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

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dc.contributor.authorWang, Meiyao
dc.contributor.authorMisakian, Martin
dc.contributor.authorHe, Hua-Jun
dc.contributor.authorBajcsy, Peter
dc.contributor.authorAbbasi, Fatima
dc.contributor.authorDavis, Jeffrey M
dc.contributor.authorCole, Kenneth D
dc.contributor.authorTurko, Illarion V
dc.contributor.authorWang, Lili
dc.date.accessioned2021-08-30T20:50:54Z
dc.date.available2021-08-30T20:50:54Z
dc.date.issued2014-12-11
dc.description.abstractIn our previous study that characterized different human CD4+ lymphocyte preparations, it was found that both commercially available cryopreserved peripheral blood mononuclear cells (PBMC) and a commercially available lyophilized PBMC (Cyto-Trol™) preparation fulfilled a set of criteria for serving as biological calibrators for quantitative flow cytometry. However, the biomarker CD4 protein expression level measured for T helper cells from Cyto-Trol was about 16% lower than those for cryopreserved PBMC and fresh whole blood using 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 lyophilized control cells. Targeted multiple reaction monitoring (MRM) mass spectrometry (MS) is used to quantify the copy number of CD4 receptor protein per CD4+ lymphocyte. Scanning electron microscopy (SEM) is utilized to assist searching the underlying reasons for the observed difference in CD4 receptor copy number per cell determined by MRM MS and CD4 expression measured previously by flow cytometry. The copy number of CD4 receptor proteins on the surface of the CD4+ lymphocyte in cryopreserved PBMCs and in lyophilized control cells is determined to be (1.45 ± 0.09) × 105 and (0.85 ± 0.11) × 105, respectively, averaged over four signature peptides using MRM MS. In comparison with cryopreserved PBMCs, there are more variations in the CD4 copy number in lyophilized control cells determined based on each signature peptide. SEM images of CD4+ lymphocytes from lyophilized control cells are very different when compared to the CD4+ T cells from whole blood and cryopreserved PBMC. Because of the lyophilization process applied to Cyto-Trol control cells, a lower CD4 density value, defined as the copy number of CD4 receptors per CD4+ lymphocyte, averaged over three different production lots is most likely explained by the loss of the CD4 receptors on 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 nearly 50% lower CD4 receptor density value for cryopreserved PBMC determined from flow cytometry compared to the value obtained from MRM MS.en_US
dc.description.urihttps://doi.org/10.1186/1559-0275-11-43
dc.identifierhttps://doi.org/10.13016/acib-qhws
dc.identifier.citationWang, M., Misakian, M., He, HJ. et al. Quantifying CD4 receptor protein in two human CD4+ lymphocyte preparations for quantitative flow cytometry. Clin Proteom 11, 43 (2014).en_US
dc.identifier.urihttp://hdl.handle.net/1903/27663
dc.language.isoen_USen_US
dc.publisherSpringer Natureen_US
dc.relation.isAvailableAtCollege of Computer, Mathematical & Physical Sciencesen_us
dc.relation.isAvailableAtDigital Repository at the University of Marylanden_us
dc.relation.isAvailableAtBiologyen_us
dc.relation.isAvailableAtUniversity of Maryland (College Park, MD)en_us
dc.subjectCryopreserved peripheral blood mononuclear cells (PBMC)en_US
dc.subjectLyophilized Cyto-Trol™en_US
dc.subjectCD4 receptor densityen_US
dc.subjectFlow cytometryen_US
dc.subjectMultiple reaction monitoring (MRM) mass spectrometry (MS)en_US
dc.subjectScanning electron microscopy (SEM)en_US
dc.subjectMicrovillien_US
dc.subjectCell surface areaen_US
dc.titleQuantifying CD4 receptor protein in two human CD4+ lymphocyte preparations for quantitative flow cytometryen_US
dc.typeArticleen_US

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