Performance of calibration standards for antigen quantitation with flow cytometry in chronic lymphocytic leukemia.
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.
Rossmann
Eva D
ED
Department of Oncology (Radiumhemmet), Karolinska University Hospital, Stockholm, Sweden. eva.rossmann@karolinska.se
Lenkei
Rodica
R
Lundin
Jeanette
J
Mellstedt
Håkan
H
Osterborg
Anders
A
D000328:Adult; D000368:Aged; D018951:Antigens, CD20 / Q000378:metabolism; D001402:B-Lymphocytes / Q000276:immunology* / Q000473:pathology; D017252:CD3 Complex / Q000378:metabolism; D015704:CD4 Antigens / Q000378:metabolism; D002138:Calibration / Q000592:standards; D016022:Case-Control Studies; D005260:Female; D005434:Flow Cytometry / Q000379:methods* / Q000592:standards; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000276:immunology* / Q000473:pathology; D008297:Male; D008863:Microspheres; D008875:Middle Aged; D051918:Sialic Acid Binding Ig-like Lectin 2 / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology* / Q000473:pathology
D018951:Antigens, CD20; D017252:CD3 Complex; D015704:CD4 Antigens; D051918:Sialic Acid Binding Ig-like Lectin 2