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

Abstract

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™ (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. Statistically significant correlations were found between the values obtained using various standards. The present study is the first to report down‐regulation of CD3 antigen on T cells from patients with CLL. Conclusions: This study emphasizes the relevance of quantitative measurement of fluorescence intensity by flow cytometry as a standardized approach to measure and interpret the expression of some CLL markers and reduce variability of results obtained at different sites in multi‐center clinical studies. © 2007 Clinical Cytometry Society