title: Quantifying CD4 receptor protein in two human CD4+ lymphocyte preparations for quantitative flow cytometry.
abstract: BACKGROUND
  In 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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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) × 10(5) and (0.85 ± 0.11) × 10(5), 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Institute for Bioscience and Biotechnology Research, University of Maryland, 9600 Gudelsky Drive, Rockville, MD 20850 USA ; Biomolecular Measurement Division, National Institute of Standards and Technology (NIST), Gaithersburg, MD 20899 USA.
authors.forename: Meiyao
authors.initials: M
authors.lastname: Wang
authors.affiliation: Quantum Measurements Division, NIST, 100 Bureau Drive, Stop 8312, Gaithersburg, MD 20899 USA.
authors.forename: Martin
authors.initials: M
authors.lastname: Misakian
authors.affiliation: Biosystems and Biomaterials Division, NIST, 100 Bureau Drive, Stop 8312, Gaithersburg, MD 20899 USA.
authors.forename: Hua-Jun
authors.initials: HJ
authors.lastname: He
authors.affiliation: Software and Systems Division, NIST, 100 Bureau Drive, Stop 8312, Gaithersburg, MD 20899 USA.
authors.forename: Peter
authors.initials: P
authors.lastname: Bajcsy
authors.affiliation: Laboratory of Stem Cell Biology, Cellular and Tissue Therapy Branch, Division of Cell and Gene Therapies, CBER FDA, 8800 Rockville Pike, Bethesda, MD 20892 USA.
authors.forename: Fatima
authors.initials: F
authors.lastname: Abbasi
authors.affiliation: Materials Measurement Science Division, NIST, 100 Bureau Drive, Stop 8312, Gaithersburg, MD 20899 USA.
authors.forename: Jeffrey M
authors.initials: JM
authors.lastname: Davis
authors.affiliation: Biosystems and Biomaterials Division, NIST, 100 Bureau Drive, Stop 8312, Gaithersburg, MD 20899 USA.
authors.forename: Kenneth D
authors.initials: KD
authors.lastname: Cole
authors.affiliation: Institute for Bioscience and Biotechnology Research, University of Maryland, 9600 Gudelsky Drive, Rockville, MD 20850 USA ; Biomolecular Measurement Division, National Institute of Standards and Technology (NIST), Gaithersburg, MD 20899 USA.
authors.forename: Illarion V
authors.initials: IV
authors.lastname: Turko
authors.affiliation: Biosystems and Biomaterials Division, NIST, 100 Bureau Drive, Stop 8312, Gaithersburg, MD 20899 USA.
authors.forename: Lili
authors.initials: L
authors.lastname: Wang
keywords: CD4 receptor density; Cell surface area; Cryopreserved peripheral blood mononuclear cells (PBMC); Flow cytometry; Lyophilized Cyto-Trol™; Microvilli; Multiple reaction monitoring (MRM) mass spectrometry (MS); Scanning electron microscopy (SEM)