Cells

Research

15 pages, 3818 KiB

Open AccessArticle

Flow Cytometric Detection of Waterborne Bacteria Metabolic Response to Anthropogenic Chemical Inputs to Aquatic Ecosystems

by Jill A. Jenkins, Scott V. Mize, Darren Johnson and Bonnie L. Brown

Cells 2025, 14(5), 352; https://doi.org/10.3390/cells14050352 - 28 Feb 2025

Viewed by 438

Abstract

Typical investigations into the biological consequences of suspected xenobiotics or nutrients introduced in watersheds include analytical chemistry screens of environmental samples—such as periphyton responses or studies of fish condition—which are all costly in terms of equipment, reagents, time, and human resources. An alternative [...] Read more.

Typical investigations into the biological consequences of suspected xenobiotics or nutrients introduced in watersheds include analytical chemistry screens of environmental samples—such as periphyton responses or studies of fish condition—which are all costly in terms of equipment, reagents, time, and human resources. An alternative is to assess pollutant effects on waterborne bacteria. A flow cytometric method was developed to yield rapid, same-day results that could be used to proactively screen for suspected chemical inputs into watersheds using water sampling methods that are identical to those in standard use. The analytical methods are microbe cultivation-independent, for use with waterborne bacteria that are typically viable but not culturable. The procedure is quick and inexpensive, generating measures of bacterial esterase that reflect metabolic activity and are sensitive and statistically robust. After phosphate-EDTA incubation to increase cell wall permeability, staining was performed with 5(6) carboxyfluorescein diacetate (enzyme activity) and propidium iodide (cell viability) with three bacterial species in exponential phase growth having been incubated with organic wastewater compounds (atrazine, pharmaceuticals [17α-ethynylestradiol and trenbolone], and antimicrobials [tylosin and butylparaben]). This method successfully detected metabolic changes in all bacterial species, with atrazine inducing the greatest change. Additional fluorescent stains can target specific microbial structures or functions of interest in a particular watershed. This biotechnology can inform analytical chemistry and study of biota at sites of interest and has the potential to be automated. Full article

(This article belongs to the Special Issue The Applications of Flow Cytometry: Advances, Challenges, and Trends)

► Show Figures

Graphical abstract

13 pages, 3393 KiB

Open AccessArticle

Imaging Flow Cytometric Identification of Chromosomal Defects in Paediatric Acute Lymphoblastic Leukaemia

by Ana P. A. Simpson, Carly E. George, Henry Y. L. Hui, Ravi Doddi, Rishi S. Kotecha, Kathy A. Fuller and Wendy N. Erber

Cells 2025, 14(2), 114; https://doi.org/10.3390/cells14020114 - 14 Jan 2025

Viewed by 1235

Abstract

Acute lymphoblastic leukaemia is the most common childhood malignancy that remains a leading cause of death in childhood. It may be characterised by multiple known recurrent genetic aberrations that inform prognosis, the most common being hyperdiploidy and t(12;21) ETV6::RUNX1. We aimed to [...] Read more.

Acute lymphoblastic leukaemia is the most common childhood malignancy that remains a leading cause of death in childhood. It may be characterised by multiple known recurrent genetic aberrations that inform prognosis, the most common being hyperdiploidy and t(12;21) ETV6::RUNX1. We aimed to assess the applicability of a new imaging flow cytometry methodology that incorporates cell morphology, immunophenotype, and fluorescence in situ hybridisation (FISH) to identify aneuploidy of chromosomes 4 and 21 and the translocation ETV6::RUNX1. We evaluated this new “immuno-flowFISH” platform on 39 cases of paediatric ALL of B-lineage known to have aneuploidy of chromosomes 4 and 21 and the translocation ETV6::RUNX1. After identifying the leukaemic population based on immunophenotype (i.e., expression of CD34, CD10, and CD19 antigens), we assessed for copy numbers of loci for the centromeres of chromosomes 4 and 21 and the ETV6 and RUNX1 regions using fluorophore-labelled DNA probes in more than 1000 cells per sample. Trisomy 4 and 21, tetrasomy 21, and translocations of ETV6::RUNX1, as well as gains and losses of ETV6 and RUNX1, could all be identified based on FISH spot counts and digital imagery. There was variability in clonal makeup in individual cases, suggesting the presence of sub-clones. Copy number alterations and translocations could be detected even when the cell population comprised less than 1% of cells and included cells with a mature B-cell phenotype, i.e., CD19-positive, lacking CD34 and CD10. In this proof-of-principle study of 39 cases, this sensitive and specific semi-automated high-throughput imaging flow cytometric immuno-flowFISH method has been able to show that alterations in ploidy and ETV6::RUNX1 could be detected in the 39 cases of paediatric ALL. This imaging flow cytometric FISH method has potential applications for diagnosis and monitoring disease and marrow regeneration (i.e., distinguishing residual ALL from regenerating haematogones) following chemotherapy. Full article

(This article belongs to the Special Issue The Applications of Flow Cytometry: Advances, Challenges, and Trends)

► Show Figures

Figure 1

12 pages, 2706 KiB

Open AccessArticle

Flow Cytometry Evaluation of Blood-Cell-Bound Surface FVIII in Hemophilia A and Thrombosis

by Anjud Al-Mohannadi, Reem Mohammed Yahia, Hani Bibawi, Che-Ann Lachica, Watfa Ahmed, Igor Pavlovski, Giusy Gentilcore, Elkhansa Elbukhari Elgaali, Anila Ejaz, Areeg Ahmed, Mohammed Elanbari, Zainab Awada, Mohammed J. Al-Kubaisi, Muhammad Elnaggar, Ayman Saleh, Chiara Cugno and Sara Deola

Cells 2025, 14(2), 73; https://doi.org/10.3390/cells14020073 - 8 Jan 2025

Viewed by 1102

Abstract

Hemophilia A (HA) is associated with FVIII coagulation insufficiency or inactivity leading to excessive bleeding. Elevated FVIII, on the contrary, is associated with thrombophilia, thrombosis, myocardial infarctions, and stroke. Active FVIII (aFVIII) uses its C2 domain to bind to blood cells’ membranes, consequently [...] Read more.

Hemophilia A (HA) is associated with FVIII coagulation insufficiency or inactivity leading to excessive bleeding. Elevated FVIII, on the contrary, is associated with thrombophilia, thrombosis, myocardial infarctions, and stroke. Active FVIII (aFVIII) uses its C2 domain to bind to blood cells’ membranes, consequently carrying out its coagulative function. We developed a reliable flow cytometry (FC) method for FVIII detection that can be utilized for assessing surface-bound FVIII on leukocytes in different coagulation/clinical states; we analyzed 49 pediatric subjects, encompassing patients with HA, other coagulopathies, venous thrombosis, and normal coagulation. Interestingly, the total leukocyte surface FVIII showed a declining trend across thrombosis, normal, and hypo-coagulation states. As expected, the leukocytes of HA patients displayed significantly lower levels of cellular-surface FVIII in comparison to patients with thrombosis. However, no significant correlation was observed between circulating levels of FVIII in plasma and the levels of FVIII bound to leukocytes, indicating that the differences in FVIII surface binding are not directly proportional to the availability of FVIII in the circulation and suggesting a specific binding mechanism governing the interaction between FVIII and leukocytes. Intriguingly, when analyzing the distinct blood subpopulations, we observed that surface FVIII levels were significantly elevated in classical monocytes of thrombosis patients compared to HA patients, healthy controls, and patients with other coagulopathies. Our study highlights the reliability of our FC platform in assessing FVIII abundance on leukocytes’ membranes across coagulation states. Monocytes, particularly in cases of thrombosis, exhibit active binding of FVIII on their surface, suggesting a potential role in the pathophysiology of thrombosis that requires further investigation. Full article

(This article belongs to the Special Issue The Applications of Flow Cytometry: Advances, Challenges, and Trends)

► Show Figures

Graphical abstract

14 pages, 8652 KiB

Open AccessArticle

Validation of a Spectral Flow Cytometry Single-Tube Panel for the Clinical Diagnosis and Follow-Up of Children and Adolescents with B-Cell Acute Lymphoblastic Leukemia

by Gonzalo García-Aguilera, Ana Castillo-Robleda, Alejandro Sanz and Manuel Ramírez

Cells 2024, 13(22), 1891; https://doi.org/10.3390/cells13221891 - 15 Nov 2024

Viewed by 1650

Abstract

The ability of flow cytometry to identify and quantify the presence of cell populations defined by their expression profile of specific markers has made this technique a powerful and routinary tool in clinical diagnostic practice. Specifically in the field of hematological malignancies, flow [...] Read more.

The ability of flow cytometry to identify and quantify the presence of cell populations defined by their expression profile of specific markers has made this technique a powerful and routinary tool in clinical diagnostic practice. Specifically in the field of hematological malignancies, flow cytometry allows the identification of the correct type and lineage of each patient’s disease and also sensitively quantifies the presence of the disease at precise moments during treatment, that is, levels of measurable residual disease (MRD). The quantification of MRD by flow cytometry has allowed the adaptation of tailored therapies to patients, contributing to the improvement of the results of the different protocols in recent decades. In this context, our objective in the present work was to evaluate the potential impact that spectral flow cytometry can provide compared to conventional cytometry, which is the one usually used in clinics. We present here a comparative study of both technologies, spectral versus conventional flow cytometry, in primary samples corresponding to the diagnosis and follow-up of children and adolescents with acute lymphoblastic leukemia. Our initial experience demonstrates the feasibility of incorporating spectral flow cytometry into the routine workflow of a reference laboratory. Full article

(This article belongs to the Special Issue The Applications of Flow Cytometry: Advances, Challenges, and Trends)

► Show Figures

Figure 1

28 pages, 7251 KiB

Open AccessEditor’s ChoiceArticle

The Power of Reagent Titration in Flow Cytometry

by Diana L. Bonilla, Alberta Paul, Jesus Gil-Pulido, Lily M. Park and Maria C. Jaimes

Cells 2024, 13(20), 1677; https://doi.org/10.3390/cells13201677 - 11 Oct 2024

Viewed by 7098

Abstract

Flow cytometry facilitates the detection of multiple cell parameters simultaneously with a high level of resolution and throughput, enabling in-depth immunological evaluations. High data resolution in flow cytometry depends on multiple factors, including the concentration of reagents used in the staining protocol, and [...] Read more.

Flow cytometry facilitates the detection of multiple cell parameters simultaneously with a high level of resolution and throughput, enabling in-depth immunological evaluations. High data resolution in flow cytometry depends on multiple factors, including the concentration of reagents used in the staining protocol, and reagent validation and titration should be the first step in any assay optimization. Titration is the process of finding the concentration of the reagent that best resolves a positive signal from the background, with the saturation of all binding sites, and minimal antibody excess. The titration process involves the evaluation of serial reagent dilutions in cells expressing the antigen target for the tested antibody. The concentration of antibody that provides the highest signal to noise ratio is calculated by plotting the percentage of positive cells and the intensity of the fluorescence of the stained cells with respect to the negative events, in a concentration–response curve. The determination of the optimal antibody concentration is necessary to ensure reliable and reproducible results and is required for each sample type, reagent clone and lot, as well as the methods used for cell collection, staining, and storage conditions. If the antibody dilution is too low, the signal will be too weak to be accurately determined, leading to suboptimal data resolution, high variability across measurements, and the underestimation of the frequency of cells expressing a specific marker. The use of excess antibodies could lead to non-specific binding, reagent misuse, and detector overloading with the signal off scale and higher spillover spreading. In this publication, we summarized the titration fundamentals and best practices, and evaluated the impact of using a different instrument, sample, staining, acquisition, and analysis conditions in the selection of the optimal titer and population resolution. Full article

(This article belongs to the Special Issue The Applications of Flow Cytometry: Advances, Challenges, and Trends)

► Show Figures

Figure 1

Review

Jump to: Research, Other

20 pages, 6473 KiB

Open AccessReview

Recent Technologies on 2D and 3D Imaging Flow Cytometry

by Masashi Ugawa and Sadao Ota

Cells 2024, 13(24), 2073; https://doi.org/10.3390/cells13242073 - 16 Dec 2024

Viewed by 2020

Abstract

Imaging flow cytometry is a technology that performs microscopy image analysis of cells within flow cytometry and allows high-throughput, high-content cell analysis based on their intracellular molecular distribution and/or cellular morphology. While the technology has been available for a couple of decades, it [...] Read more.

Imaging flow cytometry is a technology that performs microscopy image analysis of cells within flow cytometry and allows high-throughput, high-content cell analysis based on their intracellular molecular distribution and/or cellular morphology. While the technology has been available for a couple of decades, it has recently gained significant attention as technical limitations for higher throughput, sorting capability, and additional imaging dimensions have been overcome with various approaches. These evolutions have enabled imaging flow cytometry to offer a variety of solutions for life science and medicine that are not possible with conventional flow cytometry or microscopy-based screening. It is anticipated that the extent of applications will expand in the upcoming years as the technology becomes more accessible through dissemination. In this review, we will cover the technical advances that have led to this new generation of imaging flow cytometry, focusing on the advantages and limitations of each technique. Full article

(This article belongs to the Special Issue The Applications of Flow Cytometry: Advances, Challenges, and Trends)

► Show Figures

Figure 1

34 pages, 2584 KiB

Open AccessReview

Advances and Challenges in Sepsis Management: Modern Tools and Future Directions

by Elena Santacroce, Miriam D’Angerio, Alin Liviu Ciobanu, Linda Masini, Domenico Lo Tartaro, Irene Coloretti, Stefano Busani, Ignacio Rubio, Marianna Meschiari, Erica Franceschini, Cristina Mussini, Massimo Girardis, Lara Gibellini, Andrea Cossarizza and Sara De Biasi

Cells 2024, 13(5), 439; https://doi.org/10.3390/cells13050439 - 2 Mar 2024

Cited by 17 | Viewed by 14989

Abstract

Sepsis, a critical condition marked by systemic inflammation, profoundly impacts both innate and adaptive immunity, often resulting in lymphopenia. This immune alteration can spare regulatory T cells (Tregs) but significantly affects other lymphocyte subsets, leading to diminished effector functions, altered cytokine profiles, and [...] Read more.

Sepsis, a critical condition marked by systemic inflammation, profoundly impacts both innate and adaptive immunity, often resulting in lymphopenia. This immune alteration can spare regulatory T cells (Tregs) but significantly affects other lymphocyte subsets, leading to diminished effector functions, altered cytokine profiles, and metabolic changes. The complexity of sepsis stems not only from its pathophysiology but also from the heterogeneity of patient responses, posing significant challenges in developing universally effective therapies. This review emphasizes the importance of phenotyping in sepsis to enhance patient-specific diagnostic and therapeutic strategies. Phenotyping immune cells, which categorizes patients based on clinical and immunological characteristics, is pivotal for tailoring treatment approaches. Flow cytometry emerges as a crucial tool in this endeavor, offering rapid, low cost and detailed analysis of immune cell populations and their functional states. Indeed, this technology facilitates the understanding of immune dysfunctions in sepsis and contributes to the identification of novel biomarkers. Our review underscores the potential of integrating flow cytometry with omics data, machine learning and clinical observations to refine sepsis management, highlighting the shift towards personalized medicine in critical care. This approach could lead to more precise interventions, improving outcomes in this heterogeneously affected patient population. Full article

(This article belongs to the Special Issue The Applications of Flow Cytometry: Advances, Challenges, and Trends)

► Show Figures

Graphical abstract

Other

Jump to: Research, Review

8 pages, 3442 KiB

Open AccessBrief Report

Flow Cytometry as the Tool to Define Peripheral Blood Leukocyte Signatures in Acute EBV Infection

by Pragya Singh, Manisha Gadgeel, Batool AlQanber, Ahmad Farooqi and Süreyya Savaşan

Cells 2024, 13(11), 963; https://doi.org/10.3390/cells13110963 - 3 Jun 2024

Viewed by 1642

Abstract

Primary Epstein–Barr virus (EBV) infection which can manifest as infectious mononucleosis (IM) is commonly acquired during childhood. EBV primarily invades B cells leading to a lytic reaction; the control of the infection is handled by natural killer and T cells in immunocompetent individuals. [...] Read more.

Primary Epstein–Barr virus (EBV) infection which can manifest as infectious mononucleosis (IM) is commonly acquired during childhood. EBV primarily invades B cells leading to a lytic reaction; the control of the infection is handled by natural killer and T cells in immunocompetent individuals. The infection has a wide spectrum of clinical findings and can lead to serious complications in patients with certain underlying immunological dysfunctions. We retrospectively investigated peripheral white blood cell populations’ surface marker characteristics in IM using a comprehensive flow cytometry marker panel. Twenty-one cases of IM and seventeen EBV-seropositive cases without IM serving as controls were included. We observed novel alterations in lymphocyte, neutrophil, and monocyte populations. In addition to increased activated cytotoxic T cells and low B cells, we demonstrated high T-large granular lymphocyte (T-LGL) populations in IM cases. Furthermore, despite T cells’ increased HLA-DR expression, another activation marker, CD11b, was lower in T-LGL populations. Monocytes showed increased CD16 expression; CD64 was higher in neutrophils. Our findings point to monocyte and neutrophil activation which may account for acute clinical features and may contribute to the understanding of IM immunobiology. Furthermore, they may serve as a useful tool in investigating inherited and post-transplant conditions characterized by deficiencies in controlling EBV infection. Full article

(This article belongs to the Special Issue The Applications of Flow Cytometry: Advances, Challenges, and Trends)

► Show Figures

Figure 1

Journal Menu

Journal Browser

The Applications of Flow Cytometry: Advances, Challenges, and Trends

Share This Special Issue

Special Issue Editor

Special Issue Information

Keywords

Benefits of Publishing in a Special Issue

Published Papers (8 papers)

Research

Review

Other

Further Information

Guidelines

MDPI Initiatives

Follow MDPI