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Flow Cytometry: Basic Principles and Applications

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A special issue of Cells (ISSN 2073-4409). This special issue belongs to the section "Cell Methods".

Deadline for manuscript submissions: closed (20 April 2023) | Viewed by 16195

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Special Issue Editors

Prof. Dr. Leo Koenderman

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Guest Editor

Department of Respiratory Medicine, University Medical Center Utrecht, 3584CX Utrecht, The Netherlands
Interests: flow cytometry; neutrophil; infection; immunity; eosinophils

Dr. G. H. Tinnevelt

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Guest Editor

Institute for Molecules and Materials (Analytical Chemistry), Radboud University, P.O. Box 9010, 6500 GL Nijmegen, The Netherlands
Interests: flow cytometry; data analysis; chemometrics; matlab; bioinformatics

Dr. Nienke Vrisekoop

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Guest Editor

Department of Respiratory Medicine, University Medical Center Utrecht, 3584CX Utrecht, The Netherlands
Interests: flow cytometry; phagocyte biology; intravital imaging; neutrophil

Special Issue Information

Dear Colleagues,

Flow cytometry is an indispensable technique in clinical diagnostics and immunological and hematological research. This Special Issue in Cells will highlight the powerful applications of this technique as well as the pitfalls in both data acquisition and interpretation. Therefore, several aspects will be discussed in detail: Firstly, data pre-processing with a focus on: i. correct settings of the hardware and ii. correct transformation of the data to avoid erroneous results caused by inadequate processing. Secondly, the vast increase in fluorescent labels comes with essential and complex issues associated with correct compensation. Thirdly, as the data obtained via cytometry are multidimensional in nature, the choice of dimension reduction algorithms is key, as their use depends on the application. Finally, the translation of algorithms described in complex software environments such as R, Matlab and Python must be applied to easy-to-use and intuitive graphical user interfaces, aiding care professionals’ clinical decision making with limited background in flow cytometry. Several articles will describe newly developed applications both based on multicolor fluorescence and multi-mass cytometry. Last but not least, an article will deal with recent developments of point-of-care, 24/7, fully automated flow cytometry that can be implemented outside the walls of a large hospital.

Prof. Dr. Leo Koenderman
Dr. G. H. Tinnevelt
Dr. Nienke Vrisekoop
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Cells is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

multi-color flow cytometry
multi-dimensional analysis
compensation
transformation
automation
point-of-care
mass cytometry
software
immune monitoring and minimal residual disease.

Published Papers (4 papers)

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Research

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18 pages, 2713 KiB

Open AccessArticle

Development of an Automated Online Flow Cytometry Method to Quantify Cell Density and Fingerprint Bacterial Communities

by Juan López-Gálvez, Konstanze Schiessl, Michael D. Besmer, Carmen Bruckmann, Hauke Harms and Susann Müller

Cells 2023, 12(12), 1559; https://doi.org/10.3390/cells12121559 - 6 Jun 2023

Cited by 2 | Viewed by 2748

Abstract

Cell density is an important factor in all microbiome research, where interactions are of interest. It is also the most important parameter for the operation and control of most biotechnological processes. In the past, cell density determination was often performed offline and manually, resulting in a delay between sampling and immediate data processing, preventing quick action. While there are now some online methods for rapid and automated cell density determination, they are unable to distinguish between the different cell types in bacterial communities. To address this gap, an online automated flow cytometry procedure is proposed for real-time high-resolution analysis of bacterial communities. On the one hand, it allows for the online automated calculation of cell concentrations and, on the other, for the differentiation between different cell subsets of a bacterial community. To achieve this, the OC-300 automation device (onCyt Microbiology, Zürich, Switzerland) was coupled with the flow cytometer CytoFLEX (Beckman Coulter, Brea, USA). The OC-300 performs the automatic sampling, dilution, fixation and 4′,6-diamidino-2-phenylindole (DAPI) staining of a bacterial sample before sending it to the CytoFLEX for measurement. It is demonstrated that this method can reproducibly measure both cell density and fingerprint-like patterns of bacterial communities, generating suitable data for powerful automated data analysis and interpretation pipelines. In particular, the automated, high-resolution partitioning of clustered data into cell subsets opens up the possibility of correlation analysis to identify the operational or abiotic/biotic causes of community disturbances or state changes, which can influence the interaction potential of organisms in microbiomes or even affect the performance of individual organisms. Full article

(This article belongs to the Special Issue Flow Cytometry: Basic Principles and Applications)

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19 pages, 2967 KiB

Open AccessFeature PaperArticle

Identification of CD203c as a New Basophil-Specific Flow-Marker in Ph⁺ Chronic Myeloid Leukemia

by Irina Sadovnik, Daniel Ivanov, Dubravka Smiljkovic, Gabriele Stefanzl, Lina Degenfeld-Schonburg, Susanne Herndlhofer, Gregor Eisenwort, Alexander W. Hauswirth, Thamer Sliwa, Felix Keil, Wolfgang R. Sperr and Peter Valent

Cells 2023, 12(1), 3; https://doi.org/10.3390/cells12010003 - 20 Dec 2022

Cited by 1 | Viewed by 1980

Abstract

Basophilia is a crucial prognostic variable in Ph-chromosome-positive chronic myeloid leukemia (CML). The ectoenzyme CD203c is an activation-linked surface antigen that is expressed specifically on basophil-committed progenitor cells and mature basophils. We examined the expression of CD203c on progenitors and/or basophils in 21 healthy donors and 44 patients with CML. As expected, the numbers of CD203c⁺ blood leukocytes were significantly higher in CML patients compared to controls (percentage of CD203c⁺ cells among viable cells in CML at diagnosis: 4.19 ± 3.68% vs. controls: 0.53 ± 0.23%, p < 0.05). Moreover, CML basophils expressed higher levels of CD203c compared to normal basophils (median staining-index in CML at diagnosis: 29.41 ± 19.14 versus controls: 20.44 ± 13.45). We also found that the numbers and percentage of circulating CD203c⁺ cells at diagnosis correlate with the disease-related risk-profile. Incubation of CML basophils with an anti-IgE-antibody resulted in further upregulation of CD203c. After successful treatment with imatinib and/or other BCR::ABL1 inhibitors leading to major or complete molecular responses, the numbers of CD203c⁺ basophils decreased substantially in our CML patients compared to pre-treatment values. Together, CD203c is overexpressed on CML basophils, is further upregulated by IgE receptor cross-linking, and may serve as a biomarker to quantify basophilia in patients with CML at diagnosis and during therapy. Full article

(This article belongs to the Special Issue Flow Cytometry: Basic Principles and Applications)

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20 pages, 7917 KiB

Open AccessArticle

Comparison of Whole Blood Cryopreservation Methods for Extensive Flow Cytometry Immunophenotyping

by Valentina Serra, Valeria Orrù, Sandra Lai, Monia Lobina, Maristella Steri, Francesco Cucca and Edoardo Fiorillo

Cells 2022, 11(9), 1527; https://doi.org/10.3390/cells11091527 - 2 May 2022

Cited by 4 | Viewed by 3733

Abstract

Fresh blood immunophenotyping by flow cytometry, based on the reliable simultaneous detection of several markers in a cell, is the method of choice to study the circulating human immune system. Especially in large and multicenter studies, high sample quality is difficult to achieve, and adequate collection and storage of samples with fine-tuned whole blood cryopreservation is mandatory. Here, we compared the quality of immunophenotypic data obtained from fresh blood with those obtained after five cryopreservation methods by quantifying the levels of 41 immune cell populations. They comprised B and T lymphocyte subsets and their maturation stages, as well as monocytes and granulocytes. Three methods used fixative solutions and two other methods used dimethyl sulfoxide solutions to preserve cell viability. The fixative methods prevented detection of markers critical for identification of B and T cell subsets, including CD27, CXCR3, and CCR6. The other two methods permitted reliable discrimination of most immune-cell populations in thawed samples, though some cell frequencies varied compared to the corresponding fresh sample. Of those two methods, the one preserving blood in media containing dimethyl sulfoxide produced results that were most similar to those with fresh samples. Full article

(This article belongs to the Special Issue Flow Cytometry: Basic Principles and Applications)

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Review

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30 pages, 2484 KiB

Open AccessReview

Flow Cytometry: The Next Revolution

by J. Paul Robinson, Raluca Ostafe, Sharath Narayana Iyengar, Bartek Rajwa and Rainer Fischer

Cells 2023, 12(14), 1875; https://doi.org/10.3390/cells12141875 - 17 Jul 2023

Cited by 12 | Viewed by 6371

Abstract

Unmasking the subtleties of the immune system requires both a comprehensive knowledge base and the ability to interrogate that system with intimate sensitivity. That task, to a considerable extent, has been handled by an iterative expansion in flow cytometry methods, both in technological capability and also in accompanying advances in informatics. As the field of fluorescence-based cytomics matured, it reached a technological barrier at around 30 parameter analyses, which stalled the field until spectral flow cytometry created a fundamental transformation that will likely lead to the potential of 100 simultaneous parameter analyses within a few years. The simultaneous advance in informatics has now become a watershed moment for the field as it competes with mature systematic approaches such as genomics and proteomics, allowing cytomics to take a seat at the multi-omics table. In addition, recent technological advances try to combine the speed of flow systems with other detection methods, in addition to fluorescence alone, which will make flow-based instruments even more indispensable in any biological laboratory. This paper outlines current approaches in cell analysis and detection methods, discusses traditional and microfluidic sorting approaches as well as next-generation instruments, and provides an early look at future opportunities that are likely to arise. Full article

(This article belongs to the Special Issue Flow Cytometry: Basic Principles and Applications)

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