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

Cytometry B Clin Cytom. 2007 Nov;72(6):450-7. doi: 10.1002/cyto.b.20359.


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 approximately 15% higher than ABC(QSC), whereas ABC(Q-MESF) was approximately 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.

Publication types

  • Comparative Study

MeSH terms

  • Adult
  • Aged
  • Antigens, CD20 / metabolism
  • B-Lymphocytes / immunology*
  • B-Lymphocytes / pathology
  • CD3 Complex / metabolism
  • CD4 Antigens / metabolism
  • Calibration / standards
  • Case-Control Studies
  • Female
  • Flow Cytometry / methods*
  • Flow Cytometry / standards
  • Humans
  • Leukemia, Lymphocytic, Chronic, B-Cell / immunology*
  • Leukemia, Lymphocytic, Chronic, B-Cell / pathology
  • Male
  • Microspheres
  • Middle Aged
  • Sialic Acid Binding Ig-like Lectin 2 / metabolism
  • T-Lymphocytes / immunology*
  • T-Lymphocytes / pathology


  • Antigens, CD20
  • CD3 Complex
  • CD4 Antigens
  • Sialic Acid Binding Ig-like Lectin 2