Research

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19 pages, 2318 KiB

Open AccessArticle

Assessment of Automated Flow Cytometry Data Analysis Tools within Cell and Gene Therapy Manufacturing

by Melissa Cheung, Jonathan J. Campbell, Robert J. Thomas, Julian Braybrook and Jon Petzing

Int. J. Mol. Sci. 2022, 23(6), 3224; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23063224 - 17 Mar 2022

Cited by 5 | Viewed by 4179

Flow cytometry is widely used within the manufacturing of cell and gene therapies to measure and characterise cells. Conventional manual data analysis relies heavily on operator judgement, presenting a major source of variation that can adversely impact the quality and predictive potential of [...] Read more.

Flow cytometry is widely used within the manufacturing of cell and gene therapies to measure and characterise cells. Conventional manual data analysis relies heavily on operator judgement, presenting a major source of variation that can adversely impact the quality and predictive potential of therapies given to patients. Computational tools have the capacity to minimise operator variation and bias in flow cytometry data analysis; however, in many cases, confidence in these technologies has yet to be fully established mirrored by aspects of regulatory concern. Here, we employed synthetic flow cytometry datasets containing controlled population characteristics of separation, and normal/skew distributions to investigate the accuracy and reproducibility of six cell population identification tools, each of which implement different unsupervised clustering algorithms: Flock2, flowMeans, FlowSOM, PhenoGraph, SPADE3 and SWIFT (density-based, k-means, self-organising map, k-nearest neighbour, deterministic k-means, and model-based clustering, respectively). We found that outputs from software analysing the same reference synthetic dataset vary considerably and accuracy deteriorates as the cluster separation index falls below zero. Consequently, as clusters begin to merge, the flowMeans and Flock2 software platforms struggle to identify target clusters more than other platforms. Moreover, the presence of skewed cell populations resulted in poor performance from SWIFT, though FlowSOM, PhenoGraph and SPADE3 were relatively unaffected in comparison. These findings illustrate how novel flow cytometry synthetic datasets can be utilised to validate a range of automated cell identification methods, leading to enhanced confidence in the data quality of automated cell characterisations and enumerations. Full article

(This article belongs to the Special Issue Flow Cytometry and Its Applications to Molecular Biology and Diagnosis)

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12 pages, 1522 KiB

Open AccessArticle

Identification of Spiro-Fused [3-azabicyclo[3.1.0]hexane]oxindoles as Potential Antitumor Agents: Initial In Vitro Evaluation of Anti-Proliferative Effect and Actin Cytoskeleton Transformation in 3T3 and 3T3-SV40 Fibroblast

by Nickolay A. Knyazev, Stanislav V. Shmakov, Sofya A. Pechkovskaya, Alexander S. Filatov, Alexander V. Stepakov, Vitali M. Boitsov and Natalia A. Filatova

Int. J. Mol. Sci. 2021, 22(15), 8264; https://doi.org/10.3390/ijms22158264 - 31 Jul 2021

Cited by 9 | Viewed by 1643

Abstract

Novel heterocyclic compounds containing 3-spiro[3-azabicyclo[3.1.0]hexane]oxindole framework (4a, 4b and 4c) have been studied as potential antitumor agents. The in silico ADMET (adsorption, distribution, metabolism, excretion and toxicity) analysis was performed on 4a–c compounds with promising antiproliferative activity, previously [...] Read more.

Novel heterocyclic compounds containing 3-spiro[3-azabicyclo[3.1.0]hexane]oxindole framework (4a, 4b and 4c) have been studied as potential antitumor agents. The in silico ADMET (adsorption, distribution, metabolism, excretion and toxicity) analysis was performed on 4a–c compounds with promising antiproliferative activity, previously synthetized and screened against human erythroleukemic cell line K562 tumor cell line. Cytotoxicity of 4a–c against murine fibroblast 3T3 and SV-40 transformed murine fibroblast 3T3-SV40 cell lines were evaluated. The 4a and 4c compounds were cytotoxic against 3T3-SV40 cells in comparison with those of 3T3. In agreement with the DNA cytometry studies, the tested compounds have achieved significant cell-cycle perturbation with higher accumulation of cells in G0/G1 phase. Using confocal microscopy, we found that with 4a and 4c treatment of 3T3 cells, actin filaments disappeared, and granular actin was distributed diffusely in the cytoplasm in 82–97% of cells. The number of 3T3-SV40 cells with stress fibers increased to 7–30% against 2% in control. We discovered that transformed 3T3-SV40 cells after treatment with compounds 4a and 4c significantly reduced the number of cells with filopodium-like membrane protrusions (from 86 % in control cells to 6–18% after treatment), which indirectly suggests a decrease in cell motility. We can conclude that the studied compounds 4a and 4c have a cytostatic effect, which can lead to a decrease in the number of filopodium-like membrane protrusions. Full article

(This article belongs to the Special Issue Flow Cytometry and Its Applications to Molecular Biology and Diagnosis)

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12 pages, 2805 KiB

Open AccessArticle

Radiation Response of Cervical Cancer Stem Cells Is Associated with Pretreatment Proportion of These Cells and Physical Status of HPV DNA

by Irina Zamulaeva, Elena Selivanova, Olga Matchuk, Valentina Kiseleva, Liana Mkrtchyan and Ludmila Krikunova

Int. J. Mol. Sci. 2021, 22(3), 1445; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22031445 - 01 Feb 2021

Cited by 6 | Viewed by 2340

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⁺CD24^low 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

(This article belongs to the Special Issue Flow Cytometry and Its Applications to Molecular Biology and Diagnosis)

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17 pages, 5814 KiB

Open AccessArticle

Centrifugation Removes a Population of Large Vesicles, or “Macroparticles,” Intermediate in Size to RBCs and Microvesicles

by Michael C. Larson, Neil Hogg and Cheryl A. Hillery

Int. J. Mol. Sci. 2021, 22(3), 1243; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22031243 - 27 Jan 2021

Cited by 3 | Viewed by 1880

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

(This article belongs to the Special Issue Flow Cytometry and Its Applications to Molecular Biology and Diagnosis)

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18 pages, 14245 KiB

Open AccessArticle

Time-Lapse Flow Cytometry: A Robust Tool to Assess Physiological Parameters Related to the Fertilizing Capability of Human Sperm

by Arturo Matamoros-Volante, Valeria Castillo-Viveros, Paulina Torres-Rodríguez, Marcela B. Treviño and Claudia L. Treviño

Int. J. Mol. Sci. 2021, 22(1), 93; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22010093 - 24 Dec 2020

Cited by 3 | Viewed by 3557

Abstract

Plasma membrane (PM) hyperpolarization, increased intracellular pH (pH_i), 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 (pH_i), 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 (E_m) and pH_i changes, and progesterone-induced [Ca²⁺]_i increases. Our results show that TLFC is a robust method to measure absolute E_m and pH_i 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, pH_i 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

(This article belongs to the Special Issue Flow Cytometry and Its Applications to Molecular Biology and Diagnosis)

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Graphical abstract

15 pages, 1235 KiB

Open AccessArticle

Diameters and Fluorescence Calibration for Extracellular Vesicle Analyses by Flow Cytometry

by Pasquale Simeone, Christian Celia, Giuseppina Bologna, Eva Ercolino, Laura Pierdomenico, Felisa Cilurzo, Rossella Grande, Francesca Diomede, Simone Vespa, Barbara Canonico, Michele Guescini, Vilberto Stocchi, Lavinia Vittoria Lotti, Maria Teresa Guagnano, Luisa Stellin, Stefano Papa, Oriana Trubiani, Marco Marchisio, Sebastiano Miscia and Paola Lanuti

Int. J. Mol. Sci. 2020, 21(21), 7885; https://doi.org/10.3390/ijms21217885 - 23 Oct 2020

Cited by 35 | Viewed by 3938

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

(This article belongs to the Special Issue Flow Cytometry and Its Applications to Molecular Biology and Diagnosis)

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Review

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20 pages, 2475 KiB

Open AccessReview

Emerging Role of T-cell Receptor Constant β Chain-1 (TRBC1) Expression in the Flow Cytometric Diagnosis of T-cell Malignancies

by Pedro Horna, Min Shi, Horatiu Olteanu and Ulrika Johansson

Int. J. Mol. Sci. 2021, 22(4), 1817; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22041817 - 12 Feb 2021

Cited by 23 | Viewed by 9060

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

(This article belongs to the Special Issue Flow Cytometry and Its Applications to Molecular Biology and Diagnosis)

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27 pages, 40457 KiB

Open AccessReview

Contributions of Flow Cytometry to the Molecular Study of Spermatogenesis in Mammals

by Rosana Rodríguez-Casuriaga and Adriana Geisinger

Int. J. Mol. Sci. 2021, 22(3), 1151; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22031151 - 25 Jan 2021

Cited by 6 | Viewed by 3880

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

(This article belongs to the Special Issue Flow Cytometry and Its Applications to Molecular Biology and Diagnosis)

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