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Quantification of Cells with Specific Phenotypes II: Determination of CD4 Expression Level on Lyophilized Human PBMC Surface Labeled with Anti-CD4 FITC Antibody



Lili Wang, Adolfas K. Gaigalas


The study was carried out under the auspices of the Bioanlysis Working Group (BAWG) of the Comité Consultatif pour la Quantité de Matière (CCQM) and was piloted jointly by the National Institute of Biological Standards and Control (NIBSC) in South Mimms (UK), PTB (Physikalisch-Technische Bundesanstalt, Germany) and NIST (National Institute of Standards and Technology, USA). The objective of CCQM pilot study was to demonstrate participant capability in flow cytometric measurements of CD4+ cell concentration and the level of CD4 antigen expression on lyophilized human peripheral blood mononuclear cells (PBMC) surface labelled with anti-CD4 FITC and to document the variability and measurement uncertainty. This report focuses on the measurements of CD4 expression level and associated measurement uncertainties. Sixteen laboratories from eight countries participated in this pilot study. Twelve different flow cytometer platforms were used and evaluated. A standard measurement procedure provided to participants includes the construction of a fluorescence intensity calibration for the FITC channel of a flow cytometer using commercially available calibration microbeads. The equivalent fluorescein fluorophore (EFF) values of the five fluorescent calibration microbead populations were assigned at NIST using fluorescein SRM 1932 as the primary standard reference solution. On the basis of the intensity calibration, the EFF value was reported as a measure of CD4 expression level for a single dilution of each of the three PBMC sample vials provided to each participant. Both the results of analysis of the data by each participant, and the results of centralized analysis of all participant’s raw data are reported. The study design will be adopted for the development of future biological cell reference materials. Possible improvements of the standard measurement procedure are discussed.
Cytometry Part A


CD4 expression, peripheral blood mononuclear cells (PBMC), surface labelled, lyophilized PBMC, FITC, equivalent fluorescein fluorophore (EFF), quantitative flow cytometry, calibration, standard measurement procedure, measurement uncertainty, different flow cytometer platforms.


Wang, L. and Gaigalas, A. (2015), Quantification of Cells with Specific Phenotypes II: Determination of CD4 Expression Level on Lyophilized Human PBMC Surface Labeled with Anti-CD4 FITC Antibody, Cytometry Part A, [online], (Accessed May 25, 2024)


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Created March 10, 2015, Updated February 19, 2017