Comparison of peripheral blood mononuclear cell isolation techniques and the impact of cryopreservation on human lymphoc
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ORIGINAL ARTICLE
Comparison of peripheral blood mononuclear cell isolation techniques and the impact of cryopreservation on human lymphocytes expressing CD39 and CD73 Ross J. Turner 1,2,3 & Nicholas J. Geraghty 1,2 & Jonathan G. Williams 1,2 & Diane Ly 1,2 & Daniel Brungs 1,3,4 & Martin G. Carolan 1,3,4 & Thomas V. Guy 1 & Debbie Watson 1,2,3 & Jeremiah F. de Leon 4,5 & Ronald Sluyter 1,2,3 Received: 21 April 2020 / Accepted: 7 July 2020 # Springer Nature B.V. 2020
Abstract 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. Keywords Ecto-enzymes . ENTPD1 . NT5E . Purinergic signalling . Purinergic receptor . Liquid biopsy
Introduction
* Ronald Sluyter [email protected] 1
Illawarra Health and Medical Research Institute, Wollongong, NSW 2522, Australia
2
Molecular Horizons and School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, NSW 2522, Australia
3
CONCERT - Centre for Oncology Education and Research Translation, Liverpool, NSW 2170, Australia
4
Illawarra Cancer Care Centre, Wollongong Hospital, Wollongong, NSW 2500, Australia
5
GenesisCare, St Vincent’s Clinic, Darlinghurst, NSW 2010, Australia
CD39 and CD73 are ectonucleotidases expressed on the surface of immune and other cells types and together mediate the hydrolysis of extracellular adenosine triphosphate (ATP) into adenosine [1, 2]. Specifically, CD39, an ectonucleoside triphosphate diphosphohydrolase, hydrolyses ATP and adenosine diphosphate (ADP
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