Flow cytometric quantitation of immunofluorescence intensity: Problems and perspectives

Gratama, Jan W.; D'Hautcourt, Jean-Luc; Mandy, Francis; Rothe, Gregor; Barnett, David; Jánossy, G.; Papa, Stefano; Schmitz, Gerd; Lenkei, Rodiça

doi:10.1002/(sici)1097-0320(19981001)33:2<166::aid-cyto11>3.0.co;2-s

Cited by 136 publications

(106 citation statements)

References 21 publications

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“…The calibrators can represent an error source caused by possible lot-to-lot variations and instability (40,41). Receptor number overestimations, caused by suboptimal attachment of mAbs to the QSC beads versus cells had been previously reported (34).…”

Section: Groupmentioning

confidence: 94%

Performance of calibration standards for antigen quantitation with flow cytometry in chronic lymphocytic leukemia

Rossmann

Lenkei

Lundin

et al. 2007

Cytometry Part B Clinical

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Background: The fluorescence intensities of CD3, CD4 on T cells and CD20, CD22 molecules on B cells were quantitatively measured on lymphocytes from chronic lymphocytic leukemia (CLL) patients and healthy donors.Methods: The performance of three different types of microbeads was compared, i.e. Quantum molecules of equivalent soluble fluorochrome (Q-MESF), Quantum simply cellular (QSC), and QuantiBRITE TM (QB). As all PE-conjugates had a F/P ratio of 1:1, the MESF units represented also the antibody binding capacity (ABC).Results: The ABCs of CD4 and CD20 antigens estimated with QSC (ABC QSC ) were higher than those assigned with QB (ABC QB ) with an average difference 49%. Higher numbers of antigenic sites were obtained with Q-MESF than with QSC for CD20 antigen. On the contrary, CD4 antigenic sites numbers estimated with QSC were higher than those estimated with Q-MESF. ABC values estimated with Quantum MESF PE (ABC Q-MESF ) were *15% higher than ABC QSC , whereas ABC Q-MESF was *49% higher than ABC QB INTRODUCTIONQuantitative measurements of fluorescence intensity by flow cytometry (QFCM) assess the numbers of monoclonal antibody (mAb) molecules bound to a cell. The fluorescence intensity (FI) of staining correlates to the number of fluorochrome conjugated mAb molecules bound to the cell, and consequently, the amount of receptors on the cell. Thus, the number of antigen receptors can be determined by QFCM, through estimation of the exact numbers of bound fluorescent antibody molecules, provided that appropriate standards and fluorescence controls are tested in parallel and reagents in saturating amounts are used. In addition, the process of quantitation facilitates standardization and quality control (1).

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Section: Groupmentioning

confidence: 94%

Performance of calibration standards for antigen quantitation with flow cytometry in chronic lymphocytic leukemia

Rossmann

Lenkei

Lundin

et al. 2007

Cytometry Part B Clinical

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“…Of these, the indirect immunofluorescence tests, such as the Quantitative Indirect ImmunoFluorescence (QIFI) test (40), is the most reliable because the first antibody used to bind to the membrane antigens at saturating concentrations remains unconjugated and therefore unmodified; the fluorochrome-labeled second antibody, used at saturating level, is then a universal reagent used in every assay. Thus, the results observed are comparable and allow the construction of "league tables" for the expression of different membrane antigens on different cell types (39,40). For example, the CD45 antigens on lymphocytes are expressed at four times higher levels (at 200 ϫ 10 3 molecules/cell) than CD4 antigens on "helper-type" T cells (50 ϫ 10 3 molecules/cell) and 10 times higher than CD19 antigens on B lymphocytes (20 ϫ 10 3 molecules/cell; Fig.…”

Section: Standards For Quantitative Flow Cytometrymentioning

confidence: 56%

“…These premises can be studied further by methods that quantitate antibody-binding capacity (ABC) expressed as molecules per cell (39). Of these, the indirect immunofluorescence tests, such as the Quantitative Indirect ImmunoFluorescence (QIFI) test (40), is the most reliable because the first antibody used to bind to the membrane antigens at saturating concentrations remains unconjugated and therefore unmodified; the fluorochrome-labeled second antibody, used at saturating level, is then a universal reagent used in every assay.…”

Section: Standards For Quantitative Flow Cytometrymentioning

confidence: 99%

Clinical flow cytometry, a hypothesis‐driven discipline of modern cytomics

Jánossy

2004

Cytometry Pt A

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“…Importantly, labeling with a directly fluorochrome-conjugated antibody is feasible in the case of highly-expressed surface antigens such as CD8, CD45RO on T-cells, or CD14 on monocytes, among others (17). This further simplifies staining and facilitates polychromatic analysis, as it also could be achieved with quantum dot labeled reagents (18).…”

Section: Four-color Analysismentioning

confidence: 99%

Quantitative histology by multicolor slide‐based cytometry

et al. 2004

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BackgroundIn lymphatic organs, the quantitative analysis of the spatial distribution of leukocytes by tissue cytometry would give relevant information about alterations during diseases (leukemia, HIV, AIDS) and their therapeutic regimen, as well as in experimental settings.MethodsWe have developed a semiautomated analysis method for laser scanning cytometry (LSC) termed “multiple thresholding,” which is suitable for archived or fresh biopsy material of human lymph nodes and tonsils. Sections are stained with PI for nuclear DNA and up to four antigens using direct or indirect immunofluorescence staining. Measurement is triggered on DNA‐fluorescence (argon laser, Ar) or on specific cell labeling. Due to the heterogeneity of cell density, measurements are performed repeatedly at different threshold levels (low threshold: regions of low cellular density, germinal center; high threshold: dense regions, mantle zone). Data are acquired by single‐ (Ar) or dual‐laser excitation (Ar‐HeNe) in order to analyze single‐ (FITC) up to four‐color (FITC/PE/PECy5/APC) stained specimen.ResultsPercentage and cellular density of cell‐subsets is quantified in different microanatomical regions of the specimen. These data were highly correlated with manual scoring of identical specimens (r2 = 0.96, P < 0.0001). With LSC, semiautomated operator‐independent immunophenotyping in tissue sections of lymphatic organs with up to three antibodies simultaneously is possible.ConclusionsWe expect this tissue cytometric approach to yield new insight into processes during diseases and help to quantify the success of therapeutic interventions. © 2004 Wiley‐Liss, Inc.

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Flow cytometric quantitation of immunofluorescence intensity: Problems and perspectives

Cited by 136 publications

References 21 publications

Performance of calibration standards for antigen quantitation with flow cytometry in chronic lymphocytic leukemia

Performance of calibration standards for antigen quantitation with flow cytometry in chronic lymphocytic leukemia

Clinical flow cytometry, a hypothesis‐driven discipline of modern cytomics

Quantitative histology by multicolor slide‐based cytometry

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