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Quantifying CD4 receptor protein in two human CD4+ lymphocyte preparations for quantitative flow cytometry



Meiyao M. Wang, Martin Misakian, Hua-Jun He, Peter Bajcsy, Jeffrey M. Davis, Kenneth D. Cole, Illarion Turko, Lili Wang, Fatima Abbasi


For quantitative flow cytometry, a biological cell reference material with a known biomarker expression level is needed to transform a linear arbitrary fluorescence intensity scale obtained with fluorescent microspheres to an antibody bound per cell (ABC) scale. In our previous study of characterizing different human CD4+ lymphocyte preparations, it was found that both commercially available cryopreserved peripheral blood mononuclear cells (PBMC) and a commercially available stabilized and lyophilized PBMC (Cyto-TrolTM) preparation reasonably fulfilled a set of criteria for serving as biological calibrators. However, the biomarker CD4 expression level measured for T helper cells from stabilized cells was about 16% lower than those for cryopreserved PBMC and fresh Whole Blood using both flow cytometry and mass cytometry. A primary reason was hypothesized to be due to steric interference in anti- CD4 antibody binding to the smaller sized stabilized cells. In this study, both multiple reaction monitoring mass spectrometry (MRM-MS) and scanning electron microscopy (SEM) as well as flow cytometry are used to determine likely underlying reasons for the observed difference in the CD4 expression. Because of the stabilization process, a lower CD4 density value, defined as the number of CD4 receptors per CD4+ lymphocyte, from the stabilized cells is most likely explained by the loss of the CD4 receptor proteins suggested by damaged and/or broken microvilli where CD4 receptors reside. Steric hindrance of antibody binding and the association of CD4 receptors with other biomolecules likely contribute significantly to the close to 50% lower CD4 receptor density value for PBMC determined from flow cytometry compared to the value obtained from MRM MS.
Clinical Proteomics


cryopreserved peripheral blood mononuclear cells (PBMC), stabilized control cells, Whole Blood, CD4 expression level, CD4 receptor density, flow cytometry, multiple reaction monitoring mass spectrometry (MRM MS) scanning electron microscopy (SEM), microvilli, lyophilization, surface roughness, probability distribution function (PDF)


Wang, M. , Misakian, M. , He, H. , Bajcsy, P. , Davis, J. , Cole, K. , Turko, I. , Wang, L. and Abbasi, F. (2014), Quantifying CD4 receptor protein in two human CD4+ lymphocyte preparations for quantitative flow cytometry, Clinical Proteomics, [online],, (Accessed June 13, 2024)


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Created December 10, 2014, Updated October 12, 2021