Comparison of peripheral blood mononuclear cell isolation techniques and the impact of cryopreservation on human lymphocytes expressing CD39 and CD73

Authors

Ross J. Turner, University of WollongongFollow
Nicholas Geraghty, University of WollongongFollow
Jonathan Williams, University of WollongongFollow
Diane T. Ly, University of WollongongFollow
Daniel Brungs, University of WollongongFollow
Martin G. Carolan, University of WollongongFollow
Thomas Guy, University of WollongongFollow
Debbie Watson, University of WollongongFollow
Jeremiah de Leon
Ronald Sluyter, University of WollongongFollow

RIS ID

144759

Publication Details

Turner, R., Geraghty, N., Williams, J., Ly, D., Brungs, D., Carolan, M., Guy, T., Watson, D., de Leon, J. & Sluyter, R. (2020). Comparison of peripheral blood mononuclear cell isolation techniques and the impact of cryopreservation on human lymphocytes expressing CD39 and CD73. Purinergic Signalling,

Abstract

© 2020, Springer Nature B.V. CD39 and CD73 are ecto-nucleotidases present on human peripheral blood mononuclear cells (PBMCs) and are emerging biomarkers on these cells in various disorders including cancer. Many factors influence PBMC quality, so it is essential to validate sample processing methods prior to incorporation in clinical studies. This study examined the impact of both PBMC cryopreservation and PBMC isolation using SepMate density gradient centrifugation on CD39 and CD73 expressing subsets. First, PBMCs were isolated from the peripheral blood of 11 healthy donors by routine Ficoll-Paque density gradient centrifugation, cryopreserved and compared with freshly isolated PBMCs by flow cytometry. The proportions of T and B cells expressing combinations of CD39 and CD73 were relatively stable over 6-month cryopreservation, although some T cell combinations revealed small but significant changes. Second, peripheral blood was collected from six healthy donors to compare PBMCs isolated by SepMate or Ficoll-Paque density gradient centrifugation. Compared with Ficoll-Paque, the more rapid SepMate method yielded 9.1% less PBMCs but did not alter cell viability or proportions of T and B cells expressing combinations of CD39 and CD73. The present study reveals that cryopreservation is suitable for studying T and B cells expressing combinations of CD39 and CD73. However, caution should be exercised when observing small differences in these cryopreserved subsets between different cohorts. Further, SepMate and Ficoll-Paque methods of PBMC isolation show similar results for T and B cell subset analysis; however, SepMate is a faster and easier approach.