Performance of calibration standards for antigen quantitation with flow cytometry

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).

Section: Discussionmentioning

confidence: 99%

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

Rossmann

Lenkei

Lundin

et al. 2007

“…DAKO Fluorospheres TM (type IIIa beads) with assigned values of molecules of equivalent soluble fluorochrome were used for intracenter longitudinal monitoring of instrument performance stability (17). One hundred thirty-seven measurements of these beads (which contain blank as well as quadruple-fluorescence beads with five different emission intensities) were performed during the study period.…”

Section: Flow Cytometrymentioning

confidence: 99%

Modulation of antigen expression in B‐cell precursor acute lymphoblastic leukemia during induction therapy is partly transient: Evidence for a drug‐induced regulatory phenomenon. Results of the AIEOP‐BFM‐ALL‐FLOW‐MRD‐Study Group

Dworzak¹,

Gaipa

Schumich³

et al. 2010

Background: Changes of antigen expression on residual blast cells of acute lymphoblastic leukemia (ALL) occur during induction treatment. Many markers used for phenotyping and minimal residual disease (MRD) monitoring are affected. Glucocorticoid (GC)-induced expression modulation has been causally suspected, however, subclone selection may also cause the phenomenon.Methods: We investigated this by following the phenotypic evolution of leukemic cells with flow cytometry from diagnosis to four time points during and after GC containing chemotherapy in the 20 (of 360 consecutive) B-cell precursor patients with ALL who had persistent MRD throughout.Results: The early expression changes of CD10 and CD34 were reversible after stop of GC containing chemotherapy. Modulation of CD20 and CD45 occurred mostly during the GC phase, whereas CD11a also changed later on. Blast cells at diagnosis falling into gates designed according to ''shifted'' phenotypes from follow-up did not form clusters and were frequently less numerous than later on.Conclusions: Our data support the idea that drug-induced modulation rather than selection causes the phenomenon. The good message for MRD assessment is that modulation is transient in at least two (CD10 and CD34) of the five prominent antigens investigated and reverts to initial aberrant patterns after stop of GC therapy, whereas CD20 expression gains new aberrations exploitable for MRD detection.

“…In contrast, other markers are expressed consistently by human MCs, but quantitative differences exist between normal and pathologic MCs with abnormally high-CD59, CD63, and CD69 -or low-CD117-amounts being detected in mastocytosis. For the first group of markers, objective and reproducible criteria for positivity are required; for the second group, accurate quantitative estimation of the amount of antigen expressed per cell is a prerequisite for obtaining reproducible results (9,16,17).…”

Section: Information Provided By the Flow Cytometric Immunophenotypicmentioning

confidence: 99%

Immunophenotypic analysis of mast cells in mastocytosis: When and how to do it. Proposals of the Spanish Network on Mastocytosis (REMA)

Escribano¹,

Díaz‐Agustín²,

López

et al. 2004

136

131

Background: Mastocytosis is a term used for a heterogeneous group of disorders characterized by an abnormal proliferation and accumulation of mast cells (MCs) in one or multiple tissues including skin, bone marrow, liver, spleen, and lymph nodes, among others.Methods: In recent years, multiparameter flow cytometric studies have shown that pathologic MCs from patients with mastocytosis display unique aberrant immunophenotypic characteristics as compared with normal MCs.Results: Among other features, pathologic MCs show aberrant expression of CD25 and CD2 antigens and abnormally high levels of the CD11c and CD35 complement receptors, the CD59 complement regulatory molecule, the CD63 lysosomal membrane antigen, and the CD69 early-activation antigen. In addition, MCs from mastocytosis express abnormally low levels of CD117 and unexpectedly high light scatter and autofluorescence characteristics.Conclusions: These aberrant immunophenotypic features are of great relevance for the assessment of tissue involvement in mastocytosis with consequences in the diagnosis, classification, and follow-up of the disease and in its differential diagnosis with other entities. In this paper we provide the reader with information for the objective and reproducible identification of pathologic MCs by using quantitative multiparametric flow cytometry, information for their phenotypic characterization, and the criteria currently used for a correct interpretation of the immunophenotypic results obtained.