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Special Issue "Flow Cytometry and Its Applications to Molecular Biology and Diagnosis"

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Immunology".

Deadline for manuscript submissions: closed (30 November 2020).

Special Issue Editors

Prof. Dr. Stefano Papa
Website
Guest Editor
Department of Biomolecular Sciences, University of Urbino Carlo Bo, 61029 Urbino, Italy
Interests: Flow Cytometry; Apoptosis; Exosomes; NK cells; Melatonin
Special Issues and Collections in MDPI journals
Dr. Paula Fernandez
Website
Guest Editor
Institute for Laboratory Medicine, Kantonsspital Aarau, Aarau, Switzerland
Interests: flow cytometry; hematology; immunology; clinical diagnostics
Special Issues and Collections in MDPI journals
Prof. Dr. Claudio Ortolani
Website
Guest Editor
Department of Biomolecular Sciences, University of Urbino, Urbino, Italy
Interests: flow cytometry; hematology; immunology; clinical diagnostics
Special Issues and Collections in MDPI journals

Special Issue Information

Dear Colleagues,

Flow Cytometry is the most powerful technology to investigate antigen expression at single cell level, both in living and fixed cells as well as in subcellular bodies like exosomes. The recent evolution of optical benches for the analysis of fluorescence intensity combined with the new spectral cytometry has increased the performance towards an 18-signal analysis in the diagnostic lab. Thanks to multispectral technology, instruments with improved performance allow the number of parameters to increase beyond forty.

This Special Issue will focus upon flow analysis applied to both cellular and subcellular levels, in order to suggest new protocols for these investigations and new findings that could combine research practice with diagnosis in clinical immunology and hematology.

The COVID-19 pandemic has placed flow cytometry at center stage again. The Guest Editors ask researchers and clinicians to suggest and prepare comprehensive reviews regarding the application of flow cytometry in the relevant molecular biology. Moreover, we are inviting colleagues interested in the main aspects of flow analysis applications to contribute to this Special Issue by submitting original papers or short communications.

Prof. Dr. Stefano Papa
Dr. Paula Fernandez
Prof. Dr. Claudio Ortolani
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 papers will be 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. International Journal of Molecular Sciences is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. There is an Article Processing Charge (APC) for publication in this open access journal. For details about the APC please see here. 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

  • Flow Cytometry
  • Apoptosis
  • Autophagy
  • Oxidative stress
  • Lysosome pathway
  • Hematological diseases
  • Immunodeficiency
  • COVID-19 immunodepression
  • Immune response

Published Papers (6 papers)

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Research

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Open AccessArticle
Radiation Response of Cervical Cancer Stem Cells Is Associated with Pretreatment Proportion of These Cells and Physical Status of HPV DNA
Int. J. Mol. Sci. 2021, 22(3), 1445; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22031445 - 01 Feb 2021
Abstract
Radio- and chemoresistance of cancer stem cells (CSCs) is considered as one of the possible causes of adverse results of chemoradiotherapy for various malignancies, including cervical cancer. However, little is known about quantitative changes in the CSC subpopulation in the course of treatment [...] Read more.
Radio- and chemoresistance of cancer stem cells (CSCs) is considered as one of the possible causes of adverse results of chemoradiotherapy for various malignancies, including cervical cancer. However, little is known about quantitative changes in the CSC subpopulation in the course of treatment and mechanisms for individual response of CSCs to therapy. The purpose of the study was to evaluate the association of radiation response of cervical CSCs with clinical and morphological parameters of disease and features of human papillomavirus (HPV) infection. The proportion of CD44+CD24low CSCs was determined by flow cytometry in cervical scrapings from 55 patients with squamous cell carcinoma of uterine cervix before treatment and after fractionated irradiation at a total dose of 10 Gy. Real-time PCR assay was used to evaluate molecular parameters of HPV DNA. Post-radiation increase in the CSC proportion was found in 47.3% of patients. Clinical and morphological parameters (stage, status of lymph node involvement, and histological type) were not significantly correlated with radiation changes in the CSC proportion. Single- and multifactor analyses revealed two independent indicators affecting the radiation response of CSCs: initial proportion of CSCs and physical status of HPV DNA (R = 0.86, p = 0.001 for the multiple regression model in the whole). Full article
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Open AccessArticle
Centrifugation Removes a Population of Large Vesicles, or “Macroparticles,” Intermediate in Size to RBCs and Microvesicles
Int. J. Mol. Sci. 2021, 22(3), 1243; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22031243 - 27 Jan 2021
Abstract
Microparticles or microvesicles (MPs/MVs) are sub-cellular vesicles with a growing number of known biological functions. Microvesicles from a variety of parent cells within the vascular system increase in numerous pathological states. Red blood cell-derived MVs (RMVs) are relatively less studied than other types [...] Read more.
Microparticles or microvesicles (MPs/MVs) are sub-cellular vesicles with a growing number of known biological functions. Microvesicles from a variety of parent cells within the vascular system increase in numerous pathological states. Red blood cell-derived MVs (RMVs) are relatively less studied than other types of circulating MVs despite red blood cells (RBCs) being the most abundant intravascular cell. This may be in part due the echoes of past misconceptions that RBCs were merely floating anucleate bags of hemoglobin rather than dynamic and responsive cells. The initial aim of this study was to maximize the concentration of RMVs derived from various blood or blood products by focusing on the optimal isolation conditions without creating more MVs from artificial manipulation. We found that allowing RBCs to sediment overnight resulted in a continuum in size of RBC membrane-containing fragments or vesicles extending beyond the 1 µm size limit suggested by many as the maximal size of an MV. Additionally, dilution and centrifugation factors were studied that altered the resultant MV population concentration. The heterogeneous size of RMVs was confirmed in mice models of hemolytic anemia. This methodological finding establishes a new paradigm in that it blurs the line between RBC, fragment, and RMV as well as suggests that the concentration of circulating RMVs may be widely underestimated given that centrifugation removes the majority of such RBC-derived membrane-containing particles. Full article
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Open AccessArticle
Time-Lapse Flow Cytometry: A Robust Tool to Assess Physiological Parameters Related to the Fertilizing Capability of Human Sperm
Int. J. Mol. Sci. 2021, 22(1), 93; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22010093 - 24 Dec 2020
Abstract
Plasma membrane (PM) hyperpolarization, increased intracellular pH (pHi), and changes in intracellular calcium concentration ([Ca2+]i) are physiological events that occur during human sperm capacitation. These parameters are potential predictors of successful outcomes for men undergoing artificial reproduction [...] Read more.
Plasma membrane (PM) hyperpolarization, increased intracellular pH (pHi), and changes in intracellular calcium concentration ([Ca2+]i) are physiological events that occur during human sperm capacitation. These parameters are potential predictors of successful outcomes for men undergoing artificial reproduction techniques (ARTs), but methods currently available for their determination pose various technical challenges and limitations. Here, we developed a novel strategy employing time-lapse flow cytometry (TLFC) to determine capacitation-related membrane potential (Em) and pHi changes, and progesterone-induced [Ca2+]i increases. Our results show that TLFC is a robust method to measure absolute Em and pHi values and to qualitatively evaluate [Ca2+]i changes. To support the usefulness of our methodology, we used sperm from two types of normozoospermic donors: known paternity (subjects with self-reported paternity) and no-known paternity (subjects without self-reported paternity and no known fertility problems). We found relevant differences between them. The incidences of membrane hyperpolarization, pHi alkalinization, and increased [Ca2+]i were consistently high among known paternity samples (100%, 100%, and 86%, respectively), while they varied widely among no-known paternity samples (44%, 17%, and 45%, respectively). Our results indicate that TLFC is a powerful tool to analyze key physiological parameters of human sperm, which pending clinical validation, could potentially be employed as fertility predictors. Full article
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Open AccessArticle
Diameters and Fluorescence Calibration for Extracellular Vesicle Analyses by Flow Cytometry
Int. J. Mol. Sci. 2020, 21(21), 7885; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21217885 - 23 Oct 2020
Cited by 5
Abstract
Extracellular vesicles (EVs) play a crucial role in the intercellular crosstalk. Mesenchymal stem cell-derived EVs (MSC-EVs), displaying promising therapeutic roles, contribute to the strong rationale for developing EVs as an alternative therapeutic option. EV analysis still represents one of the major issues to [...] Read more.
Extracellular vesicles (EVs) play a crucial role in the intercellular crosstalk. Mesenchymal stem cell-derived EVs (MSC-EVs), displaying promising therapeutic roles, contribute to the strong rationale for developing EVs as an alternative therapeutic option. EV analysis still represents one of the major issues to be solved in order to translate the use of MSC-EV detection in clinical settings. Even if flow cytometry (FC) has been largely applied for EV studies, the lack of consensus on protocols for FC detection of EVs generated controversy. Standard FC procedures, based on scatter measurements, only allows the detection of the “tip of the iceberg” of all EVs. We applied an alternative FC approach based on the use of a trigger threshold on a fluorescence channel. The EV numbers obtained by the application of the fluorescence triggering resulted significantly higher in respect to them obtained from the same samples acquired by placing the threshold on the side scatter (SSC) channel. The analysis of EV concentrations carried out by three different standardized flow cytometers allowed us to achieve a high level of reproducibility (CV < 20%). By applying the here-reported method highly reproducible results in terms of EV analysis and concentration measurements were obtained. Full article
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Review

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Open AccessReview
Emerging Role of T-cell Receptor Constant β Chain-1 (TRBC1) Expression in the Flow Cytometric Diagnosis of T-cell Malignancies
Int. J. Mol. Sci. 2021, 22(4), 1817; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22041817 - 12 Feb 2021
Abstract
T-cell clonality testing is integral to the diagnostic work-up of T-cell malignancies; however, current methods lack specificity and sensitivity, which can make the diagnostic process difficult. The recent discovery of a monoclonal antibody (mAb) specific for human TRBC1 will greatly improve the outlook [...] Read more.
T-cell clonality testing is integral to the diagnostic work-up of T-cell malignancies; however, current methods lack specificity and sensitivity, which can make the diagnostic process difficult. The recent discovery of a monoclonal antibody (mAb) specific for human TRBC1 will greatly improve the outlook for T-cell malignancy diagnostics. The anti-TRBC1 mAb can be used in flow cytometry immunophenotyping assays to provide a low-cost, robust, and highly specific test that detects clonality of immunophenotypically distinct T-cell populations. Recent studies demonstrate the clinical utility of this approach in several contexts; use of this antibody in appropriately designed flow cytometry panels improves detection of circulating disease in patients with cutaneous T-cell lymphoma, eliminates the need for molecular clonality testing in the context of large granular lymphocyte leukemia, and provides more conclusive results in the context of many other T-cell disorders. It is worth noting that the increased ability to detect discrete clonal T-cell populations means that identification of T-cell clones of uncertain clinical significance (T-CUS) will become more common. This review discusses this new antibody and describes how it defines clonal T-cells. We present and discuss assay design and summarize findings to date about the use of flow cytometry TRBC1 analysis in the field of diagnostics, including lymph node and fluid sample investigations. We also make suggestions about how to apply the assay results in clinical work-ups, including how to interpret and report findings of T-CUS. Finally, we highlight areas that we think will benefit from further research. Full article
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Open AccessReview
Contributions of Flow Cytometry to the Molecular Study of Spermatogenesis in Mammals
Int. J. Mol. Sci. 2021, 22(3), 1151; https://doi.org/10.3390/ijms22031151 - 25 Jan 2021
Abstract
Mammalian testes are very heterogeneous organs, with a high number of different cell types. Testicular heterogeneity, together with the lack of reliable in vitro culture systems of spermatogenic cells, have been an obstacle for the characterization of the molecular bases of the unique [...] Read more.
Mammalian testes are very heterogeneous organs, with a high number of different cell types. Testicular heterogeneity, together with the lack of reliable in vitro culture systems of spermatogenic cells, have been an obstacle for the characterization of the molecular bases of the unique events that take place along the different spermatogenic stages. In this context, flow cytometry has become an invaluable tool for the analysis of testicular heterogeneity, and for the purification of stage-specific spermatogenic cell populations, both for basic research and for clinical applications. In this review, we highlight the importance of flow cytometry for the advances on the knowledge of the molecular groundwork of spermatogenesis in mammals. Moreover, we provide examples of different approaches to the study of spermatogenesis that have benefited from flow cytometry, including the characterization of mutant phenotypes, transcriptomics, epigenetic and genome-wide chromatin studies, and the attempts to establish cell culture systems for research and/or clinical aims such as infertility treatment. Full article
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