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Flow Cytometry: Applications and Challenges
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Flow Cytometry: Applications and Challenges

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 September 2025 | Viewed by 3868

Special Issue Editor

Dr. Alessandra Falda

E-Mail Website
Guest Editor
Laboratory Medicine Unit, Integrated Diagnostic Services—DIDAS, Padova University Hospital, 35128 Padova, Italy
Interests: flow cytometry; hematology; morphology; molecular tests; laboratory medicine; clinical chemistry; cell biology

Special Issue Information

Dear Colleagues,

Nowadays, despite molecular tests and imaging techniques, flow cytometry continues to capture attention. In particular, non-conventional flow cytometry is becoming increasingly intriguing, like imaging flow cytometry and mass cytometry.

Artificial intelligence and precision medicine are pushing medical knowledge to the extreme. In the context of integration, flow cytometry contributes to understanding processes, including functional ones, in both oncological and non-oncological pathologies. The wealth of information we gain from proteomics, genetic sequencing, and high-sensitivity multiparametric flow cytometry has led to new insights into the immune system and signaling pathways, allowing us to better understand diseases. Furthermore, the evaluation of minimal residual disease is also guaranteed by flow cytometry to personalize treatments. Other challenges involve studying the flow cytometry of less invasive materials (such as saliva or sputum) to obtain early information on various pathologies. In recent years, the study of the intestinal microbiota, microenvironment, and extracellular vesicles, with the use of numerous color panels, allowed us to gain insights into the immune system’s modulations, oncological and infectious diseases, and biological therapies, also helping to understand new therapeutic targets.

We welcome submissions, including original papers, and reviews, to explore how flow cytometry can enhance our understanding of pathologies.

Dr. Alessandra Falda
Guest Editor

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. 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
  • non-conventional flow cytometry
  • hematology
  • morphology
  • molecular tests
  • cell biology
  • immunology
  • target therapy
  • microbiology
  • antigens
  • functional mechanisms
  • new technologies

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Published Papers (3 papers)

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Research

19 pages, 3292 KiB  
Article
Analysis of Antigen Expression in T-Cell Acute Lymphoblastic Leukemia by Multicolor Flow Cytometry: Implications for the Detection of Measurable Residual Disease
by Alexandra Semchenkova, Ekaterina Mikhailova, Irina Demina, Julia Roumiantseva, Alexander Karachunskiy, Galina Novichkova and Alexander Popov
Int. J. Mol. Sci. 2025, 26(5), 2002; https://doi.org/10.3390/ijms26052002 - 25 Feb 2025
Viewed by 532
Abstract
Multicolor flow cytometry (MFC) is a key method for assessing measurable residual disease (MRD) in acute lymphoblastic leukemia (ALL). However, very few approaches were developed for MRD in T-cell ALL (T-ALL). To identify MRD markers suitable for T-ALL, we analyzed the expression of [...] Read more.
Multicolor flow cytometry (MFC) is a key method for assessing measurable residual disease (MRD) in acute lymphoblastic leukemia (ALL). However, very few approaches were developed for MRD in T-cell ALL (T-ALL). To identify MRD markers suitable for T-ALL, we analyzed the expression of CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD34, CD45, CD48, CD56, CD99, and HLA-DR in T-ALL patients at diagnosis. The median fluorescence intensities (MFIs) of surface CD3, CD4, CD5, CD7, CD8, CD45, CD48, CD99, and CD16+CD56 were also evaluated at Day 15 and the end-of-induction (EOI). The MFC data from 198 pediatric T-ALL patients were analyzed retrospectively. At diagnosis, the most common antigens were identified, and the MFI of T-lineage antigens in blasts was compared to that in T lymphocytes. At follow-up, the MFIs of the proposed MRD markers were compared to those observed at diagnosis. The most common T-ALL antigens were CD7 (100.0%), intracellular CD3 (100.0%), CD45 (98.5%), and CD5 (90.9%). The MFIs of T-lineage antigens in blasts differed significantly from those in T lymphocytes. By the EOI, a substantial modulation of sCD3, CD4, CD5, CD7, CD8, and CD45 was observed. CD48 and CD99 were the most stable markers. The proposed MRD markers (sCD3, CD4, CD5, CD7, CD8, CD45, CD48, CD99, CD16+CD56) enabled MFC-MRD monitoring in virtually all T-ALL patients. Full article
(This article belongs to the Special Issue Flow Cytometry: Applications and Challenges)
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17 pages, 1836 KiB  
Article
Clustering Algorithm-Driven Detection of TRBC1-Restricted Clonal T-Cell Populations Produces Better Results than Manual Gating Analysis
by Simon Buček, Andreja Brožič, Simona Miceska, Gorana Gašljević and Veronika Kloboves Prevodnik
Int. J. Mol. Sci. 2025, 26(1), 170; https://doi.org/10.3390/ijms26010170 - 28 Dec 2024
Viewed by 850
Abstract
Flow cytometric (FC) immunophenotyping and T-cell receptor (TCR) gene rearrangement studies are essential ancillary methods for the characterisation of T-cell lymphomas. Traditional manual gating and polymerase chain reaction (PCR)-based analyses can be labour-intensive, operator-dependent, and have limitations in terms of sensitivity and specificity. [...] Read more.
Flow cytometric (FC) immunophenotyping and T-cell receptor (TCR) gene rearrangement studies are essential ancillary methods for the characterisation of T-cell lymphomas. Traditional manual gating and polymerase chain reaction (PCR)-based analyses can be labour-intensive, operator-dependent, and have limitations in terms of sensitivity and specificity. The objective of our study was to investigate the efficacy of the Phenograph and t-SNE algorithms together with an antibody specific for the TCR β-chain constant region 1 (TRBC1) to identify monoclonal T-cell populations. FC- and PCR-based clonality analyses were performed on 275 samples of T-cell lymphomas, B-cell lymphomas, and reactive lymphocytic proliferations. Monotypic T-cell populations were identified in 65.1% of samples by manual gating and 72.4% by algorithm-driven analysis, while PCR-based analysis detected clonal T cells in 68.0%. Of the 262 monotypic populations identified, 46.6% were classified as T-cell lymphomas and 53.4% as T-cell populations of uncertain significance (T-CUS). Algorithm-driven gating identified monotypic populations that were overlooked by manual gating or PCR-based methods. The study highlights the difficulty in distinguishing monotypic populations as T-cell lymphoma or T-CUS. Further research is needed to establish criteria for distinguishing between these populations and to improve FC diagnostic accuracy. Full article
(This article belongs to the Special Issue Flow Cytometry: Applications and Challenges)
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13 pages, 9132 KiB  
Article
Fluorescent Aerolysin (FLAER) Binding Is Abnormally Low in the Clonal Precursors of Acute Leukemias, with Binding Particularly Low or Absent in Acute Promyelocytic Leukemia
by María Beatriz Álvarez Flores, María Sopeña Corvinos, Raquel Guillén Santos and Fernando Cava Valenciano
Int. J. Mol. Sci. 2024, 25(22), 11898; https://doi.org/10.3390/ijms252211898 - 5 Nov 2024
Cited by 1 | Viewed by 1653
Abstract
Flow cytometry plays a fundamental role in the diagnosis of leukemias and lymphomas, as well as in the follow-up and evaluation of minimally measurable disease after treatment. In some instances, such as in the case of acute promyelocytic leukemia (APL), rapid diagnosis is [...] Read more.
Flow cytometry plays a fundamental role in the diagnosis of leukemias and lymphomas, as well as in the follow-up and evaluation of minimally measurable disease after treatment. In some instances, such as in the case of acute promyelocytic leukemia (APL), rapid diagnosis is required to avoid death due to serious blood clotting or bleeding complications. Given that promyelocytes do not express the glycophosphatidylinositol (GPI)-anchored protein CD16 and that deficient CD16 expression is a feature of some CD16 polymorphisms and paroxysmal nocturnal hemoglobinuria (PNH), we included the GPI anchor probe FLAER aerolysin in the APL flow cytometry probe panel. Initial tests showed that FLAER binding was absent in pathological promyelocytes from APL patients but was consistently detected with high intensity in healthy promyelocytes from control bone marrow. FLAER binding was studied in 71 hematologic malignancies. Appropriate control cells were obtained from 16 bone marrow samples from patients with idiopathic thrombocytopenic purpura and non-infiltrated non-Hodgkin’s lymphoma. Compared with the positive FLAER signal in promyelocytes from healthy bone marrow, malignant promyelocytes from APL patients showed weak or negative FLAER binding. The FLAER signal in APL promyelocytes was also lower than that in control myeloid progenitors and precursors from patients with other forms of acute myeloid leukemia (AML), B-cell acute lymphoblastic leukemia, or myelodysplastic syndrome. Minimal measurable disease studies performed in APL patients after treatment found normal promyelocyte expression when minimal measurable disease was negative and FLAER-negative promyelocytes when disease relapse was detected. The inclusion of FLAER in the flow cytometry diagnosis and follow-up of APL could be very helpful. Decreased FLAER binding was found in all cases of APL, confirmed by the detection of the PML-RARA fusion transcript and, to a lesser extent, in the other AMLs studied. This study also revealed FLAER differences in other acute leukemias and even between different precursors (myeloid and lymphoid) from healthy controls. However, the reason for FLAER’s non-binding to the malignant precursors of these leukemias remains unknown, and future studies should explore the possible relation with an immune escape phenomenon in these leukemias. Full article
(This article belongs to the Special Issue Flow Cytometry: Applications and Challenges)
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