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Normothermic ex vivo perfusion of the limb allograft depletes donor leukocytes prior to transplantation

  • KR Amin
    Affiliations
    The Ex-Vivo Lab, Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom

    Blond McIndoe Laboratories, Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom
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  • JP Stone
    Affiliations
    3F66, Block 3, Alderley Park, Nether Alderley, Cheshire, SK10 4TG

    The Ex-Vivo Lab, Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom

    The Transplant Centre, Manchester University NHS Foundation Trust, Manchester M23 9LT, United Kingdom
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  • JC Kerr
    Affiliations
    3F66, Block 3, Alderley Park, Nether Alderley, Cheshire, SK10 4TG

    The Ex-Vivo Lab, Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom
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  • JK Wong
    Affiliations
    Blond McIndoe Laboratories, Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom

    Department of Plastic Surgery & Burns, Wythenshawe Hospital, Manchester University NHS Foundation Trust, Manchester, United Kingdom
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  • JE Fildes
    Correspondence
    Corresponding author at: The Ex-Vivo Research Laboratory, Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester Academic Health Science Centre, Manchester M13 9NT, UK.
    Affiliations
    3F66, Block 3, Alderley Park, Nether Alderley, Cheshire, SK10 4TG

    The Ex-Vivo Lab, Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom

    The Transplant Centre, Manchester University NHS Foundation Trust, Manchester M23 9LT, United Kingdom
    Search for articles by this author
Published:April 11, 2021DOI:https://doi.org/10.1016/j.bjps.2021.03.071

      Summary

      Introduction

      The donor immune compartment plays a central role in graft rejection of the vascularised composite allograft (VCA) by contributing to ‘direct presentation’. Using our limb ex vivo normothermic machine perfusion (EVNP) protocol designed for prolonged allograft preservation, this study aimed to assess whether donor leukocytes responsible for allograft rejection are mobilised from the donor compartment.

      Methods

      Five genetically different pig forelimbs underwent perfusion via the brachial and radial collateral artery for 6 h after 2 h of cold storage. Oxygenated haemodilute leucocyte-deplete blood was recirculated at normothermia using an extracorporeal perfusion system. Tissue perfusion was evaluated clinically and biochemically via blood perfusate. The temporal kinetics of donor leucocyte extravasation, cytokine secretion and cell-free DNA was characterised in the circulating perfusate.

      Results

      Flow cytometry revealed increasing populations of viable leukocytes over time, reaching 49 billion leukocytes by 6 h. T (3.0 × 109 cells) and B cells (3.1 × 108 cells) lymphocytes, monocytes (2.7 × 109 cells), granulocytes (8.1 × 109 cells), NK (6.3 × 108) and γδ (8.1 × 108) cells were all identified. Regulatory T cells comprised a minor population (1.6 × 107 cells). There was a cumulative increase in pro-inflammatory cytokines suggesting that the donor limb has the capacity to elicit significant inflammatory responses that could contribute to leucocyte activation and diapedesis.

      Conclusion

      EVNP not only acts as a preservation tool, but could also be utilized to immunodeplete the VCA allograft prior to transplantation. This has clinical implications to mitigate acute rejection and prevent graft dysfunction and supports the future application of machine perfusion in graft preservation and immune modulation.

      Keywords

      Abbreviations:

      APCs (antigen presenting cells), DCs (dendritic cells), EVNP (ex vivo normothermic machine perfusion), gDNA (genomic DNA), mtDNA (mitochondrial DNA), NMP-70 (normothermic perfusion with a mean arterial pressure of 70 mmHg), PVR (peripheral vascular resistance), SCS (static cold storage), VCA (vascularised composite allograft), WIT (warm ischaemic time), MHC (major histocompatibility complex), INF-γ (interferon-γ), TNF-α (tumour necrosis factor-α), IL (interleukin), GM-CSF (granulocyte macrophage colony-stimulating factor), qPCR (quantitative polymerase chain reaction)
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