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Trends and Prospects of Flow Cytometry in Cell and Molecular Biology

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

Deadline for manuscript submissions: 20 December 2024 | Viewed by 19286

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Prof. Dr. Claudio Ortolani

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Department of Biomolecular Sciences, University of Urbino, Urbino, Italy
Interests: flow cytometry; hematology; immunology; clinical diagnostics
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Prof. Dr. José-Enrique O’Connor

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Department of Biochemistry, School of Medicine, University of Valencia, 46010 Valencia, Spain
Interests: flow cytometry; cell biology; metabolism; biochemistry; immunology; stem cell biology; macrophage
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Dr. Monia Lenzi

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Department of Pharmacy and Biotechnology, Alma Mater Studiorum University of Bologna, 40126 Bologna, Italy
Interests: chemoprevention; genotoxicity; in vitro toxicology; bioactive molecules; natural compounds; flow cytometry
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Special Issue Information

Dear Colleagues,

Flow Cytometry is a methodology we consider mature but paradoxically keeps evolving rapidly, continuously offering new challenges even to those who think they know it deeply.

As proof of what has just been said, we could recall the new experimental fields for its application that have emerged in the last period.

Born as a technique for automatically evaluating cell features, such as physical parameters and the content of nucleic acids, it has become a multifaceted tool in a series of different sectors and an invaluable instrument in studying the biochemistry and molecular features of the cells.

On the one hand, the continuously growing availability of a panoply of fluorescent probes allows us to investigate a series of cellular activities in a new way; on the other hand, the appearance of new technologies, such as Raman cytometry and others, promises the arrival of an era in which it will be possible to acquire information on metabolism and cell functions through a label-free approach.

The ambition of this issue of the Journal is to take stock of what is happening in this field and help us understand the new directions toward which Flow Cytometry is moving.

I'm confident that, with the help of all of you, this special number of IJMS will be a success.

Prof. Dr. Claudio Ortolani
Prof. Dr. José-Enrique O’Connor
Dr. Monia Lenzi
Guest Editors

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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.

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Keywords

biochemistry
molecular biology
cell function
cell metabolism
new cytometries
new applications
new methods
microbiology

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Published Papers (11 papers)

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Research

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16 pages, 1815 KiB

Open AccessArticle

A New Easy-to-Perform Flow Cytometry Assay for Determining Bacterial- and Viral-Infection-Induced Polymorphonuclear Neutrophil and Monocyte Membrane Marker Modulation in Febrile Patients

by Marilena La Sorda, Desy De Lorenzis, Alessandra Battaglia, Barbara Fiori, Rosalia Graffeo, Rosaria Santangelo, Tiziana D’Inzeo, Gennaro De Pascale, Giovanni Schinzari, Romina Rose Pedone, Ernesto Rossi, Maurizio Sanguinetti, Michela Sali and Andrea Fattorossi

Int. J. Mol. Sci. 2024, 25(21), 11632; https://doi.org/10.3390/ijms252111632 - 29 Oct 2024

Viewed by 541

Abstract

We developed a flow cytometry (FC) assay enabling the rapid and accurate identification of bacterial and viral infections using whole blood samples. The streamlined flow cytometry assay is designed to be user-friendly, making it accessible even for operators with limited experience in FC techniques. The key components of the assay focus on the expression levels of specific surface markers—CD64 on polymorphonuclear neutrophils (PMN) as a marker for bacterial infection, and CD169 on monocytes (MO) for viral infection. The strong performance indicated by an area under the receiver operating characteristic (ROC) curve of 0.94 for both PMN CD64 positive predictive value (PPV) 97.96% and negative predictive value (NPV) 76.67%, and MO CD169 PPV 82.6% and NPV 86.9%, highlight the assay’s robustness in differentiating between bacterial and viral infections accurately. The FC assay includes the assessment of immune system status through HLA-DR and IL-1R2 modulation in MO, providing a useful insight into the patients’ immune response. The significant increase in the frequency of MO exhibiting reduced HLA-DR expression and elevated IL-1R2 levels in infected patients (compared to healthy controls) underscores the potential of these markers as indicators of infection severity. Although the overall correlation between HLA-DR and IL-1R2 expression levels was not significant across all patients, there was a trend in patients with more severe disease suggesting that these markers may have the potential to assist in stratifying patient risk. The present FC assay has the potential to become routine in the clinical microbiology laboratory community and to be helpful in guiding clinical decision making. Full article

(This article belongs to the Special Issue Trends and Prospects of Flow Cytometry in Cell and Molecular Biology)

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18 pages, 3486 KiB

Open AccessArticle

A Label-Free Optical Flow Cytometry Based-Method for Rapid Assay of Disinfectants’ Bactericidal Activity

by Andreea Maria Pîndaru, Luminița Măruțescu, Marcela Popa and Mariana Carmen Chifiriuc

Int. J. Mol. Sci. 2024, 25(13), 7158; https://doi.org/10.3390/ijms25137158 - 28 Jun 2024

Viewed by 971

Abstract

Selecting the appropriate disinfectant to control and prevent healthcare-associated infections (HAIs) is a challenging task for environmental health experts due to the large number of available disinfectant products. This study aimed to develop a label-free flow cytometry (FCM) method for the rapid evaluation of bactericidal activity and to compare its efficacy with that of standard qualitative/quantitative suspension tests. The bactericidal efficiency of eight commercial disinfectants containing quaternary ammonium compounds (QACs) was evaluated against four strains recommended by EN 13727 (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus hirae) and four multidrug-resistant pathogens. The proposed FCM protocol measures changes in scattered light and counts following disinfectant exposure, neutralization, and culture steps. Unlike other available FCM-based methods, this approach does not rely on autofluorescence measurements, impedance cytometry, or fluorescent dyes. The FCM scattered light signals revealed both decreased count rates and morphological changes after treatment with minimum inhibitory concentrations (MICs) and higher concentrations for all tested bacteria. The results from the FCM measurements showed excellent correlation with those from standard assays, providing a rapid tool for monitoring the susceptibility profile of clinical, multidrug-resistant pathogens to chemical disinfectants, which could support infection prevention and control procedures for healthcare environments. This label-free FCM protocol offers a novel and rapid tool for environmental health experts, aiding in the optimization of disinfectant selection for the prevention and control of HAIs. Full article

(This article belongs to the Special Issue Trends and Prospects of Flow Cytometry in Cell and Molecular Biology)

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15 pages, 3884 KiB

Open AccessArticle

Investigating Vα7.2⁺/CD161⁻ T Cell and MAIT Cell Profiles Using Flow Cytometry in Healthy Subjects and Subjects with Atopic Dermatitis

by Parvind Singh, Krisztian Gaspar, Andrea Szegedi, Laszlo Sajtos, Sandor Barath and Zsuzsanna Hevessy

Int. J. Mol. Sci. 2024, 25(6), 3486; https://doi.org/10.3390/ijms25063486 - 20 Mar 2024

Cited by 1 | Viewed by 1546

Abstract

This study investigates the roles of mucosal-associated invariant T (MAIT) cells and Vα7.2⁺/CD161⁻ T cells in skin diseases, focusing on atopic dermatitis. MAIT cells, crucial for bridging innate and adaptive immunity, were analyzed alongside Vα7.2⁺/CD161⁻ T cells in peripheral blood samples from 14 atopic dermatitis patients and 10 healthy controls. Flow cytometry and machine learning algorithms were employed for a comprehensive analysis. The results indicate a significant decrease in MAIT cells and CD69 subsets in atopic dermatitis, coupled with elevated CD38 and polyfunctional MAIT cells producing TNFα and Granzyme B (TNFα⁺/GzB⁺). Vα7.2⁺/CD161⁻ T cells in atopic dermatitis exhibited a decrease in CD8 and IFNγ-producing subsets but an increase in CD38 activated and IL-22-producing subsets. These results highlight the distinctive features of MAIT cells and Vα7.2⁺/CD161⁻ T cells and their different roles in the pathogenesis of atopic dermatitis and provide insights into their potential roles in immune-mediated skin diseases. Full article

(This article belongs to the Special Issue Trends and Prospects of Flow Cytometry in Cell and Molecular Biology)

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21 pages, 5694 KiB

Open AccessArticle

Phenotypic Analysis of Hematopoietic Stem and Progenitor Cell Populations in Acute Myeloid Leukemia Based on Spectral Flow Cytometry, a 20-Color Panel, and Unsupervised Learning Algorithms

by Thomas Matthes

Int. J. Mol. Sci. 2024, 25(5), 2847; https://doi.org/10.3390/ijms25052847 - 29 Feb 2024

Viewed by 1729

Abstract

The analysis of hematopoietic stem and progenitor cell populations (HSPCs) is fundamental in the understanding of normal hematopoiesis as well as in the management of malignant diseases, such as leukemias, and in their diagnosis and follow-up, particularly the measurement of treatment efficiency with the detection of measurable residual disease (MRD). In this study, I designed a 20-color flow cytometry panel tailored for the comprehensive analysis of HSPCs using a spectral cytometer. My investigation encompassed the examination of forty-six samples derived from both normal human bone marrows (BMs) and patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) along with those subjected to chemotherapy and BM transplantation. By comparing my findings to those obtained through conventional flow cytometric analyses utilizing multiple tubes, I demonstrate that my innovative 20-color approach enables a more in-depth exploration of HSPC subpopulations and the detection of MRD with at least comparable sensitivity. Furthermore, leveraging advanced analytical tools such as t-SNE and FlowSOM learning algorithms, I conduct extensive cross-sample comparisons with two-dimensional gating approaches. My results underscore the efficacy of these two methods as powerful unsupervised alternatives for manual HSPC subpopulation analysis. I expect that in the future, complex multi-dimensional flow cytometric data analyses, such as those employed in this study, will be increasingly used in hematologic diagnostics. Full article

(This article belongs to the Special Issue Trends and Prospects of Flow Cytometry in Cell and Molecular Biology)

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18 pages, 2099 KiB

Open AccessArticle

Persistence of Chronic Lymphocytic Leukemia Stem-like Populations under Simultaneous In Vitro Treatment with Curcumin, Fludarabine, and Ibrutinib: Implications for Therapy Resistance

by Àngel Bistué-Rovira, Laura G. Rico, Jorge Bardina, Jordi Juncà, Isabel Granada, Jolene A. Bradford, Michael D. Ward, Roser Salvia, Francesc Solé and Jordi Petriz

Int. J. Mol. Sci. 2024, 25(4), 1994; https://doi.org/10.3390/ijms25041994 - 7 Feb 2024

Cited by 1 | Viewed by 1633

Abstract

Leukemic stem cells (LSCs) possess similar characteristics to normal hematopoietic stem cells, including self-renewal capacity, quiescence, ability to initiate leukemia, and drug resistance. These cells play a significant role in leukemia relapse, persisting even after apparent remission. LSCs were first described in 1994 by Lapidot et al. Although they have been extensively studied in acute leukemia, more LSC research is still needed in chronic lymphocytic leukemia (CLL) to understand if reduced apoptosis in mature cells should still be considered as the major cause of this disease. Here, we provide new evidence suggesting the existence of stem-like cell populations in CLL, which may help to understand the disease as well as to develop effective treatments. In this study, we identified a potential leukemic stem cell subpopulation using the tetraploid CLL cell line I83. This subpopulation is characterized by diploid cells that were capable of generating the I83 tetraploid population. Furthermore, we adapted a novel flow cytometry analysis protocol to detect CLL subpopulations with stem cell properties in peripheral blood samples and primary cultures from CLL patients. These cells were identified by their co-expression of CD19 and CD5, characteristic markers of CLL cells. As previously described, increased alkaline phosphatase (ALP) activity is indicative of stemness and pluripotency. Moreover, we used this method to investigate the potential synergistic effect of curcumin in combination with fludarabine and ibrutinib to deplete this subpopulation. Our results confirmed the effectiveness of this ALP-based analysis protocol in detecting and monitoring leukemic stem-like cells in CLL. This analysis also identified limitations in eradicating these populations using in vitro testing. Furthermore, our findings demonstrated that curcumin significantly enhanced the effects of fludarabine and ibrutinib on the leukemic fraction, exhibiting synergistic effects (combination drug index, CDI 0.97 and 0.37, respectively). Our results lend support to the existence of potential stem-like populations in CLL cell lines, and to the idea that curcumin could serve as an effective adjuvant in therapies aimed at eliminating these populations and improving treatment efficacy. Full article

(This article belongs to the Special Issue Trends and Prospects of Flow Cytometry in Cell and Molecular Biology)

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Review

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18 pages, 7782 KiB

Open AccessReview

Environmental Diagnosis through a Flow Cytometric Approach

by Giovanna Panza, Fabrizio Frontalini, Caterina Ciacci, Giuseppe Protano, Mariele Montanari, Daniele Lopez, Francesco Nannoni, Stefano Papa, Claudio Ortolani, Federica Rebecchi, Vieri Fusi, Riccardo Santolini and Barbara Canonico

Int. J. Mol. Sci. 2024, 25(20), 11069; https://doi.org/10.3390/ijms252011069 - 15 Oct 2024

Viewed by 639

Abstract

In an era when ecological and environmental needs and responsibilities apply pressure on the world’s countries and sustainability takes centre stage, ecologic/environmental (E/E) laboratories stand as beacons of scientific inquiry, innovating, optimising, and applying various tests for a better knowledge of our natural resources and the quality status of ecosystems. The purpose of this review is to provide an overview of the use of flow cytometry (FC) as a tool for assessing environmental quality, mainly using living organisms and their biological changes as bioindicators. Cytometric approaches applied to both marine and terrestrial ecosystems ensure the detection of biochemical and functional status of the cells composing either an organ thereof or the organism itself. In addition to cytometric evaluations of the biotic matrix, a brief overview of the techniques for the environmental assessment of biotic and abiotic matrices using mass spectrometry is given. The technique involving the continuous monitoring of the chemical and physical parameters of water, sediment, and soil is basically incapable of detecting any additive and synergetic effects of toxicants on living organisms. Therefore, techniques employing bioindicators provide valuable information for environmental diagnosis, and several studies have demonstrated the strong relationship between specific environmental data and cell/organ behaviour. Full article

(This article belongs to the Special Issue Trends and Prospects of Flow Cytometry in Cell and Molecular Biology)

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12 pages, 1043 KiB

Open AccessReview

Using Spectral Flow Cytometry for CAR T-Cell Clinical Trials: Game Changing Technologies Enabling Novel Therapies

by Thomas C. Beadnell, Susmita Jasti, Ruqi Wang, Bruce H. Davis and Virginia Litwin

Int. J. Mol. Sci. 2024, 25(19), 10263; https://doi.org/10.3390/ijms251910263 - 24 Sep 2024

Viewed by 1243

Abstract

Monitoring chimeric antigen redirected (CAR) T-cells post-infusion in clinical trials is a specialized application of flow cytometry. Unlike the CAR T-cell monitoring for individual patients conducted in clinical laboratories, the data generated during a clinical trial will be used not only to monitor the therapeutic response of a single patient, but determine the success of the therapy itself, or even of an entire class of therapeutic compounds. The data, typically acquired at multiple testing laboratories, will be compiled into a single database. The data may also be used for mathematical modeling of cellular kinetics or to identify predictive biomarkers. With the expanded context of use, a robust, standardized assay is mandatory in order to generate a valuable and reliable data set. Hence, the requirements for assay validation, traceable calibration, technology transfer, cross-instrument standardization and regulatory compliance are high. Full article

(This article belongs to the Special Issue Trends and Prospects of Flow Cytometry in Cell and Molecular Biology)

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19 pages, 2925 KiB

Open AccessReview

The Evolving Landscape of Flowcytometric Minimal Residual Disease Monitoring in B-Cell Precursor Acute Lymphoblastic Leukemia

by Martijn W. C. Verbeek and Vincent H. J. van der Velden

Int. J. Mol. Sci. 2024, 25(9), 4881; https://doi.org/10.3390/ijms25094881 - 30 Apr 2024

Viewed by 2407

Abstract

Detection of minimal residual disease (MRD) is a major independent prognostic marker in the clinical management of pediatric and adult B-cell precursor Acute Lymphoblastic Leukemia (BCP-ALL), and risk stratification nowadays heavily relies on MRD diagnostics. MRD can be detected using flow cytometry based on aberrant expression of markers (antigens) during malignant B-cell maturation. Recent advances highlight the significance of novel markers (e.g., CD58, CD81, CD304, CD73, CD66c, and CD123), improving MRD identification. Second and next-generation flow cytometry, such as the EuroFlow consortium’s eight-color protocol, can achieve sensitivities down to 10⁻⁵ (comparable with the PCR-based method) if sufficient cells are acquired. The introduction of targeted therapies (especially those targeting CD19, such as blinatumomab or CAR-T19) introduces several challenges for flow cytometric MRD analysis, such as the occurrence of CD19-negative relapses. Therefore, innovative flow cytometry panels, including alternative B-cell markers (e.g., CD22 and CD24), have been designed. (Semi-)automated MRD assessment, employing machine learning algorithms and clustering tools, shows promise but does not yet allow robust and sensitive automated analysis of MRD. Future directions involve integrating artificial intelligence, further automation, and exploring multicolor spectral flow cytometry to standardize MRD assessment and enhance diagnostic and prognostic robustness of MRD diagnostics in BCP-ALL. Full article

(This article belongs to the Special Issue Trends and Prospects of Flow Cytometry in Cell and Molecular Biology)

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15 pages, 7680 KiB

Open AccessReview

SLAM Family Receptors in B Cell Chronic Lymphoproliferative Disorders

by Dominik Kľoc, Slavomír Kurhajec, Mykhailo Huniadi, Ján Sýkora, Tomáš Guman and Marek Šarišský

Int. J. Mol. Sci. 2024, 25(7), 4014; https://doi.org/10.3390/ijms25074014 - 4 Apr 2024

Cited by 1 | Viewed by 1786

Abstract

The signaling lymphocytic activation molecule (SLAM) receptor family (SLAMF) consists of nine glycoproteins that belong to the CD2 superfamily of immunoglobulin (Ig) domain-containing molecules. SLAMF receptors modulate the differentiation and activation of a wide range of immune cells. Individual SLAMF receptors are expressed on the surface of hematopoietic stem cells, hematopoietic progenitor cells, B cells, T cells, NK cells, NKT cells, monocytes, macrophages, dendritic cells, neutrophils, and platelets. The expression of SLAMF receptors was studied during normal B cell maturation. Several SLAMF receptors were also detected in cancer cell lines of B-lymphoid origin and in pathological B cells from patients with B cell chronic lymphoproliferative disorders (B-CLPD), the most frequent hematological malignancies in adults. This review summarizes current knowledge on the expression of SLAMF receptors and their adaptor proteins SAP and EAT-2 in B-CLPD. Several SLAMF receptors could be regarded as potential diagnostic and differential diagnostic markers, prognostic factors, and targets for the development of novel drugs for patients with B-CLPD. Full article

(This article belongs to the Special Issue Trends and Prospects of Flow Cytometry in Cell and Molecular Biology)

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19 pages, 1198 KiB

Open AccessReview

Applications of Flow Cytometry in Drug Discovery and Translational Research

by Sumana Ullas and Charles Sinclair

Int. J. Mol. Sci. 2024, 25(7), 3851; https://doi.org/10.3390/ijms25073851 - 29 Mar 2024

Cited by 2 | Viewed by 3550

Abstract

Flow cytometry is a mainstay technique in cell biology research, where it is used for phenotypic analysis of mixed cell populations. Quantitative approaches have unlocked a deeper value of flow cytometry in drug discovery research. As the number of drug modalities and druggable mechanisms increases, there is an increasing drive to identify meaningful biomarkers, evaluate the relationship between pharmacokinetics and pharmacodynamics (PK/PD), and translate these insights into the evaluation of patients enrolled in early clinical trials. In this review, we discuss emerging roles for flow cytometry in the translational setting that supports the transition and evaluation of novel compounds in the clinic. Full article

(This article belongs to the Special Issue Trends and Prospects of Flow Cytometry in Cell and Molecular Biology)

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Other

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17 pages, 11028 KiB

Open AccessCase Report

Flow Cytometry Analysis in Breast Implant-Associated Anaplastic Large Cell Lymphoma: Three Case Reports

by Veronica Davanzo, Alessandra Falda, Paola Fogar, Kathrin Ludwig, Jenny Zuin, Maria Cristina Toffanin, Marco Pizzi, Angelo Paolo Dei Tos and Daniela Basso

Int. J. Mol. Sci. 2024, 25(6), 3518; https://doi.org/10.3390/ijms25063518 - 20 Mar 2024

Viewed by 1285

Abstract

Breast Implant-Associated-Anaplastic Large Cell Lymphoma (BIA-ALCL) is a rare T-cell non-Hodgkin lymphoma associated with breast prosthetic implants and represents a diagnostic challenge. The National Comprehensive Cancer Network (NCCN) guidelines, updated in 2024, recommend for diagnosis an integrated work-up that should include cell morphology, CD30 immunohistochemistry (IHC), and flow cytometry (FCM). CD30 IHC, although the test of choice for BIA-ALCL diagnosis, is not pathognomonic, and this supports the recommendation to apply a multidisciplinary approach. A close collaboration between pathologists and laboratory professionals allowed the diagnosis of three BIA-ALCLs, presented as case reports, within a series of 35 patients subjected to periprosthetic effusions aspiration from 2018 to 2023. In one case, rare neoplastic cells were identified by FCM, and this result was essential in leading the anatomopathological picture as indicative of this neoplasm. In fact, the distinction between a lymphomatous infiltrate from reactive cells may be very complex in the cytopathology and IHC setting when neoplastic cells are rare. On the other hand, one limitation of FCM analysis is the need for fresh samples. In this study, we provide evidence that a dedicated fixative allows the maintenance of an unaltered CD30 expression on the cell surface for up to 72 h. Full article

(This article belongs to the Special Issue Trends and Prospects of Flow Cytometry in Cell and Molecular Biology)

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