Development of Clinical Standards for Flow Cytometry

Schwartz, A.; Fernández-Repollet, Emma

doi:10.1111/j.1749-6632.1993.tb38760.x

Cited by 91 publications

(78 citation statements)

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“…Quantitative flow cytometry requires a comprehensive calibration procedure to monitor instrument performance and sample preparation as well as to ensure accurate and clinically relevant flow cytometric data (9,10). For a calibration standard to be useful in studies that may cover a range of environmental conditions, it is necessary to evaluate it under such conditions to ensure that it responds in a fashion similar to the samples being measured.…”

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confidence: 99%

Quantification of EGFP expression on Molt‐4 T cells using calibration standards

et al. 2004

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Background: Enhanced green fluorescent protein (EGFP) is used extensively to assess gene expression on cells; however, quantification of this expression by flow cytometry has been limited by the unavailability of calibration standards. Thus, we characterized the response of an experimental set of EGFP calibration standards to environmental changes and then quantitate the expression of EGFP, in molecules of equivalent soluble fluorochrome (MESF) units, of a transfected Molt-4 T cell line by flow cytometry. Methods: Characterization of the EGFP standards: EGFP standards were equilibrated in suspension solutions having a pH range of 5.0 -9.0, temperatures of 37-80°C, and osmolalities of 100 -600 mOsm/kg. Quantification of EGFP on cells: For transfections, Molt-4 T cells were incubated with two different concentrations (0.2 g and 0.4 g) of pEGFP-N2 vector and the EGFP expression was quantified after 48 h by flow cytometry using the EGFP standards and by the cytofluor technique using a standard curve of known EGFP solutions. Results: The fluorescence intensity of the EGFP standards increased from pH 5.0 to 9.0 and remained relatively

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confidence: 99%

Quantification of EGFP expression on Molt‐4 T cells using calibration standards

et al. 2004

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“…The determination of the fluorescence intensity may add bi-ologically relevant information, because fluorescence intensity is directly related to the number of binding antibodies and, thus, to the level of antigen expression ( 15). This information could have direct implications with respect to the diagnosis, prognosis, and treatment of pathological conditions.…”

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Peripheral blood cell preparation influences the level of expression of leukocyte cell surface markers as assessed with quantitative multicolor flow cytometry

1995

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We have compared the influence of sample preparation upon the level of surface expression of T, B, and NK cell-related antigens as assessed by flow cytometry. Lysed whole blood (WBL), Ficoll-Paque separated peripheral blood lymphocyte (F-PBL), and frozen peripheral blood lymphocyte (Fr-PBL) were analyzed via single-and multicolor flow cytometry. The percentage of positive cells expressing the individual cell surface markers was not affected by the procedure for preparation of WBL, F-PBL, and Fr-PBL. In contrast, the fluorescence intensity level of individual cell surface markers varied depending on cell preparation. By using Quantum Simply Cellular (QSC) microbeads, the antibody binding capacity (ABC) of single-color stained cells was quantified and compared. The amount of monoclonal antibody (MAb) anti-CD3-FITC bound to Fr-PBL (mean ABC = 137,040) was significantly higher (P < 0.001) that the amounts bound to WBL (mean ABC = 112,410) and F-PBL (mean ABC = 107,738). In multicolor analysis, the fluorescence intensity of CD3-FITC and CD4-FITC was significantly higher on Fr-PBL than on WBL and F-PBL; CD8-PE and CD20-PerCP was significantly higher on WBL. Furthermore, the intensity of CD3 and CD4 was different on T-cell subsets. The intensity of CD3 staining in three-color analysis was lower than with single-color staining using the same fluorochrome.We conclude that particularly the method of cell preparation but also the selection of MAb combinations may influence the level of staining of certain lymphocyte antigens. This may be of relevance in the analysis of cellular activation and regulation of differentiation.

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“…Given the value of α and assuming that the maximal fluorescence intensity observed in Figure 3 represents full labeling, one can evaluate n without any fitting and obtain the similar result of n = 67.2·10 3 . This standard approach to antigen quantitation confirms the fitting results, but it does not provide the other parameters of interest.…”

Section: Reaction Rate Constant Instead Of Calibration?mentioning

confidence: 99%

“…From the binding rate constant k + , it was possible to estimate the radius b of the binding site (a circular approximation of the shape of the site placed on a spherical reagent) using following expression [12]: (3) where η is the viscosity of the media, k B is the Boltzmann constant, T is the temperature; R 1 and R 2 are radii, N 1 and N 2 are valences of the first and second reactants, correspondingly. It should be noted that equation (3) is applied if bond formation is diffusion limited, -this is not necessary the general case, but this is true for many particular antibodies.…”

Section: Human T Cellsmentioning

confidence: 99%

“…However, at a minimum, a calibration procedure with carefully prepared reagents is needed to convert the intensity of the fluorescence signal to the number of target antigens [2]. For instance, among the currently marketed technologies there are three that are well known: Quantum Simply Cellular beads (QSC) designed to bind any fluorochrome-labeled murine monoclonal antibody [3]; Quantitative Immunofluorescence Intensity beads (QIFI kit) [4] for indirect immunofluorescence; and the Quanti-BRITE assay [5]. Although the calibration bead-based technologies seem to be a straightforward and easyto-use approach for quantitative fluorescence flow cytometry, the on-site comparison of these three technologies [2] has revealed their limitations.…”

Section: Introductionmentioning

confidence: 99%

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Dynamic quantification of antigen molecules with flow cytometry

Moskalensky

Chernyshev

Yurkin

et al. 2015

Journal of Immunological Methods

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Traditional methods for estimating the number of expressed molecules, based on the detection of target antigens bound with fluorescently labeled antibodies, assume that the antigen-antibody reaction reaches equilibrium. A calibration procedure is used to convert the intensity of the fluorescence signal to the number of target molecules. Along with the different limitations of every calibration system, this substantially limits the applicability of the traditional approaches especially in the case of low affinity antibodies.We address this problem here with studies in which we demonstrate a new approach to the antigen molecule quantification problem. Instead of using a static calibration system, we analyzed mean fluorescence values over time by flow cytometry during antibody-antigen binding. Experimental data obtained with an LSRII cytometer were fitted by a diffusion-reaction mathematical model using the Levenberg-Marquardt nonlinear least squares curve-fitting algorithm in order to obtain the number of target antigen molecules per cell. Results were compared with the Quanti-BRITE calibration system. We conclude that, instead of using experiment-specific calibration, the value of the binding rate constant for each particular antibody-antigen reaction can be used to quantify antigen molecules with flow cytometry. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript fluorescent label, medium viscosity and temperature). This approach is independent of specially prepared calibration beads, antibody reagents and the specific dye and can be applied to both low and high affinity antibodies, under both saturating and non-saturating binding conditions. The method was demonstrated on a human blood sample dataset investigating CD8α antigen on T cells in stable binding conditions.

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Development of Clinical Standards for Flow Cytometry

Cited by 91 publications

References 1 publication

Quantification of EGFP expression on Molt‐4 T cells using calibration standards

Quantification of EGFP expression on Molt‐4 T cells using calibration standards

Peripheral blood cell preparation influences the level of expression of leukocyte cell surface markers as assessed with quantitative multicolor flow cytometry

Dynamic quantification of antigen molecules with flow cytometry

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