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 V. 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.