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

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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.

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Acknowledgements

The authors thank all blood donors involved in this study and the Illawarra Health and Medical Research Institute technical officers for technical support.

Funding

This project was funded by Molecular Horizons (University of Wollongong, Wollongong) and the Radiation Oncology Trust Fund (Illawarra Cancer Care Centre, Wollongong).

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Correspondence to Ronald Sluyter.

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Ross J. Turner declares that he has no conflict of interest.

Nicholas J. Geraghty declares that he has no conflict of interest.

Jonathan G. Williams declares that he has no conflict of interest.

Diane Ly declares that she has no conflict of interest.

Daniel Brungs declares that he has no conflict of interest.

Martin G. Carolan declares that he has no conflict of interest.

Thomas V. Guy declares that he has no conflict of interest.

Debbie Watson declares that she has no conflict of interest.

Jeremiah F. de Leon declares that he has no conflict of interest.

Ronald Sluyter declares that he has no conflict of interest.

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All experiments with human blood were conducted in accordance with approval by the University of Wollongong Human Research Ethics Committee (Wollongong, Australia).

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Turner, R.J., Geraghty, N.J., Williams, J.G. et al. Comparison of peripheral blood mononuclear cell isolation techniques and the impact of cryopreservation on human lymphocytes expressing CD39 and CD73. Purinergic Signalling 16, 389–401 (2020). https://doi.org/10.1007/s11302-020-09714-1

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