen alone. Thanks to this automatic data analysis it was possible to identify, in an unbiased way, different B cell populations and also intermediate stages of cell differentiation elicited by immunization, thus providing a signature of B cell recall response that can be hardly obtained with the classical bidimensional gating analysis. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

    更新日期:2019-11-11
  • Machine Learning of Discriminative Gate Locations for Clinical Diagnosis
    Cytom. Part A (IF 3.433) Pub Date : 2019-11-05
    Disi Ji, Preston Putzel, Yu Qian, Ivan Chang, Aishwarya Mandava, Richard H. Scheuermann, Jack D. Bui, Huan‐You Wang, Padhraic Smyth

    High‐throughput single‐cell cytometry technologies have significantly improved our understanding of cellular phenotypes to support translational research and the clinical diagnosis of hematological and immunological diseases. However, subjective and ad hoc manual gating analysis does not adequately handle the increasing volume and heterogeneity of cytometry data for optimal diagnosis. Prior work has shown that machine learning can be applied to classify cytometry samples effectively. However, many of the machine learning classification results are either difficult to interpret without using characteristics of cell populations to make the classification, or suboptimal due to the use of inaccurate cell population characteristics derived from gating boundaries. To date, little has been done to optimize both the gating boundaries and the diagnostic accuracy simultaneously. In this work, we describe a fully discriminative machine learning approach that can simultaneously learn feature representations (e.g., combinations of coordinates of gating boundaries) and classifier parameters for optimizing clinical diagnosis from cytometry measurements. The approach starts from an initial gating position and then refines the position of the gating boundaries by gradient descent until a set of globally‐optimized gates across different samples are achieved. The learning procedure is constrained by regularization terms encoding domain knowledge that encourage the algorithm to seek interpretable results. We evaluate the proposed approach using both simulated and real data, producing classification results on par with those generated via human expertise, in terms of both the positions of the gating boundaries and the diagnostic accuracy. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

    更新日期:2019-11-06
  • Classification of Human White Blood Cells Using Machine Learning for Stain‐Free Imaging Flow Cytometry
    Cytom. Part A (IF 3.433) Pub Date : 2019-11-05
    Maxim Lippeveld, Carly Knill, Emma Ladlow, Andrew Fuller, Louise J Michaelis, Yvan Saeys, Andrew Filby, Daniel Peralta
    更新日期:2019-11-05
  • Single Cell Phenotypic Profiling of 27 DLBCL Cases Reveals Marked Intertumoral and Intratumoral Heterogeneity
    Cytom. Part A (IF 3.433) Pub Date : 2019-10-22
    Michael D. Nissen, Manabu Kusakabe, Xuehai Wang, Guillermo Simkin, Deanne Gracias, Kateryna Tyshchenko, Ainsleigh Hill, Justin Meskas, Stacy Hung, Elizabeth A. Chavez, Daisuke Ennishi, Tomohiro Aoki, Clementine Sarkozy, Joseph M. Connors, Pedro Farinha, Graham W. Slack, Randy D. Gascoyne, Ryan R. Brinkman, David W. Scott, Christian Steidl, Andrew P. Weng

    Diffuse large B‐cell lymphoma (DLBCL) is the most common histologic subtype of non‐Hodgkin lymphoma and is notorious for its clinical heterogeneity. Patient outcomes can be predicted by cell‐of‐origin (COO) classification, demonstrating that the underlying transcriptional signature of malignant B‐cells informs biological behavior in the context of standard combination chemotherapy regimens. In the current study, we used mass cytometry (CyTOF) to examine tumor phenotypes at the protein level with single cell resolution in a collection of 27 diagnostic DLBCL biopsy specimens from treatment naïve patients. We found that malignant B‐cells from each patient occupied unique regions in 37‐dimensional phenotypic space with no apparent clustering of samples into discrete subtypes. Interestingly, variable MHC class II expression was found to be the greatest contributor to phenotypic diversity. Within individual tumors, a subset of cases showed multiple phenotypic subpopulations, and in one case, we were able to demonstrate direct correspondence between protein‐level phenotypic subsets and DNA mutation‐defined subclones. In summary, CyTOF analysis can resolve both intertumoral and intratumoral heterogeneity among primary samples and reveals that each case of DLBCL is unique and may be comprised of multiple, genetically distinct subclones. © 2019 International Society for Advancement of Cytometry

    更新日期:2019-11-04
  • Microfluidic Cytometry for High‐Throughput Characterization of Single Cell Cytoplasmic Viscosity Using Crossing Constriction Channels
    Cytom. Part A (IF 3.433) Pub Date : 2019-10-22
    Ke Wang, Xiaohao Sun, Yi Zhang, Yuanchen Wei, Deyong Chen, Hengan Wu, Zijian Song, Rong Long, Junbo Wang, Jian Chen

    This article presents an approach of microfluidic flow cytometry capable of continuously characterizing cytoplasmic viscosities of single cells. The microfluidic system consists of a major constriction channel and a side constriction channel perpendicularly crossing each other. Cells are forced to rapidly travel through the major channel and are partially aspirated into the side channel when passing the channel junction. Numerical simulations were conducted to model the time dependence of the aspiration length into the side channel, which enables the measurement of cytoplasmic viscosity by fitting the model results to experimental data. As a demonstration for high‐throughput measurement, the cytoplasmic viscosities of HL‐60 cells that were native or treated by N‐Formylmethionine‐leucyl‐phenylalanine (fMLP) were quantified with sample sizes as large as thousands of cells. Both the average and median cytoplasmic viscosities of native HL‐60 cells were found to be about 10% smaller than those of fMLP‐treated HL‐60 cells, consistent with previous observations that fMLP treatment can increase the rigidity of white blood cells. Furthermore, the microfluidic system was used to process granulocytes from three donors (sample size >1,000 cells for each donor). The results revealed that the cytoplasmic viscosity of granulocytes from one donor was significantly higher than the other two, which may result from the fact that this donor just recovered from an inflammation. In summary, the developed microfluidic system can collect cytoplasmic viscosities from thousands of cells and may function as an enabling tool in the field of single‐cell analysis. © 2019 International Society for Advancement of Cytometry

    更新日期:2019-11-04
  • CytoNorm: A Normalization Algorithm for Cytometry Data
    Cytom. Part A (IF 3.433) Pub Date : 2019-10-21
    Sofie Van Gassen, Brice Gaudilliere, Martin S. Angst, Yvan Saeys, Nima Aghaeepour

    High‐dimensional flow cytometry has matured to a level that enables deep phenotyping of cellular systems at a clinical scale. The resulting high‐content data sets allow characterizing the human immune system at unprecedented single cell resolution. However, the results are highly dependent on sample preparation and measurements might drift over time. While various controls exist for assessment and improvement of data quality in a single sample, the challenges of cross‐sample normalization attempts have been limited to aligning marker distributions across subjects. These approaches, inspired by bulk genomics and proteomics assays, ignore the single‐cell nature of the data and risk the removal of biologically relevant signals. This work proposes CytoNorm, a normalization algorithm to ensure internal consistency between clinical samples based on shared controls across various study batches. Data from the shared controls is used to learn the appropriate transformations for each batch (e.g., each analysis day). Importantly, some sources of technical variation are strongly influenced by the amount of protein expressed on specific cell types, requiring several population‐specific transformations to normalize cells from a heterogeneous sample. To address this, our approach first identifies the overall cellular distribution using a clustering step, and calculates subset‐specific transformations on the control samples by computing their quantile distributions and aligning them with splines. These transformations are then applied to all other clinical samples in the batch to remove the batch‐specific variations. We evaluated the algorithm on a customized data set with two shared controls across batches. One control sample was used for calculation of the normalization transformations and the second control was used as a blinded test set and evaluated with Earth Mover's distance. Additional results are provided using two real‐world clinical data sets. Overall, our method compared favorably to standard normalization procedures. The algorithm is implemented in the R package “CytoNorm” and available via the following link: www.github.com/saeyslab/CytoNorm © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

    更新日期:2019-11-04
  • Collagen Organization Within the Cartilage of Trpv4−/− Mice Studied with Two‐Photon Microscopy and Polarized Second Harmonic Generation
    Cytom. Part A (IF 3.433) Pub Date : 2019-10-11
    M. Rocio Servin‐Vences, Kate Poole, Anje Sporbert, Gary R. Lewin, Anca Margineanu
    更新日期:2019-11-04
  • Apoptotic Cell Exclusion and Bias‐Free Single‐Cell Selection Are Important Quality Control Requirements for Successful Single‐Cell Sequencing Applications
    Cytom. Part A (IF 3.433) Pub Date : 2019-10-11
    Diana Ordoñez‐Rueda, Bianka Baying, Dinko Pavlinic, Luca Alessandri, Yvonne Yeboah, Jonathan J.M. Landry, Raffaele Calogero, Vladimir Benes, Malte Paulsen

    Single‐cell sequencing experiments are a new mainstay in biology and have been advancing science especially in the biomedical field. The high pressure to integrate the technology into daily laboratory live requires solid knowledge with respect to potential limitations and precautions to be taken care of before applying it to complex research questions. In the past, we have identified two issues with quality measures neglected by the growing community involving SmartSeq and droplet or micro‐well‐based scRNASeq methods (1) how to ensure that only single cells are introduced without biasing on light scattering when handling complex cell mixtures and organ preparations or (2) how best to control for (pro‐)apoptotic cell contaminations in single‐cell sequencing approaches. Sighting of concurrent literature involving single‐cell sequencing technologies revealed that these topics are generally neglected or simply approached in silico but not at the bench before generating single‐cell data sets. We fear that those important quality aspects are overlooked due to reduced awareness of their importance for guaranteeing the quality of experiments. In this Cytometry rigor issue, we provide experimentally supported guidance on how to circumvent those critical shortcomings in order to promote a better use of the fantastic single‐cell sequencing toolbox in biology. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

    更新日期:2019-11-04
  • Reproducibility of Flow Cytometry Through Standardization: Opportunities and Challenges
    Cytom. Part A (IF 3.433) Pub Date : 2019-10-08
    Tomas Kalina

    There is an agreement in the field that interlaboratory reproducibility of flow cytometry measurements as well as the whole studies might be improved by a consensual use of methodological approach. Typically, a consensus is made on a crucial markers needed in the immunostaining panel, sometimes on the particular fluorochrome conjugates and rarely on a complete set of methods for sample preparation. The term “standardization” is used to describe the complete set of methodical steps, while “harmonization” is used for partial agreement on the method. Standardization can provide a platform for improved reproducibility of cytometry results over prolonged periods of time, across different sites and across different instruments. For the purpose of structured discussion, several desired aims are described: common interpretation of the immunophenotype definition of a target subset, accurate quantification, reproducible pattern of a multicolor immunophenotype, and reproducible intensity of all measured parameters. An overview of how standardization was approached by several large consortia is provided: EuroFlow, The ONE Study, Human Immunology Project Consortium (HIPC), and several other groups. Their particular aims and the tools adopted to reach those aims are noted. How those standardization efforts were adopted in the field and how the resulting outcome was evaluated is reviewed. Multiple challenges in the instrument hardware design, instrument setup tools, reagent design, and quality features need to be addressed to achieve optimal standardization. Furthermore, the aims of different studies vary, and thus, the reasonable requirements for standardization differ. A framework of reference for the reasonable outcomes of different approaches is offered. Finally, it is argued that complete standardization is important not only for the reproducibility of measurements but also for education, for quality assessment and for algorithmic data analysis. The different standardized approaches can and in fact should serve as benchmarking reference tools for the development of future flow cytometry studies. © 2019 International Society for Advancement of Cytometry

    更新日期:2019-11-04
  • Relevance of Antibody Validation for Flow Cytometry
    Cytom. Part A (IF 3.433) Pub Date : 2019-10-02
    Tomas Kalina, Kelly Lundsten, Pablo Engel

    Antibody reagents are the key components of multiparametric flow cytometry analysis. Their quality performance is an absolute requirement for reproducible flow cytometry experiments. While there is an enormous body of antibody reagents available, there is still a lack of consensus about which criteria should be evaluated to select antibody reagents with the proper performance, how to validate antibody reagents for flow cytometry, and how to interpret the validation results. The achievements of cytometry moved the field to a higher number of measured parameters, large data sets, and computational data analysis approaches. These advancements pose an increased demand for antibody reagent performance quality. This review summarizes the codevelopment of cytometry, antibody development, and validation strategies. It discusses the diverse issues of the specificity, cross‐reactivity, epitope, titration, and reproducibility features of antibody reagents, and this review discusses the validation principles and methods that are currently available and those that are emerging. We argue that significant efforts should be invested by antibody users, developers, manufacturers, and publishers to increase the quality and reproducibility of published studies. More validation data should be presented by all stakeholders; however, the data should be presented in sufficient experimental detail to foster reproducibility, and community effort shall lead to the public availability of large data sets that can serve as a benchmark for antibody performance. © 2019 International Society for Advancement of Cytometry

    更新日期:2019-11-04
  • Estimation of the Mitochondrial Membrane Potential Using Fluorescence Lifetime Imaging Microscopy
    Cytom. Part A (IF 3.433) Pub Date : 2019-09-05
    Irina A. Okkelman, Dmitri B. Papkovsky, Ruslan I. Dmitriev
    更新日期:2019-11-04
  • Diffusion Mapping of Eosinophil‐Activation State
    Cytom. Part A (IF 3.433) Pub Date : 2019-08-31
    Justyna Piasecka, Catherine A. Thornton, Paul Rees, Huw D. Summers
    更新日期:2019-11-04
  • Rigor and Reproducibility of Cytometry Practices for Immuno‐Oncology: A multifaceted challenge
    Cytom. Part A (IF 3.433) Pub Date : 2019-08-27
    Tamara J. Laskowski, Amy L. Hazen, Renata S. Collazo, David Haviland

    The rapid advancement of immunotherapy strategies has created a need for technologies that can reliably and reproducibly identify rare populations, detect subtle changes in modulatory signals, and assess antigenic expression patterns that are time‐sensitive. Accomplishing these tasks requires careful planning and the employment of tools that provide greater sensitivity and specificity without demanding extensive time. Flow Cytometry has earned its place as a preferred analysis platform. This technology offers a flexible path to the interrogation of protein expression patterns and detection of functional properties in cell populations of interest. Mass Cytometry is a newcomer technology that has generated significant interest in the field. By incorporating mass spectrometry analysis to the traditional principles of flow cytometry, this innovative tool promises to significantly expand the ability to detect multiple proteins on a single cell. The use of these technologies in a manner that is consistent and reproducible through multiple sample sets demands careful attention to experiment design, reagent selection, and instrumentation. Whether applying flow or mass cytometry, reaching successful, reliable results involves many factors. Sample preparation, antibody titrations, and appropriate controls are major biological considerations that impact cytometric analysis. Additionally, instrument voltages, lasers, and run quality assessments are essential for ensuring comparability and reproducibility between analyses. In this article, we aim to discuss the critical aspects that impact flow cytometry, and will touch on important considerations for mass cytometry as well. Focusing on their relevance to immunotherapy studies, we will address the importance of appropriate sample processing and will discuss how selection of suitable panels, controls, and antibodies must follow a carefully designed plan. We will also comment on how educated use of instrumentation plays a significant role in the reliability and reproducibility of results.Through this work, we hope to contribute to the effort toward establishing higher standards for rigor and reproducibility of cytometry practices by researchers, operators, and general cytometry users employing cytometry‐based assays in their work. © 2019 International Society for Advancement of Cytometry

    更新日期:2019-11-04
  • Single‐Cell Intravital Microscopy of Trastuzumab Quantifies Heterogeneous in vivo Kinetics
    Cytom. Part A (IF 3.433) Pub Date : 2019-08-19
    Ran Li, Adel Attari, Mark Prytyskach, Michelle A. Garlin, Ralph Weissleder, Miles A. Miller

    Cell‐to‐cell heterogeneity can substantially impact drug response, especially for monoclonal antibody (mAb) therapies that may exhibit variability in both delivery (pharmacokinetics) and action (pharmacodynamics) within solid tumors. However, it has traditionally been difficult to examine the kinetics of mAb delivery at a single‐cell level and in a manner that enables controlled dissection of target‐dependent and ‐independent behaviors. To address this issue, here we developed an in vivo confocal (intravital) microscopy approach to study single‐cell mAb pharmacology in a mosaic xenograft comprising a mixture of cancer cells with variable expression of the receptor HER2. As a proof‐of‐principle, we applied this model to trastuzumab therapy, a HER2‐targeted mAb widely used for treating breast and gastric cancer patients. Trastuzumab accumulated to a higher degree in HER2‐over expressing tumor cells compared to HER2‐low tumor cells (~5:1 ratio at 24 h after administration) but importantly, the majority actually accumulated in tumor‐associated phagocytes. For example, 24 h after IV administration over 50% of tumoral trastuzumab was found in phagocytes whereas at 48 h it was >80%. Altogether, these results reveal the dynamics of how phagocytes influence mAb behavior in vivo, and demonstrate an application of intravital microscopy for quantitative single‐cell measurement of mAb distribution and retention in tumors with heterogeneous target expression. © 2019 International Society for Advancement of Cytometry

    更新日期:2019-11-04
  • FCMPASS Software Aids Extracellular Vesicle Light Scatter Standardization
    Cytom. Part A (IF 3.433) Pub Date : 2019-06-28
    Joshua A. Welsh, Peter Horak, James S. Wilkinson, Verity J. Ford, Jennifer C. Jones, David Smith, Judith A. Holloway, Nicola A. Englyst

    The study of extracellular vesicles (EVs) is a rapidly growing field due to their great potential in many areas of clinical medicine including diagnostics, prognostics, theranostics, and therapeutics. Flow cytometry is currently one of the most popular methods of analyzing EVs due to it being a high‐throughput, multiparametric technique, that is readily available in the majority of research labs. Despite its wide use, few commercial flow cytometers are designed specifically for the detection of EVs. Many flow cytometers used for EV analysis are working at their detection limits and are unable to detect the majority of EVs. Currently, very little standardization exists for EV flow cytometry, which is an issue because flow cytometers vary considerably in the way they collect scattered or fluorescent light from particles being interrogated. This makes published research hard to interpret, compare, and in some cases, impossible to reproduce. Here we demonstrate a method of flow cytometer light scatter standardization, utilizing flow cytometer postacquisition analysis software (FCMPASS). FCMPASS is built upon Mie theory and enables the approximation of flow cytometer geometric parameters either by analyzing beads of known diameter and refractive index or by inputting the collection angle if known. The software is then able to create a scatter‐diameter curve and scatter‐refractive index curve that enables researchers to convert scattering data and instrument sensitivity into standardized units. Furthermore, with the correct controls, light scatter data can be converted to diameter distributions or refractive index distributions. FCMPASS therefore offers a freely available and ergonomic method of standardizing and further extending EV characterization using flow cytometry. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

    更新日期:2019-11-04
  • Volume 95A, Number 10, October 2019 Cover Image
    Cytom. Part A (IF 3.433) Pub Date : 2019-10-16

    Applying flow cytometry to personalized medicine: measurement of receptor occupancy for optimal concentration of therapeutic antibodies or biopharmaceuticals. See articles by Huse https://onlinelibrary.wiley.com/doi/10.1002/cyto.a.23784, Bringeland et al. https://onlinelibrary.wiley.com/doi/10.1002/cyto.a.23723 and Pluim et al. https://onlinelibrary.wiley.com/doi/full/10.1002/cyto.a.23873.

    更新日期:2019-11-04
  • Multiparameter Flow Cytometry Assay for Quantification of Immune Cell Subsets, PD‐1 Expression Levels and PD‐1 Receptor Occupancy by Nivolumab and Pembrolizumab
    Cytom. Part A (IF 3.433) Pub Date : 2019-08-12
    Dick Pluim, Willeke Ros, Iris H. C. Miedema, Jos H. Beijnen, Jan H. M. Schellens

    We report the development and validation of a 12 parameter immunofluorescence flow cytometry method for the sensitive determination of cell concentrations, their expression of PD‐1, and PD‐1 receptor occupancy. Cell subsets include CD4+ and CD8+‐T‐cells, B‐cells, natural killer cells, classical‐, intermediate‐ and non‐classical monocytes, and myeloid‐ and plasmacytoid dendritic cells. Cells were isolated from peripheral blood by density gradient centrifugation. The validation parameters included specificity, linearity, limit of quantification, precision, biological within‐ and between subject variations. The lower limit of quantification was 5.0% of PD‐1+ cells. Samples were stable for at least 153 days of storage at −80°C. The clinical applicability of the method was demonstrated in 11 advanced cancer patients by the successful determination of immune cell concentrations, relative number of PD‐1+ immune cells, and number of PD‐1 molecules per immune cell. Shortly after infusion of nivolumab, receptor occupancy on CD8+‐T‐cells was 98%. Similar values were found predose cycle 2, suggesting receptor occupancy remained high throughout the entire cycle. © 2019 International Society for Advancement of Cytometry

    更新日期:2019-11-04
  • Automatic Bone Marrow Cellularity Estimation in H&E Stained Whole Slide Images
    Cytom. Part A (IF 3.433) Pub Date : 2019-09-06
    Frederik Skou Nielsen, Mads Jozwiak Pedersen, Mathias Vassard Olsen, Morten Skaarup Larsen, Rasmus Røge, Alex Skovsbo Jørgensen

    Bone marrow cellularity is an important measure in diagnostic hematopathology. Currently, the gold standard for bone marrow cellularity estimation is manual inspection of hematoxylin and eosin stained whole slide images (H&E WSI) by hematopathologists. However, these assessments are subjective and subject to interobserver and intraobserver variability. This may be reduced by using a computer‐assisted estimate of bone marrow cellularity. The aim of this study was to develop a fully automated algorithm to estimate bone marrow cellularity in H&E WSI stains using bone marrow segmentation. Data consisted of eight bone marrow H&E WSIs extracted from eight subjects. An algorithm was developed to estimate the bone marrow cellularity consisting of biopsy segmentation, tissue classification, and bone marrow segmentation. Segmentations of the red and yellow bone marrow (YBM) were used to estimate the bone marrow cellularity within the WSI H&E stains. The DICE coefficient between automatic tissue segmentations and ground truth segmentations conducted by an experienced hematopathologist were used for validation. Furthermore, the agreement between the automatic and two manual cellularity estimates was assessed using Bland–Altman plots and intraclass correlation coefficients (ICC). The validation of the bone marrow segmentation demonstrated an average DICE of 0.901 and 0.920 for the red and YBM, respectively. A mean cellularity estimate difference of −0.552 and − 7.816 was obtained between the automatic cellularity estimates and two manual cellularity estimates, respectively. An ICC of 0.980 (95%CI: 0.925–0.995, P‐value: 5.51 × 10−7) was obtained between the automatic and manual cellularity estimates based on manual annotations. The study demonstrated that it was possible to obtain bone marrow cellularity estimates with a good agreement with bone marrow cellularity estimates obtained from an experienced hematopathologist. © 2019 International Society for Advancement of Cytometry

    更新日期:2019-11-04
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