CFSE flow cytometric quantification of lymphocytic proliferation in extracorporeal photopheresis: Use for quality control

Evrard, Bertrand; Dosgilbert, Annie; Jacquemot, Nathalie; Deméocq, F.; Thibault, Gilles; Chassagne, J; Berger, Marc; Tridon, A.

doi:10.1016/j.transci.2009.10.002

Cited by 16 publications

(35 citation statements)

References 15 publications

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“…First of all, the comparison of the CFSE method with the tritiated thymidine reference method shows an almost complete concordance and an excellent sensitivity. In the literature, the minimum inhibition rate reported is often 90% (14,15), However, we have translated in our Standard Operating procedures the 70% minimum inhibition rate recommended by our regulatory agency (21). However, two discordant pairs of data from the same patient (Pio Gen) were obtained: the reference method has not found proliferation of the Pre-ECP cells while the alternative method made them positive.…”

Section: Discussionmentioning

confidence: 99%

Quality control of extracorporeal photochemotherapy: Proliferation assay using CFSE validated according to ISO 15189:2007 standards

et al. 2014

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Section: Discussionmentioning

confidence: 99%

Quality control of extracorporeal photochemotherapy: Proliferation assay using CFSE validated according to ISO 15189:2007 standards

et al. 2014

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“…CSFE is distributed to both daughter cells by mitosis; therefore, CSFE fluorescence decreases with cell proliferation (13). The suspended and adherent cells were labelled by CSFE at the same time and then were plated in culture dishes.…”

Section: Suspended Cells Are Present In the Tightmentioning

confidence: 99%

A new approach to screening cancer stem cells from the U251 human glioma cell line based on cell growth state

Cao

Jiang

et al. 2012

Oncology Reports

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Abstract. Cancer stem cells (CSCs) play important roles in the biological behaviour of malignant tumours. To study their properties, they must be carefully identified and purified. Cancer cells can acquire three different morphological types during single cell cloning. A small subpopulation of clones acquires a regular and compact shape, and these clones are enriched for CSCs; however, the majority of clones have an irregular morphology with loose intercellular junctions, with fewer characteristics of CSCs. At present, the main method to isolate CSCs is to collect the regular clones in low-density culture conditions; therefore, an insufficient amount of CSCs is obtained for clonal expansion. To obtain a more sufficient amount of CSCs, the clones with an irregular and loose morphology were examined in our study. We found a small subpopulation of U251 glioma cells that arrested in the suspended state and that subsequently migrated to form new clones. The suspended cells were isolated from the irregular and loose clones. Clonogenic assays were performed in which 43.70% of the suspended cells and 32.91% of the adherent cells formed new clones. To determine the biological differences between the suspended and adherent cells, carboxyfluorescein succinimidyl ester (CFSE) labelling, MTT assays, and cell cycle assays were performed. The results demonstrated that the suspended cells had the characteristics of CSCs, including higher proliferation rates, as well as selfmaintenance and self-renewal capabilities, and they stained positively for markers of brain CSCs and had multilineage potential. Thus, we established a new and efficient approach for screening CSCs from the U251 human glioma cell line based on the cell growth state. IntroductionMalignant tumours consist of a heterogeneous population of cells that differ in their expression of markers and growth capacities (1,2). The cancer stem cell (CSC) hypothesis provides new insight into the heterogeneity of malignant tumours. Heterogeneity is determined, at least in part, by the presence of CSCs (1-6). Therefore, it is important to improve screening methods to obtain sufficient amounts of CSCs for further study of the biological treatment of malignant tumours.Stem cells and committed cells can form distinct clones in vitro (7). CSCs can be obtained by monoclonal morphology screening in some cancer cell lines. In the glioma cell line, U251, clones are characterised as tight, intermediate, or loose. The clones with a round and compact shape (tight clones) consist of CSCs, while the irregular (intermediate clones) and looseshaped clones did not show any CSC properties (8). Therefore, the intermediate-and loose-shaped clones have not been extensively studied. Similar clone patterns have been observed in malignant cell lines, such as head and neck squamous cell carcinoma, breast carcinoma and prostate carcinoma cell lines, in which only the cells of tight clones were capable of selfrenewal (9-11). These studies also indicated that the majority of the clones were intermediate ...

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“…For example, measurement of lymphocyte proliferation to mitogens, such as Phytohemagglutinin (PHA), Pokeweed mitogen (PWM) and Concanavalin A (Con A), and antigens, such as Candida albicans (CA) and Tetanus toxoid (TT) to ascertain T cell immune competence in PIDs [110] has long been performed by DNA incorporation of radiolabeled thymidine ( 3 H-T) after stimulation of peripheral blood mononuclear cells (PBMCs) with the appropriate agent. Elimination of techniques involving radioactivity is always beneficial to the clinical laboratory, and flow cytometry-based methods, primarily using the intracellular fluorescent dye, CFSE (carboxyfluorescein diacetate succinimidyl ester), are now available for measuring cellular proliferation [111-113]. However, a recent study seems to suggest that the use of CFSE to measure lymphocyte proliferation for the diagnosis of cellular PIDs would be inaccurate due to the high rate of false positive results [114].…”

Section: Cases 3 Andmentioning

confidence: 99%

Relevance of laboratory testing for the diagnosis of primary immunodeficiencies: a review of case-based examples of selected immunodeficiencies

Abraham

2011

Clin Mol Allergy

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The field of primary immunodeficiencies (PIDs) is one of several in the area of clinical immunology that has not been static, but rather has shown exponential growth due to enhanced physician, scientist and patient education and awareness, leading to identification of new diseases, new molecular diagnoses of existing clinical phenotypes, broadening of the spectrum of clinical and phenotypic presentations associated with a single or related gene defects, increased bioinformatics resources, and utilization of advanced diagnostic technology and methodology for disease diagnosis and management resulting in improved outcomes and survival. There are currently over 200 PIDs with at least 170 associated genetic defects identified, with several of these being reported in recent years. The enormous clinical and immunological heterogeneity in the PIDs makes diagnosis challenging, but there is no doubt that early and accurate diagnosis facilitates prompt intervention leading to decreased morbidity and mortality. Diagnosis of PIDs often requires correlation of data obtained from clinical and radiological findings with laboratory immunological analyses and genetic testing. The field of laboratory diagnostic immunology is also rapidly burgeoning, both in terms of novel technologies and applications, and knowledge of human immunology. Over the years, the classification of PIDs has been primarily based on the immunological defect(s) ("immunophenotype") with the relatively recent addition of genotype, though there are clinical classifications as well. There can be substantial overlap in terms of the broad immunophenotype and clinical features between PIDs, and therefore, it is relevant to refine, at a cellular and molecular level, unique immunological defects that allow for a specific and accurate diagnosis. The diagnostic testing armamentarium for PID includes flow cytometry - phenotyping and functional, cellular and molecular assays, protein analysis, and mutation identification by gene sequencing. The complexity and diversity of the laboratory diagnosis of PIDs necessitates many of the above-mentioned tests being performed in highly specialized reference laboratories. Despite these restrictions, there remains an urgent need for improved standardization and optimization of phenotypic and functional flow cytometry and protein-specific assays. A key component in the interpretation of immunological assays is the comparison of patient data to that obtained in a statistically-robust manner from age and gender-matched healthy donors. This review highlights a few of the laboratory assays available for the diagnostic work-up of broad categories of PIDs, based on immunophenotyping, followed by examples of disease-specific testing.

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CFSE flow cytometric quantification of lymphocytic proliferation in extracorporeal photopheresis: Use for quality control

Cited by 16 publications

References 15 publications

Quality control of extracorporeal photochemotherapy: Proliferation assay using CFSE validated according to ISO 15189:2007 standards

Quality control of extracorporeal photochemotherapy: Proliferation assay using CFSE validated according to ISO 15189:2007 standards

A new approach to screening cancer stem cells from the U251 human glioma cell line based on cell growth state

Relevance of laboratory testing for the diagnosis of primary immunodeficiencies: a review of case-based examples of selected immunodeficiencies

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