Human CD4+ lymphocytes for antigen quantification: Characterization using conventional flow cytometry and mass cytometry

Authors

  • Lili Wang,

    Corresponding author
    1. Biochemical Science Division, National Institute of Standards and Technology (NIST), 100 Bureau Drive, Stop 8312, Gaithersburg, Maryland 20899
    • National Institute of Standards and Technology, 100 Bureau Drive, Stop 8312, Gaithersburg, Maryland 20899-8312
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  • Fatima Abbasi,

    1. Division of Cell and Gene Therapies, Laboratory of Stem Cell Biology, Cellular and Tissue Therapy Branch, Office of Cellular, Tissues and Gene Therapies, CBER FDA, NIH Bdg 29B Rm 1NN010, 8800 Rockville Pike, Bethesda, Maryland 20892
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  • Olga Ornatsky,

    1. Department of Chemistry, University of Toronto, 80 St. George St., Toronto ON, Canada M5S 3H6
    2. DVS Sciences Inc., 70 Peninsula Cr., Richmond Hill ON, Canada L4S 1Z5
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  • Kenneth D. Cole,

    1. Biochemical Science Division, National Institute of Standards and Technology (NIST), 100 Bureau Drive, Stop 8312, Gaithersburg, Maryland 20899
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  • Martin Misakian,

    1. Biochemical Science Division, National Institute of Standards and Technology (NIST), 100 Bureau Drive, Stop 8312, Gaithersburg, Maryland 20899
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  • Adolfas K. Gaigalas,

    1. Biochemical Science Division, National Institute of Standards and Technology (NIST), 100 Bureau Drive, Stop 8312, Gaithersburg, Maryland 20899
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  • Hua-Jun He,

    1. Biochemical Science Division, National Institute of Standards and Technology (NIST), 100 Bureau Drive, Stop 8312, Gaithersburg, Maryland 20899
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  • Gerald E. Marti,

    1. Division of Immunology and Hematology Devices, Heme/Path Branch, Office of In Vitro Diagnostic Device Evaluation and Safety, CDRH FDA, Building 66, Room 4227, 10903 New Hampshire Avenue, Silver Spring, Maryland 20993
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  • Scott Tanner,

    1. Department of Chemistry, University of Toronto, 80 St. George St., Toronto ON, Canada M5S 3H6
    2. DVS Sciences Inc., 70 Peninsula Cr., Richmond Hill ON, Canada L4S 1Z5
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  • Richard Stebbings

    1. Biotherapeutics Group, National Institute for Biological Standards and Control (NIBSC), Blanche Lane, South Mimms, Potters Bar, Hertfordshire, EN6 3QG, United Kingdom
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  • This article is a US government work and, as such, is in the public domain in the United States of America.

Abstract

To transform the linear fluorescence intensity scale obtained with fluorescent microspheres to an antibody bound per cell (ABC) scale, a biological cell reference material is needed. Optimally, this material should have a reproducible and tight ABC value for the expression of a known clinical reference biomarker. In this study, we characterized commercially available cryopreserved peripheral blood mononuclear cells (PBMCs) and two lyophilized PBMC preparations, Cyto-Trol and PBMC–National Institute for Biological Standard and Control (NIBSC) relative to freshly prepared PBMC and whole blood samples. It was found that the ABC values for CD4 expression on cryopreserved PBMC were consistent with those of freshly obtained PBMC and whole blood samples. By comparison, the ABC value for CD4 expression on Cyto-Trol is lower and the value on PBMC–NIBSC is much lower than those of freshly prepared cell samples using both conventional flow cytometry and CyTOF™ mass cytometry. By performing simultaneous surface and intracellular staining measurements on these two cell samples, we found that both cell membranes are mostly intact. Moreover, CD4+ cell diameters from both lyophilized cell preparations are smaller than those of PBMC and whole blood. This could result in steric interference in antibody binding to the lyophilized cells. Further investigation of the fixation effect on the detected CD4 expression suggests that the very low ABC value obtained for CD4+ cells from lyophilized PBMC–NIBSC is largely due to paraformaldehyde fixation; this significantly decreases available antibody binding sites. This study provides confirmation that the results obtained from the newly developed mass cytometry are directly comparable to the results from conventional flow cytometry when both methods are standardized using the same ABC approach. Published 2012 Wiley Periodicals, Inc.

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