Enhanced Recovery of Donor Hearts

ENHANCED RECOVERY OF DONOR HEARTS

Hong Chee CHEW

A thesis in fulfilment of the requirements for the degree of Doctor of Philosophy

March, 2019

Faculty of Medicine

UNSW Sydney

Transplantation Laboratory

Victor Chang Cardiac Research Institute

ENHANCED RECOVERY OF DONOR HEARTS:

COMBINED EXPERIMENTAL AND CLINICAL PHASE STUDY OF DONATION AFTER CIRCULATORY DEATH (DCD) PHYSIOLOGY, PROCUREMENT AND PRESERVATION, ASSESSMENT AND OUTCOMES AFTER TRANSPLANTATION USING NORMOTHERMIC MACHINE PERFUSION.

AND DEVELOPING A RODENT DCD HEART ASSESSMENT MODEL FOR THE VALIDATION OF PERIMORTEM HEPARIN AND SUPPLEMENTED PRESERVATION SOLUTION, AND TO INVESTIGATE THE EFFECT OF AGEING ON CARDIAC PRESERVATION AND RECOVERY.

By:

Hong Chee CHEW

BSc(med), MBBS (UNSW)

Masters of Surgery (USyd)

Supervisors:

Professor Peter S. Macdonald

Assoc. Professor Kumud K. Dhital

A thesis submitted to the University of New South Wales in fulfillment of the requirements for the degree of Doctor of Philosophy

Heart Transplant Research Laboratory

The Victor Chang Cardiac Research Institute, Darlinghurst, NSW, 2010

The Department of Heart and Lung Transplantation

St Vincent’s Hospital, Darlinghurst, NSW, 2010

THE UNIVERSITY OF NEW SOUTH WALES

Thesis/Dissertation Sheet

Surname or Family Name: CHEW

First Name: Hong Chee

Abbreviation for degree as given in the University Calendar: PhD

School: School of Medicine, St Vincent’s Clinical School, Victor Chang Cardiac Research Institute

Cardiac transplantation is the gold standard treatment for end stage heart failure. One of the biggest limitation with cardiac transplantation is a limited supply of donor hearts. In an attempt to increase transplant activity, new categories of donors have been established including marginal/extended criteria brain death donors (MBD) and circulatory death donors (DCD). The main concern with these donors is the potential increased risk of primary graft dysfunction/failure which can severely impact recipient survival. As a result, novel approaches to assessment of MBD and DCD donor hearts are being evaluated. Normothermic machine perfusion (NMP) is one of such assessment tool which allows the surgical team to examine the organs prior to implantation.

The goal of this thesis is to investigate the role of NMP in DCD heart transplantation as an extension to our clinical trial which has since concluded and is now a part of routine practise. In this thesis, we examined the use of NMP for the assessment of DCD hearts, including factors affecting recipient outcomes (i.e. mechanical support, mortality and graft function). Base on these findings, we device further animal experiments in an effort to solve these issues. The key experiments we conducted include the role of bank blood as perfusate for the donor heart during NMP, and the role of antemortem heparin in the donor.

Furthermore, with our increasing experience with DCD heart transplants, we have recently increased our DCD donor age criteria. However, there remains much concern with the effect of ageing and donor graft function. In response to this, we performed rodent experiments using animals of different ages to assess the recovery of the heart and investigate the reversibility of these injury with pharmacological intervention.

Declaration relating to disposition of project thesis/dissertation

I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation.

I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts international (this is applicable to doctoral thesis only).

……………………………………… …………………………..29/03/2019 Signature Witness Date

The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restrictions for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research. FOR OFFICE USE ONLY Date of completion of requirements for Award: INCLUSION OF PUBLICATIONS STATEMENT

UNSW is supportive of candidates publishing their research results during their candidature as detailed in the UNSW Thesis Examination Procedure.

Publications can be used in their thesis in lieu of a Chapter if: • The student contributed greater than 50% of the content in the publication and is the "primary author", ie. the student was responsible primarily for the planning, execution and preparation of the work for publication • The student has approval to include the publication in their thesis in lieu of a Chapter from their supervisor and Postgraduate Coordinator. • The publication is not subject to any obligations or contractual agreements with a third party that would constrain its inclusion in the thesis

Please indicate whether this thesis contains published material or not. □ Some of the work described in this thesis has been published and it has been □ documented in the relevant Cha ters with acknowledgement - This thesis has publications (either published or submitted for publication) incorporated into it in lieu of a chapter and the details are presented below

CANDIDATE'S DECLARATION I declare that: • I have complied with the Thesis Examination Procedure • where I have used a publication in lieu of a Chapter, the listed publication(s) below meet(s) the requirements to be included in the thesis. Name Signature Date (dd/mm/yy)

Hon Chee Chew 27/03/2019

dinator's Declaration ( -�

I declare that: • the information below is accurate • where listed publication(s) have been used in lieu of Chapter(s), their use complies with the Thesis Examination Procedure • the minimum requirements for the format of the thesis have been met. PGC's Name PGC's Signature Date (dd/mm/yy) �'o(03 1°1 For each publication incorporated into the thesis in lieu of a Chapter, provide all of the requested details and signatures required

Details of publication #1: Full title: Primary Graft Dysfunction After Heart Transplantation Authors: Hong Chee Chew & Gayathri Kumarasinghe & Arjun Iyer & Mark Hicks & Ling Gao & Aoife Doyle & Andrew Jabbour & Kumud Dhital & Emily Granger & Paul Jansz & Christopher Hayward & Anne Keogh & Eugene Kotlyar & Phillip Spratt & Peter Macdonald Journal or book name: Current Transplantation Reports Volume/page numbers: 1:257-265 Date accepted/ published: 4/9/2014 Status Published x Accepted and In In progress press (submitted) The Candidate’s Contribution to the Work I wrote the paper. Location of the work in the thesis and/or how the work is incorporated in the thesis: This is a literature review related to the thesis. Primary Supervisor’s Declaration I declare that: • the information above is accurate • this has been discussed with the PGC and it is agreed that this publication can be included in this thesis in lieu of a Chapter • All of the co-authors of the publication have reviewed the above information and have agreed to its veracity by signing a ‘Co-Author Authorisation’ form. Supervisor’s name Supervisor’s signature Date (dd/mm/yy) Peter Macdonald 28/03/2019

Details of publication #2: Full title: The Donor Heart and Organ Perfusion Technology Authors: Hong Chee Chew, Peter s Macdonald, Kumud k Dhital Journal or book name: Journal of Thoracic Disease Volume/page numbers: Vol11, Supplement 6: 938-945 Date accepted/ published: 22/01/2019 Status Published x Accepted and In In progress press (submitted) The Candidate’s Contribution to the Work Wrote the paper Location of the work in the thesis and/or how the work is incorporated in the thesis: A review of organ perfusion technology which is used in the Clinical and Large animal studies in this thesis Primary Supervisor’s Declaration I declare that: • the information above is accurate • this has been discussed with the PGC and it is agreed that this publication can be included in this thesis in lieu of a Chapter • All of the co-authors of the publication have reviewed the above information and have agreed to its veracity by signing a ‘Co-Author Authorisation’ form. Supervisor’s name Supervisor’s signature Date (dd/mm/yy) Peter Macdonald 28/03/2019

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ii Details of publication #3: Full title: Pathophysiological Trends During Withdrawal of Life Support: Implications for Organ Donation After Circulatory Death Authors: Arjun Iyer, Hong Chee Chew, Ling Gao, Jeanette Villanueva, Mark Hicks, Aoife Doyle, Gayathri Kumarasinghe, Andrew Jabbour, Paul Cassius Jansz, Michael P. Feneley, Richard P. Harvey, Robert M. Graham, Kumud K. Dhital, and Peter S. Macdonald Journal or book name: Transplantation Volume/page numbers: 100:2621-2629 Date accepted/ published: Status Published x Accepted and In In progress press (submitted) The Candidate’s Contribution to the Work Performed experiments for series 2, including analysis of data and writing up of the segment. Location of the work in the thesis and/or how the work is incorporated in the thesis: Victor Chang Cardiac Research Institute, Transplantation Laboratory. This forms the basis for understanding of the physiology during DCD. Primary Supervisor’s Declaration I declare that: • the information above is accurate • this has been discussed with the PGC and it is agreed that this publication can be included in this thesis in lieu of a Chapter • All of the co-authors of the publication have reviewed the above information and have agreed to its veracity by signing a ‘Co-Author Authorisation’ form. Supervisor’s name Supervisor’s signature Date (dd/mm/yy) Peter Macdonald 28/03/2019

Details of publication #4: Full title: Donation after Circulatory Death (DCD) Heart Transplantation: Update of Current Experience and Outcomes in Australia Authors: Hong Chee CHEW, Arjun IYER, Mark CONNELLAN, Sarah SCHEUER, Jeanette VILLANUEVA, Ling GAO, Aoife DOYLE, Mark HICKS, Claudio SOTO, Andrew DINALE, Priya NAIR, Alasdair WATSON, Emily GRANGER, Paul JANSZ, Kavitha MUTHIAH, Andrew JABBOUR, Eugene KOTLYAR, Anne KEOGH, Chris HAYWARD, Robert GRAHAM, Phillip SPRATT, Peter MACDONALD, Kumud DHITAL Journal or book name: Journal of the American College of Cardiology Volume/page numbers: 73 Date accepted/ published: April 2019 Status Published X Accepted and In In progress press (submitted) The Candidate’s Contribution to the Work Performed the investigations, data management, data analysis and writeup of the paper Location of the work in the thesis and/or how the work is incorporated in the thesis: Heart Lung Clinic – this is the clinical aspect of the thesis for DCD heart transplantation Primary Supervisor’s Declaration I declare that: • the information above is accurate • this has been discussed with the PGC and it is agreed that this publication can be included in this thesis in lieu of a Chapter • All of the co-authors of the publication have reviewed the above information and have agreed to its veracity by signing a ‘Co-Author Authorisation’ form. Supervisor’s name Supervisor’s signature Date (dd/mm/yy) Peter Macdonald 28/03/2019

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iii Details of publication #5: Full title: Ex-Vivo Perfusion of Donor Hearts: The Feasibility of Banked Blood for Normothermic Machine Perfusion Authors: Hong Chee CHEW, Ling GAO, Jeanette VILLANUEVA, Aoife DOYLE, Mark HICKS, Andrew JABBOUR, Kumud K DHITAL, Peter S MACDONALD Journal or book name: OBM Transplantation Volume/page numbers: 3 (10.21926/obm.transplant.1902067) Date accepted/ published: 03/19 Status Published x Accepted and In In progress press (submitted) The Candidate’s Contribution to the Work Performed the experiments, data analysis and writeup of the paper Location of the work in the thesis and/or how the work is incorporated in the thesis: Victor Chang Cardiac Research Institute – Transplantation lab Primary Supervisor’s Declaration I declare that: • the information above is accurate • this has been discussed with the PGC and it is agreed that this publication can be included in this thesis in lieu of a Chapter • All of the co-authors of the publication have reviewed the above information and have agreed to its veracity by signing a ‘Co-Author Authorisation’ form. Supervisor’s name Supervisor’s signature Date (dd/mm/yy) Peter Macdonald 28/03/2019

Details of publication #6: Full title: Banked Blood for Normothermic Machine Perfusion of the Donor Heart: A Clinical Perspective Authors: H Chew, S Scheuer, K Dhital, P Macdonald Journal or book name: Journal of Heart and Lung Transplant Volume/page numbers: Date accepted/ published: Status Published Accepted and In In progress x press (submitted) The Candidate’s Contribution to the Work Performed the retrieval, and clinical research of the cases presented, and did the writeup. Location of the work in the thesis and/or how the work is incorporated in the thesis: St Vincent’s Hospital Primary Supervisor’s Declaration I declare that: • the information above is accurate • this has been discussed with the PGC and it is agreed that this publication can be included in this thesis in lieu of a Chapter • All of the co-authors of the publication have reviewed the above information and have agreed to its veracity by signing a ‘Co-Author Authorisation’ form. Supervisor’s name Supervisor’s signature Date (dd/mm/yy) Peter Macdonald 28/03/2019

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iv Details of publication #7: Full title: Ischaemia-Reperfusion Injury and Cardiac Preservation in a Donation after Circulatory Death (DCD) Rodent Model: Impact of Ante-mortem Heparin Authors: Hong Chee CHEW, Ling GAO, Jeanette VILLANUEVA, Aoife DOYLE, Mark HICKS, Andrew JABBOUR, Kumud K DHITAL, Peter S MACDONALD Journal or book name: Transplantation Direct Volume/page numbers: Date accepted/ published: Status Published Accepted and In In progress x press (submitted) The Candidate’s Contribution to the Work Performed and designed the experiments, data analysis and write up Location of the work in the thesis and/or how the work is incorporated in the thesis: Victor Chang Cardiac Research Institute – Rat Transplant Lab Primary Supervisor’s Declaration I declare that: • the information above is accurate • this has been discussed with the PGC and it is agreed that this publication can be included in this thesis in lieu of a Chapter • All of the co-authors of the publication have reviewed the above information and have agreed to its veracity by signing a ‘Co-Author Authorisation’ form. Supervisor’s name Supervisor’s signature Date (dd/mm/yy) Peter Macdonald 28/03/2019

Details of publication #8: Full title: Ischaemic Tolerance: The Effect of Ageing on Myocardial Susceptibility to Ischaemia in a Donation after Circulatory Death Rodent Model Authors: Hong Chee CHEW, Ling GAO, Jeanette VILLANUEVA, Aoife DOYLE, Mark HICKS, Andrew JABBOUR, Kumud K DHITAL, Peter S MACDONALD Journal or book name: Journal of Heart and Lung Transplant Volume/page numbers: Date accepted/ published: Status Published Accepted and In In progress x press (submitted) The Candidate’s Contribution to the Work Performed and designed the experiments, data analysis and write up Location of the work in the thesis and/or how the work is incorporated in the thesis: Victor Chang Cardiac Research Institute – Rat Transplant Lab Primary Supervisor’s Declaration I declare that: • the information above is accurate • this has been discussed with the PGC and it is agreed that this publication can be included in this thesis in lieu of a Chapter • All of the co-authors of the publication have reviewed the above information and have agreed to its veracity by signing a ‘Co-Author Authorisation’ form. Supervisor’s name Supervisor’s signature Date (dd/mm/yy) Peter Macdonald 28/03/2019

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v Contents ORIGINALITY STATEMENT ...... III

ACKNOWLEDGEMENTS ...... VI

PRESENTATIONS ...... VII

Oral Presentations:...... VII Poster Presentation:...... X PUBLICATIONS ...... XI

PRIZES ...... XIII

SCHOLARSHIPS ...... XIII

Chapter 1 – Cardiac Transplantation: An Update of Current Views and Practises ...... 1

1.1 History of Cardiac Transplantation ...... 1

i. The Visionaries – Drawing Science from Fiction ...... 1 ii. The Pioneers – From Bench to Bedside ...... 3 1.2 Cardiac Transplantation – Where Are We Now? ...... 5

i. Indications for Cardiac Transplantation ...... 6 ii. Current Outcomes in Transplantation ...... 9 iii. Primary Graft Failure – the Physician’s Nightmare ...... 10 iv. Key Challenges in Cardiac Transplantation ...... 13 a. Donor Scarcity ...... 15 b. Marginality ...... 15 c. Rise of the Machines ...... 17 1.3 Cardiac Transplantation in Australasia ...... 19

i. Overview of Current Practices ...... 21 ii. Demographics of Cardiac Transplantation and Organ Donation ...... 22 1.4 Donation after Circulatory Death Donors ...... 23

Direct Procurement Protocol (DPP) ...... 25 Normothermic Regional Perfusion (NRP) ...... 26 Challenges for DCD Heart Transplant ...... 29

References ...... 30

Chapter 2-7: Publications ...... 34

Chapter 2 – Publication 1 and 2 ...... 37

Foreword ...... 38

Title 1: Primary Graft Dysfunction After Heart Transplantation ...... 41

Title 2: Donor Heart and Organ Perfusion Technology ...... 51

Chapter 3 – Publication 3 ...... 60

Foreword ...... 61

Title: Pathophysiological trends during withdrawal of life support: implications for organ donation after circulatory death (DCD) ...... 63

Chapter 4 – Publication 4 ...... 73

Foreword ...... 74

Title: Donation after Circulatory Death (DCD) Heart Transplantation: Update of Current Experience and Outcomes in Australia ...... 77

Chapter 5 – Publication 5 and 6 ...... 119

Foreword ...... 120

Title 1: Ex-Vivo Perfusion of Donor Hearts: The Feasibility of Banked Blood for Normothermic Machine Perfusion ...... 122

Title 2: Banked Blood for Normothermic Machine Perfusion of the Donor Heart: A Clinical Perspective ...... 146

Chapter 6 – Publication 7 ...... 151

Foreword ...... 152

Title: Ischaemia-Reperfusion Injury and Cardiac Preservation in a Donation after Circulatory Death (DCD) Rodent Model: Impact of Ante-mortem Heparin ...... 155

Chapter 7 – Publication 8 ...... 176

Foreword ...... 177

Title: Ischaemic Tolerance: The Effect of Ageing on Myocardial Susceptibility to Ischaemia in a Donation after Circulatory Death Rodent Model ...... 179

Chapter 8 – Discussion and Conclusion ...... 199

ORIGINALITY STATEMENT

‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no material previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis.

I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project’s design and conception or in style, presentation and linguistic expression is acknowledged.’

______

Hong Chee CHEW

BSc(med), MBBS (UNSW)

Masters of Surgery (USyd)

March 2019

III

‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation.

I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International.

I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted, I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.’

Hong Chee CHEW

March 2019

AUTHENTICITY STATEMENT

‘I certify that the library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’

Hong Chee CHEW

March 2019

ACKNOWLEDGEMENTS

‘It takes a village…’ - that is my sentiment at this juncture of my clinical and research career, for indeed there have been if not ‘a village’, a multitude of individuals that have encouraged, supported, and shaped my development both as a novice researcher and clinician; trained and developed my skills as an apprentice surgeon; and ultimately served as a role model and beacon for my future aspirations both in the field of surgery and clinical research.

First and foremost, I extend my sincere gratitude and admiration to my supervisor, Professor Peter Macdonald, for giving me the opportunity to participate in the phenomenal and ground breaking project; and for his enduring patience in providing guidance, support and encouragement throughout this process which were invaluable and greatly appreciated. His passion for work and scientific vigour is truly inspiring, and has impressed upon me to become a clinician scientist in the future.

Second, I would like to thank my co-supervisor, Assoc. Professor Kumud Dhital, for having the faith in my ability to perform the ‘sometimes’ complicated experiments; for his resourcefulness which over the years has taught me much; and for all the brilliant ideas that have led to this multifaceted project. I will always remember our conversations regarding ‘culinary exploits’.

In regards to ‘the village’, I would like to thank my laboratory partners including Dr Ling Gao, Dr Jeanette Villanueva, Dr Mark Hicks and Ms Aoife Doyle. None of the experiments would have been possible without their dedication of time and effort. I appreciate all the little conversations, including scientific discussions, that have made this endeavour both rewarding and enjoyable. To the many other members from the VCCRI family, who have made me feel at home and inspired me to take a positive step towards a fitter and healthier lifestyle. My predecessors, Dr Alaisdair Watson and Dr Arjun Iyer, who at various timepoints gave me pointers and advice based on their experience working in the laboratory, and upon whose research many of my projects are built upon.

PRESENTATIONS Oral Presentations:

“Donation after Circulatory Death (DCD) Heart Transplantation: Current Clinical Experience and Update”. The Transplantation Society Congress Meeting, Madrid, Spain, July 2018

“ECMO after Donation after Circulatory Death (DCD) Heart Transplantation”. The Transplantation Society Congress Meeting, Madrid, Spain, July 2018

“Donation after Circulatory Death (DCD) Heart Transplantation in Australia: An Update of Current Practices and Outcomes”. The International Society of Heart and Lung Transplantation Annual Scientific Meeting, Nice, France, April 2018

“Post-transplant Outcomes Following Recovery of Marginal Brain Dead and Donation after Circulatory Death Hearts Using Normothermic Machine Perfusion”. The Transplantation Society of Australia and New Zealand Annual Scientific Meeting, Brisbane, April 2017

“Marginal Criteria vs. Standard Criteria Brain Dead Donor Heart Transplant: A Retrospective Single Centre Study Comparing Outcomes after Cold Static Storage”. The Transplantation Society of Australia and New Zealand Annual Scientific Meeting, Brisbane, April 2017

“Donation after Circulatory Death Heart Transplant – Experience and Outcome in Australia”. The Transplantation Society of Australia and New Zealand Annual Scientific Meeting, Brisbane, April 2017

VII

“Outcomes After Warm Machine Perfusion Recovery of Marginal Brain Dead and Donation after Circulatory Death Heart Transplantation”. International Society of Heart and Lung Transplant Annual Scientific Meeting, San Diego, April 2017

“Banked Blood in the Recovery of Donation after Circulatory Death Hearts in a Porcine Model”. Australian and New Zealand Society of Cardiothoracic Surgeons Annual Scientific Meeting, Cairns, October 2016

“Effect of Ageing on Donation after Circulatory Death Hearts in a Rodent Model”. Australian and New Zealand Society of Cardiothoracic Surgeons Annual Scientific Meeting, Cairns, October 2016

“Metabolic Profile and Functional Outcomes when using Banked Blood in Donor Porcine Heart Machine Perfusion”. Cardiac Society of Australia and New Zealand Annual Scientific Meeting, Adelaide, August 2016

“Metabolic Profile of Donation after Circulatory Death Hearts during Reconditioning”. The Transplantation Society Annual Scientific Meeting, Hong Kong, August 2016

“Outcomes of Banked Blood Normothermic Machine Perfusion of Donation after Circulatory Death in a Porcine Model”. The Transplantation Society Annual Scientific Meeting, Hong Kong, August 2016

“Metabolic profile of DCD hearts during Reconditioning”. The Transplantation Society of Australia and New Zealand Annual Scientific Meeting, Sydney, March 2016

VIII

“Retrospective Single Centre Comparison of Outcomes Between Standard Criteria and Marginal Criteria Brain Dead Heart Transplant”. The Transplantation Society of Australia and New Zealand Annual Scientific Meeting, Sydney, March 2016

“Post Asystolic Ventricular Reanimation”. (Invited Talk). International DCD Symposium, Sydney, October 2015

“Characteristic Trends in DCD withdrawal”. The Transplantation Society of Australia and New Zealand Annual Scientific Meeting, Canberra, March 2015

“Supplemented Celsior Solution Provides Superior Protection of Rat Hearts During Extended Cold Storage Compared with AL Solution”. The Transplantation Society of Australia and New Zealand Annual Scientific Meeting, Canberra, March 2015

“Reviving the Heart for Transplantation”. (Invited Talk). Sydney Adventist Hospital Cardiac Symposium, Sydney, August 2014

Poster Presentation:

“Ageing and Ischaemic Tolerance in a Rodent Donation after Circulatory Death (DCD) Model’. The International Society of Heart and Lung Transplantation Annual Scientific Meeting, Nice, France, April 2018

“Effect of Ageing on Normothermic Recovery of DCD Hearts in a Rodent Model”. The Transplantation Society of Australia and New Zealand Annual Scientific Meeting, Brisbane, April 2017

“Supplemented Celsior and Peri-mortem Heparin Enhances Recovery of Rodent DCD Hearts”. Australian and New Zealand Society of Cardiothoracic Surgeons Annual Scientific Meeting, Cairns, October 2016

“Supplemented Celsior and Peri-mortem Heparin Enhances Recovery of Rodent DCD Hearts”. The Transplantation Society Annual Scientific Meeting, Hong Kong, August 2016

“Characteristic trends in DCD Withdrawal”. International Society of Heart and Lung Transplant Annual Scientific Meeting, Washington DC, April 2016

“Retrospective Single Centre Comparison of Outcomes Between Standard Criteria and Marginal Criteria Brain Dead Heart Transplant”. International Society of Heart and Lung Transplant Annual Scientific Meeting, Washington DC, April 2016

PUBLICATIONS

Donation after Circulatory Death Heart Transplantation. K Dhital, H Chew, P Macdonald. Curr Opin Organ Transplant 2017. April 4. (doi: 10.1097/MOT.0000000000000419)

Pathophysiological Trends During Withdrawal of Life Support: Implications for Organ Donation after Circulatory Death. A Iyer, H Chew, L Gao, J Villanueva, M Hicks, K Dhital, P Macdonald. Transplantation 2016 Dec; 100(12):2621-2629

Extracorporeal Heart Perfusion before Heart Transplantation: The Heart in a Box. P Macdonald, H Chew, M Connellan, K Dhital. Curr Opin Organ Transplant 2016 Jun; 21(3):336-42

Donation after Circulatory Death for Liver Transplantation: a Meta-analysis on the Location of Life Support Withdrawal Affecting Outcomes. Y Cao, S Shahrestani, H Chew, P Macdonald, K Dhital, H Pleass. Transplantation 2016 Jun; 100(7):1513-24

Adult Heart Transplantation with Distant Procurement and Ex-vivo Preservation of Donor Hearts after Circulatory Death: A Case Series. K Dhital, A Iyer, M Connellan, H Chew, P Spratt, P Macdonald. Lancet 2015 Jun 27; 385(9987):2585- 91

Primary Graft Dysfunction After Heart Transplantation. H Chew, G Kumarasinghe, A Iyer, P Macdonald. Curr Transpl Rep 2014 Dec; 1:257-265

Size and Gender Matching in Heart Transplantation – Optimizing Donor Utilisation in an Era of Changing Donor and Recipient Characteristics. W Ziaziaris, H Chew, K Dhital, P Macdonald. Curr Transpl Rep 2014 Dec; 1:266-272

Improved Heart Function from Older Donors using Pharmacologic Conditioning Strategies. G Kumarasinghe, L Gao, M Hicks, J Villanueva, A Doyle, P Rao, M Ru, A Jabbour, A Iyer, H Chew, C Hayward, P Macdonald. JHLT 2016May; 35(5):636-

XII

PRIZES

1. The Transplantation Society Mentor-Mentee Award: TTS Congress, 2018 2. The Transplantation Society of Australia and New Zealand Travel Award: TSANZ 2017 3. International Society of Heart and Lung Transplantation Travel Grant: ISHLT 2017 4. The Transplantation Society of Australia and New Zealand Young Investigators Award: TSANZ 2017 5. University of New South Wales Postgraduate Research Scholarship: UNSW 2016 6. The Transplantation Society of Australia and New Zealand Young Investigators Award: TSANZ 2015

SCHOLARSHIPS

1. National Health and Medical Research Council: Australian Postgraduate Award (APA), 2014-2017 2. Royal Australian College of Surgeons: Eric Bishop Research Scholarship, 2016

XIII

Chapter 1 – Cardiac Transplantation: An Update of Current Views and Practises

1.1 History of Cardiac Transplantation

Heart transplantation has been performed for over 50 years, with improving

survival outcomes and longevity of the cardiac allograft. It is the gold

standard treatment of end-stage heart failure, giving patients a chance to a

new and better life. Today, over 4000 hearts are transplanted around the

world annually.

Despite its excellent outcome in the treatment of an otherwise fatal disease,

the history of heart transplant was not unmarred by failures during its

infancy. This was the result of limited understanding of acute graft rejection

and the lack of an effective immunosuppressive therapy. However, the

persistence of a few, and the discovery of Cyclosporin ultimately led to the

success witnessed today.

i. The Visionaries – Drawing Science from Fiction

Early evidence of transplantation dates back to 2000 years ago with the use

of autologous skin flaps as described by Celsus in Graeco-Roman medical

literature1. However, tissue or organ transplantation were largely

unsuccessful until circa 1900 through animal experiments by Dr Alexis Carrel,

who received a Nobel Prize in 1912 for his work in transplantation2, 3. Whilst

he was successful in ‘translocating’ organs using anastomotic techniques he devised, there was less success in transplanting donor allografts into recipient animal. It was not until the beginning of 1940 when Sir Peter

Medawar, studied the effect of the immune system on the transplanted tissue2, 4.

In relation to cardiac transplantation, the first two reported heterotopic heart transplants were published by Mann’s group from the Mayo Clinic5.

The first orthotopic transplantation of the heart was performed by

Demikhov in 1951, following which he performed twenty two similar experiments in canines with two surviving with good cardiac function for periods of 11.5 and 15.5 hours6. This was a feat, as the procedure was performed in the absence of hypothermia and without the use of cardiac bypass.

Advancement in technology in the field of heart surgery with the use of hypothermia and the heart lung machine propelled experimental work on heart transplantation significantly. In 1953, Neptune et al. performed canine orthotopic heart and lung transplant with hypothermia and was able to maintain full body circulation by the transplanted heart for up to 6 hours7.

In 1958, Goldberg et al. with the use of cardiopulmonary bypass, performed

3 orthotopic heart transplants and was able to sustain circulation off-bypass for periods between 20 minutes to two hours8.

By the 1960’s, heart transplantation had been shown to be achievable, and the first series of completely successful heart transplants was reported by

Lower and Shumway9 from the Stanford group. Further works from the same group laid the foundation of myocardial preservation, surgical techniques as well as the diagnosis and treatment of acute rejection; by the mid 1960’s, the procedure was primed for clinical translation. ii. The Pioneers – From Bench to Bedside

By 1963, a team from the University of Mississippi, was the first to undergo training for clinical heart transplantation10. In 1964, Dr James Hardy performed the first xenotransplantation using the heart of a chimpanzee.

This was a last resort, as the potential donor was unlikely to progress while the recipient was at death’s door. Despite the surgical success of the procedure, the inability of the donor heart to support the recipient circulation manifested after an hour off-bypass, resulting in death of the recipient.

The first human heart transplantation was performed by Dr Christiaan

Barnard on 3 December 1967 at Groote Schuur Hospital in Cape Town, South

Africa11. The recipient, a 54-year-old man with ischaemic cardiomyopathy,

was successfully transplanted using the heart from a 25-year-old female donor who had sustained irreversible brain injury from a motor vehicle accident. As laws pertaining to brain death had not been established, the donor was withdrawn from ventilatory support in an adjacent operating theatre prepared for retrieval procedure. Upon confirmation of death, the chest was rapidly opened, and pump-oxygenator support was initiated. The heart was cooled to a low temperature and excised. The recipient underwent concurrent surgery for removal of the heart and the donor heart implanted. The recipient survived the operation with excellent early recovery, but eventually succumbed to severe pneumonia and septicaemia

18 days later.

Three days following the first transplantation, Dr. Kantrowitz transplanted the heart of a two day old anencephalic neonate into a 17 day old baby with

Ebstein’s anomaly, in Brooklyn, New York12. However, the baby developed metabolic and respiratory acidosis resulting in death seven hours later despite resuscitative measures.

The Stanford group performed their first heart transplant on 6 January

196813. The operation was successful despite technical complexities, but the recipient’s recovery was complicated by two laparotomies secondary to

cholecystitis and upper gastrointestinal tract bleeding. The patient died of

gram negative sepsis on day 15 post-transplantation.

Following these reports, many units began performing cardiac

transplantation. A staggering 102 heart transplants were performed in 1968

following Barnard’s initial success3, 14. Predictably, the inexperienced centres

were associated with poor outcome and by 1970, a mere 18 transplants

were performed worldwide. The main issue stemmed from the poor

understanding of rejection, and the ability to diagnose and treat it. By the

early 1970s, Barnard and Shumway were the only groups that continued

with their heart transplant program.

1.2 Cardiac Transplantation – Where Are We Now?

The relative poor outcomes in the late 1960’s and early 1970’s reported

survival of 42% at 6 months and 26% at 2 years15. This was largely due to

inexperienced centres undertaking transplantation, poor donor recipient

selection, and delayed recognition and treatment of acute rejection.

However, the growing experience and further research led to improving

outcomes and better diagnosis of graft rejection.

In 1973, the Stanford group formulated a new technique, percutaneous

endomyocardial biopsy, to diagnose rejection16. By using histological

evidence from myocardium in concert with signs and symptoms of rejection,

immunosuppressive medication could be adjusted. This resulted in further improvements in patient survival17.

In 1976, J.F. Borel reported the immunosuppressive effects of cyclosporin

A18 which later led to the introduction of cyclosporin A into clinical practise by 1980. The superior immunosuppressant resulted in survival outcomes similar to current levels of acceptance and success. This has led to the revival of world-wide interest in this procedure. i. Indications for Cardiac Transplantation

Today, cardiac transplantation remains the gold standard treatment for end- stage heart failure (HF) refractory to optimal medical treatment19, providing improvements in both survival and quality of life of its recipient. It is reported up to 90% of cardiac recipients return to New York Heart

Association (NYHA) class 1 function post transplantation20 with a survival rate of ~50% at 12 years21.

Current indications for cardiac transplantation include all patients with advanced HF that have failed to improve despite optimal medical treatment and cardiac resynchronisation therapy22, 23. Any surgically amenable cardiac disease should also be addressed prior to consideration for transplantation.

Furthermore, patients with NYHA class IV HF should be evaluated by specialised HF team for management of multiorgan failure and assess

reversibility before listing for cardiac transplant24. Table 1 and 2 outlines the key indications and contraindications for transplantation.

Table 1. Indications for Cardiac Transplantation25, 26

AHA ESC 1. Cardiogenic Shock 1. Severe symptoms • Requiring continuous IV inotropic • Dyspnoea at rest or minimal exertion therapy • NYHA III/IV • Requiring IABP or LVAD 2. VO2max < 10ml/kg/min 3. NYHA III /IV despite maximal 2. Evidence of severe cardiac dysfunction: medical and resynchronisation • LVEF < 30% therapy • high LV/RV filling pressure impacting 4. Recurrent LV arrhythmias despite functional capacity implantable cardiac defibrillator, o 6min walking test < 300m anti-arrhythmic therapy, or catheter o VO2max <12-14ml/kg/min ablation • Pseudo-normal or restrictive mitral 5. End stage congenital HF with no inflow pattern on doppler evidence of pulmonary echocardiography hypertension 6. Refractory angina without medical 3. One or more hospitalisations for HF in 6 or surgical treatment options months

Table 2. Contra-indications for Cardiac Transplantation21

1. Irreversible pulmonary hypertension a. Unsuccessful vasodilator challenge during right heart catheterisation when PASP>50mmHg, TPG>15mmHg or PVR>3Wood units b. Failure of LV unloading using mechanical adjuncts, including IABP and/or LVAD 2. Age a. Carefully selected patients > 70years may be considered 3. BMI a. BMI > 35kg/m2 (recommend weight loss before listing) 4. Pre-existing neoplasms a. Cardiac transplantation considered if: i. Tumour recurrence low (based on tumour type) ii. Responsiveness to therapy iii. No metastatic disease (decision based on collaboration with oncologist) 5. Diabetes with end-organ damage a. Poor glycaemic control (HbA1c > 7.5%) despite optimal effort b. Irreversible renal dysfunction (eGFR < 20ml/min/1.73m2) c. Clinically severe cerebrovascular disease d. Peripheral vascular disease not amenable to revascularisation 6. Evidence of frailty 7. Active smoking or substance abuse despite rehabilitative efforts 8. Inadequate psychosocial structure a. History of non-compliance b. Inadequate support systems available c. Severe cognitive behavioural disability or dementia

ii. Current Outcomes in Transplantation

Since 1967, more than 113,000 adult heart transplants have been performed, based on data submitted to the International Society of Heart and Lung

Transplantation (ISHLT). Data presented in this section is based on a recent report on adult heart transplantation published in 201627.

Pre-transplant diagnosis remains largely unchanged, when compared to previous decades, with idiopathic dilated cardiomyopathy (NICM) accounting for 49% of all patients requiring cardiac transplantation, followed by ischaemic cardiomyopathy (ICM = 35%). These trends are largely similar when broken down by geographical location, with higher rates of ICM in developed countries. Furthermore, NICM accounts for more than 50% in the

18-59-year group, whilst ICM is most prevalent in the 60+ patient group.

Recipients are more likely to be on mechanical circulatory support with LVAD

(38.1% vs. 22.2%) and ECMO (1.3% vs. 0.9%) in the current period (2009-

2015) when compared to previous (2004-2008).

Baseline characteristics of donors and recipients have seen a trend towards older donors and significantly higher BMI in both donor and recipients in the recent period (2009-2015). There are significantly higher rates of recipient/donor diabetes and hypertension, but lower smoking history in

both recipient/donor in the current period. The most common cause of death in donors was traumatic head injury (42.1%) and CVA (22.6%).

Overall survival post-cardiac transplantation has improved significantly from median survival of 8.5 years (circa 1982-1991) to 11.9 years (circa 2002-

2008). Current data shows 1- and 5-year survival of 85% and 73%, with improved long term survival in recipients who are younger, male, without a history of diabetes, COPD or smoking, and whose pre-transplant diagnosis is related to congenital heart disease. Surprisingly, the use of LVAD was not associated with worse survival outcomes, but ECMO requirement pre- transplant was associated with a significantly higher early mortality post- transplant (88% vs. 57% at 1 year).

The most common cause for early mortality post-transplant is primary graft failure (40%), followed by multiple organ failure (17.8%) and infection

(13.4%). Acute rejection is most prevalent between 1-3 years (10%) post transplantation and decreases overtime. Risk for cardiac allograft vasculopathy is highest between 3-10 years post-transplant while risk of malignancy increases overtime. iii. Primary Graft Failure – the Physician’s Nightmare

One of the considerable risks associated with transplantation is primary graft dysfunction (PGD) in the immediate post-operative period. PGD occurs when

the transplanted heart fails to meet the circulatory requirement in the recipient. This may be due to single or biventricular failure of the transplanted allograft28. Despite growing experience in donor heart management, refined surgical techniques and improving technology, incidence of PGD is reported up to 30% of cases29 and is associated with a

30% one month mortality and 34.6% at one year30.

Based on current understanding, an interplay between a combination of donor, recipient and procedural risk factors result in an additive effect leading to the development of PGD. The donor heart is subjected to a series of insults, beginning from the process of brain death and its sequelae, the process of transportation in cold static storage (CS), a variable period of warm ischaemia during the process of implantation, and reperfusion injury upon re-establishment of circulation in the recipient. The effect of these insults varies between donor hearts and is dependent on the ischaemic tolerance of the organ. Appropriate organ management including preservation of donor heart, minimising ischaemic time, careful recipient selection and post-implantation management are crucial in mitigating the development of PGD.

Despite its common occurrence, the diagnosis of PGD is often difficult and confusing. This is due to the lack of a uniform definition of PGD, which potentially delays the diagnosis and management in the outset. Recent studies have suggested that early treatment of PGD and institution of ECMO is associated with improved outcome.

As outlined in the 2014 ISHLT Consensus regarding PGD, there are widely variable definitions of PGD adopted by different centres. These differences have led to difficulties in determining the true incidence of PGD post- transplant, and the substantial variations in outcome post PGD. The current accepted definition of PGD is presented in table 3.

PGD, which was previously not survivable without re-transplantation, is associated with increased mortality despite short term support with VAD and/or ECMO. Several retrospective studies have shown up to 60% mortality in patients with PGD at 1 year, with worst survival for those on mechanical support other than ECMO31-34. Furthermore, early institution of ECMO has been associated with improved survival, with no increase late mortality observed in current reports32, 35, 36.

Table 3. Classification and Grading of Graft Dysfunction

Primary Graft Dysfunction (PGD) Diagnosis to be made within 24 hours after completion of cardiac transplantation • LVEF ≤40% • Haemodynamics with

o RAP > 15mmHg Mild o PCWP > 20mmHg 2 o CI < 2.0L/min/m (more than 1 hour) requiring low dose inotropes PGD-LV • One criteria from above plus one of the following: Moderate • High-dose inotropes – inotrope scorea >10 • Newly placed IABP • Dependence of left or biventricular mechanical Severe support (i.e. ECMO, LVAD or BiVAD) • Haemodynamics with

o RAP > 15mmHg o PCWP < 15mmHg 2 o CI < 2.0L/min/m PGD-RV plus • TPG < 15mmHg and/or PASP < 50mmHg Or • Need for RVAD

Secondary Graft Dysfunction: If a discernible cause for graft dysfunction (e.g. hyper-acute rejection, pulmonary hypertension, or known surgical complication) is identified. a Inotrope score = dopamine (x1) + dobutamine (x1) + amrinone (x1) + milrinone (x15) + adrenaline(x100) + noradrenaline (x100) with each drug dose in µg/kg/min iv. Key Challenges in Cardiac Transplantation

Over the past 50 years, cardiac transplantation has undergone significant changes – from improved surgical techniques and perioperative mechanical

support technologies, to better recipient selection and perioperative management. These have resulted in the outstanding short- and long-term survival today.

Despite rising rates of heart transplantation today, and with more than 5000 heart transplants performed annually worldwide, the burden of heart failure remains high. This is in part due to the improvement in medical therapy and mechanical circulatory support strategies that are allowing these patients to survive longer and towards transplantation. This has resulted in long waiting lists in many transplant units, as the supply of donor hearts has remained stable, despite an exponential rise in heart failure patients.

According to registry data from the United Network for Organ Sharing (UNOS; http://unos.org) and Organ Procurement and Transplantation Network

(OPTN; https://optn.transplant.hrsa.gov), in 2018 there were 3467 heart procurements in the USA, but only 3408 heart transplants performed. This pales in comparison to the waitlist which has 3814 registered recipients in

2019. Median wait time has increased from 43-322days between 2003-2006 to 70-533 days between 2011-2014 (data presented reflects wait time by

ABO blood group, with AB having shortest wait-time and O having the longest wait-time).

Furthermore, data from 2018 indicates that 4.9% of patients died whilst on waiting list and 6.8% patients were removed from the wait-list due to deterioration of medical condition. Therefore, despite rising donor numbers

(3467 in 2018 vs. 2222 in 2008), there is still a shortage of donor hearts to meet current needs.

a. Donor Scarcity

One of the biggest challenges within the field is the availability of

heart donors. As mentioned above, donor scarcity remains a major

issue in heart transplantation. According to Khush et al, only 32%

of all heart donations were accepted in 201037. Donor rates have

remained stable in the early 2000s with a 15% rise in donor

numbers in 2010. Despite the modest increase in heart donors,

wait-list time is increasing, and transplant rates have dropped from

52-77% to 45-75% at 1 year (based on recipient ABO)*. This

represents the disproportionate rise of recipient to heart donors

globally.

b. Marginality

The introduction of new donor groups to address donor scarcity has

led to the inclusion of marginal criteria (MBD), and circulatory

death donors (DCD) more recently. Whilst DCD heart

transplantation is only performed in selected centres in the Europe and Australia, in the USA, the introduction of MBD or extended criteria heart donors has shown a positive trend in overall donor numbers in more recent years with increasing evidence showing comparable short and midterm survival in the MBD cohort38, 39.

Table 4 shows the current MBD criteria.

Table 4: Comparing Traditional vs. Extended Criteria DBD Heart Donors38, 40

MBD DBD (extended criteria)

Age (years) ≤55 >55

Ischaemic time (hours) <4 >4

LV ejection fraction (%) >50 40-50%

Inotropic support High (variable*)

Serology Negative Hepatitis B/C

c. Rise of the Machines

Furthermore, continued research into better alternatives for

preserving donor heart has led to the introduction of ex-situ

perfusion technology. Today, one of such devices is being utilised

in the clinical setting – the Transmedics OCS Heart™, which allows

the reanimation of arrested hearts using normothermic machine

perfusion (NMP). Steen et al. has also recently commenced a

clinical trial using hypothermic machine perfusion (HMP) to

resuscitate and bridge donor hearts to transplantation41. Whilst the

long-term outcome of these new technologies are currently being

investigated, to date NMP has shown non-inferiority when compared to cold static storage (CSS)42 but with the potential for organ recovery through a combination of physiological manipulation and pharmacotherapy.

Ultimately, the role of machine perfusion in today’s landscape of heart transplantation is to provide assurance and confidence of a viable donor heart in an increasingly older and ‘higher-risk’ (i.e.

MBD and DCD) donors.

i. Marginal Brain Dead (MBD)

Extended criteria or MBD has been introduced since 2000.

This group of donors is categorised based on older age,

borderline cardiac function on echocardiography, high

inotropic requirement and prolonged ischaemic time (see

table 4). Current outcomes are based on single centre

reports and show comparable survival to standard DBD38, 39.

However, criteria for marginality differed between individual

centres, which accounts for the variability in reported

survival between 70-93%.

The EXPAND trial, a multicentre study utilising the OCS for

marginal donor hearts that are discarded based on current

donor criteria, is underway. The trial is set for completion in

2019.

ii. Donation after Circulatory Death (DCD)

DCD heart transplantation was first performed in St Vincent’s

Hospital, Sydney in July 2014 by Dhital et al.43 The heart was

retrieved and recovered using the Transmedic OCS Heart.

Today, DCD heart transplants are performed in 5 centres in

Europe, and one unit in Australia. To date, DCD hearts are

not performed in the USA despite the use of DCD lungs being

widely accepted practice.

1.3 Cardiac Transplantation in Australasia

The relative success of heart transplantation led to the rapid adoption of this

new surgery around the world. By 23 October 1968, St Vincent’s Hospital in

Sydney was poised to perform the first heart transplant in Australia. The

recipient, a 57 year old man with ‘gross and irreversible myocardial disease

with intractable congestive cardiac failure’44 would proceed to survive the

procedure for 45 days with good cardiac function but died from an aortic

perforation around the anastomotic site.45 The second heart transplant was

performed in Melbourne twenty days later, but the recipient did not survive

the operation. Detailed publication of the first case discussed considerations

and hurdles to heart transplantation, including the ethical discussion of death, donor and recipient operation and management of rejection outlined the important barriers that placed a halt in heart transplant activity for more than a decade.

In 1974, St Vincent’s Hospital performed a second heart transplantation, with the recipient surviving for 62 days before succumbing to infection, following which the heart transplant program was voluntarily suspended until 198446.

By 1984, with improving outcomes, the team led by Dr Victor Chang at St

Vincent’s Hospital were ready to recommence the heart transplant program.

On 8 April 1984, the first heart transplant in the modern clinical era was performed in a 14 year old girl with viral cardiomyopathy46. Despite issues with graft rejection leading to re-transplantation in 1986, the girl would become the second longest surviving heart recipient 33 years down the track.

The unit remained the only transplant unit in Australasia between 1984 –

1987, until the establishment of a heart transplant program in Auckland,

New Zealand in 1987 followed by a paediatric heart transplant service in the

Royal Children’s Hospital, Melbourne in April 198847. In 1989, a second

Australian adult heart transplant unit was established at the Alfred Hospital,

Melbourne. Today, there are four adult heart transplant units across

Australia, performing close to 100 cardiac transplants a year.

i. Overview of Current Practices

Currently, both DBD and DCD donor hearts are accepted for heart transplantation. DBD including marginal criteria donors are routinely procured and preserved using cold static storage, and occasionally utilising

NMP for MBD hearts.

All DCD hearts are currently procured using NMP and will be discussed in detail in Chapter 4. Donor criteria are presented in table 5.

Table 5: Donor Criteria in NSW DBD MBD DCD

Age (years) ≤55 ≤65 ≤55

Ischaemic Time 4-6hours >4-6hours WIT* ≤30mins

LV Ejection Fraction^ >50-60 >45# >45# pre-DCD (%)

High Risk – HBV/HCV No Yes Yes

Preservation CSS CSS/NMP NMP

*WIT (Warm ischaemic time) commences from the time donor systolic blood pressure <90mmHg until institution of preservation solution

^Left ventricular (LV) ejection fraction is measured after brain death

# Hormone resuscitation is performed using T3 infusion, repeat assessment with echocardiogram is routinely performed. In MBD/DCD utilising normothermic machine perfusion (NMP) assessment can be made during machine reperfusion.

ii. Demographics of Cardiac Transplantation and Organ Donation

Since 1984, transplant activity in Australia and New Zealand has been

steadily increasing with a record 129 heart transplants performed in 2016.

Despite these increases, the heart transplant waiting list remains

overwhelmed with only 61% of listed patients receiving a heart transplant.

Approximately 10% of patients are removed from the waiting list due to

further deterioration or death.

Donors and recipients are older, with a mean donor age of 33 years and

recipient age of 45 years. The mean waiting time from listing to

transplantation is 156 days, with idiopathic dilated cardiomyopathy being

the most common pre-transplant diagnosis, followed by ischaemic heart

disease. These trends are in keeping with international data.

Since the introduction of DCD, there have been an increase in overall

donation rate and in 2018 DCD accounts for 28% of all deceased donor organ

donation.

*Data courtesy of Australian and New Zealand Cardiothoracic Organ Transplant Registry

(ANZCOTR) and International Registry of Organ Donation and Transplantation (IRODaT).

1.4 Donation after Circulatory Death Donors

The early transplantations performed by the pioneering fathers of cardiac

transplantation were technically circulatory death transplantations. Donor

and recipient operation were performed in co-located operating rooms

where donor and recipient procedures were performed simultaneously. The

instant the donor heart stopped beating, the heart was topically cooled, and

the retrieval surgeon expediently explanted the heart, which is immediately

transplanted into the recipient in an adjacent operating theatre. It was not

until the 1968’s, with the introduction of brain death legislation, that beating hearts could be harvested in a controlled manner.

More recently, with the advances in device technology, organ perfusion therapy has seen increasing utility in general transplantation. In cardiac transplant, the Transmedics OCS Heart™ is the first clinically available organ perfusion system which allows the explanted heart to be reanimated and reperfused during transportation, eliminating the risk of hypothermic injury and a non-viable heart. Whilst initially used only in BD setting, there was increasing interest in introducing this technology in the DCD setting, with the rising numbers of DCD abdominal organ donations, to alleviate the issue of organ shortage. In 2014, after years of laboratory-based research, the group in St Vincent’s Hospital in Sydney performed the first distant procurement

DCD heart transplantation. The donor heart was reperfused for at least

90mins before the decision was made to proceed with the recipient operation. Whilst mechanical circulatory support (MCS) was required in the early recovery of the recipient, the transplanted heart demonstrated normal function within 1 week of transplantation, and the recipient remains well to date.

Following the success of the first DCD heart transplantation in the current era, two units in UK (Papworth and Harefield Hospital) also successfully performed DCD heart transplantation and to date more than 100 DCD heart transplantations have been performed across six transplant units across

Europe and in Australia. Current short to medium term survival times are comparable to BD heart transplantation. There are currently two techniques of DCD heart procurement.

Direct Procurement Protocol (DPP)

The direct procurement protocol (DPP) is the most commonly practiced procurement technique for DCD hearts. Following withdrawal from circulatory and respiratory support (WCRS) and progression to asystole, the donor is rapidly transferred to the operating theatre followed by expedient sternotomy. The right atrial appendage is cannulated for blood collection, during which the aortic root is cannulated and prepared for cross-clamping.

Upon collection of 1.2-1.5L of donor blood, the cross-clamp is applied and the cardioplegia is administered. The heart is vented via the pulmonary veins and the inferior vena cava.

On completion of the cardioplegic flush, the heart is carefully explanted and immersed in cold saline. The heart is prepared and instrumented on the back table for installation onto the OCS heart as per Transmedic’s. Following

establishment of perfusion, any arrhythmia is corrected and ventricular pacing wires are stitched on. Management of the heart on OCS by achieving target coronary flow (CF) and mean aortic pressure (MAP) through altering pump flow, and pharmacological intervention with adenosine or adrenaline.

The key parameter monitored during NMP is arterial and venous lactate.

Current recommendations suggest an overall declining lactate trend with evidence of venous extraction, achieving a target lactate <5mmol/L. The heart is also assessed visually to exclude any abnormality during NMP.

Upon reaching target parameters, the heart is arrested with cold cardioplegia and disconnected from NMP for implantation.

Normothermic Regional Perfusion (NRP)

The early phase of normothermic regional perfusion (NRP) is similar to DPP.

However, after confirmation of death, the donor is centrally cannulated for extracorporeal membrane oxygenation (VA-ECMO) and the heart is reanimated in the donor. The head and neck vessels are clamped to prevent cerebral perfusion. The heart is allowed to recover on full ECMO support, after which organ assessment is performed in-vivo by using a combination of biomarkers (i.e. troponin and lactate) and functional assessment.

Functional assessment is performed using transoesophageal

echocardiography (TOE) and with a Swan Ganz catheter for right and left

heart pressures. If the recovery of the heart is deemed adequate, the heart

is arrested in a controlled fashion, and the heart is instrumented and

installed onto NMP for transportation to the recipient hospital.

The key determinants influencing the DCD retrieval pathway are both ethical and legal. Australian legislations prohibit the re-establishment of donor circulation in-vivo. Therefore, NRP will never be an accepted practice here.

While proponents of NRP argue that the establishment of donor circulation using ECMO also supports other organs which are being retrieved; Local experience using DPP has not resulted in the abandonment of lung or abdominal organs during the retrieval.

Table 6: DCD Heart Retrieval Pathways

Challenges for DCD Heart Transplant

While current outcomes for DCD heart transplantation are excellent, there is still limited uptake of DCD programs worldwide. This is largely due to the lack of consensus on the management of these hearts during NMP, and the lack of objective measures to determine organ recovery.

The purpose of this thesis is to identify key factors impacting donor heart viability in a DCD setting, and to implement recommendations or practices to improve donor heart quality.

References 1. Hassoulas J. Heart transplantation: research that led to the first human transplant in 1967. S Afr Med J. 2011;101:97-101. 2. Doyle AM, Lechler RI and Turka LA. Organ transplantation: halfway through the first century. J Am Soc Nephrol. 2004;15:2965-71. 3. DiBardino DJ. The history and development of cardiac transplantation. Tex Heart Inst J. 1999;26:198-205. 4. Tilney N. Transplant: From Myth to Reality. New Haven: Yale University Press; 2003. 5. Mann FC, Priestley JT, Markowitz JJ and Yater WM. Transplantation of the intact mammalian heart. Archives of Surgery. 1933;26:219-224. 6. Cooper DK. Experimental development of cardiac transplantation. British Medical Journal. 1968;4:174-181. 7. Neptune WB, Cookson BA, Bailey CP, Appler R and Rajkowski F. Complete homologous heart transplantation. AMA Archives of Surgery. 1953;66:174-178. 8. Golberg M, Berman EF and Akman LC. Homologous transplantation of the canine heart. J Int Coll Surg. 1958;30:575-86. 9. Lower RR and Shumway NE. Studies on orthotopic homotransplantation of the canine heart. Surg Forum. 1960;11:18-9. 10. Hardy JD, Kurrus FD, Chavez CM, Neely WA, Eraslan S, Turner MD, Fabian LW and Labecki TD. Heart Transplantation in Man. Developmental Studies and Report of a Case. JAMA. 1964;188:1132-40. 11. Brink JG and Hassoulas J. The first human heart transplant and further advances in cardiac transplantation at Groote Schuur Hospital and the University of Cape Town. Cardiovascular Journal of Africa. 2009;20:31-35. 12. Kantrowitz A, Haller JD, Joos H, Cerruti MM and Carstensen HE. Transplantation of the heart in an infant and an adult. The American Journal of Cardiology. 1968;22:782-790. 13. Stinson EB, Dong E, Jr., Schroeder JS, Harrison DC and Shumway NE. Initial clinical experience with heart transplantation. Am J Cardiol. 1968;22:791-803. 14. Patterson C and Patterson KB. The history of heart transplantation. Am J Med Sci. 1997;314:190-7. 15. Griepp RB, Stinson EB, Dong E, Jr., Clark DA and Shumway NE. Acute Rejection of the Allografted Human Heart. The Annals of Thoracic Surgery. 12:113-126. 16. Caves PK, Stinson EB, Billingham M and Shumway NE. Percutaneous Transvenous Endomyocardial Biopsy in Human Heart Recipients. The Annals of Thoracic Surgery. 16:325-336. 17. Graham AF, Rider AK, Caves PK, Stinson EB, Harrison DC, Shumway NE and Schroeder JS. Acute Rejection in the Long-Term Cardiac Transplant Survivor. Circulation. 1974;49:361. 18. Borel JF. Comparative study of in vitro and in vivo drug effects on cell-mediated cytotoxicity. Immunology. 1976;31:631-641. 19. Metra M, Ponikowski P, Dickstein K, McMurray JJV, Gavazzi A, Bergh C-H, Fraser AG, Jaarsma T, Pitsis A, Mohacsi P, Böhm M, Anker S, Dargie H, Brutsaert D, Komajda M and on behalf of the Heart Failure Association of the European Society of C. Advanced chronic heart failure: A position statement from the Study Group on Advanced Heart Failure of the Heart Failure Association of the European Society of Cardiology. European Journal of Heart Failure. 2007;9:684-694. 20. Copeland JG. Advanced medical therapy does not render heart transplantation obsolete for ambulatory end-stage heart failure patients: a debate. J Heart Lung Transplant. 2001;20:725-8. 21. Mehra MR, Canter CE, Hannan MM, Semigran MJ, Uber PA, Baran DA, Danziger-Isakov L, Kirklin JK, Kirk R, Kushwaha SS, Lund LH, Potena L, Ross HJ, Taylor DO, Verschuuren EAM and Zuckermann A. The 2016 International Society for Heart Lung Transplantation listing criteria for heart transplantation: A 10-year update. The Journal of Heart and Lung Transplantation. 2016;35:1- 23.

22. Yancy CW, Jessup M, Bozkurt B, Butler J, Casey DE, Jr., Drazner MH, Fonarow GC, Geraci SA, Horwich T, Januzzi JL, Johnson MR, Kasper EK, Levy WC, Masoudi FA, McBride PE, McMurray JJ, Mitchell JE, Peterson PN, Riegel B, Sam F, Stevenson LW, Tang WH, Tsai EJ, Wilkoff BL, American College of Cardiology F and American Heart Association Task Force on Practice G. 2013 ACCF/AHA guideline for the management of heart failure: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. J Am Coll Cardiol. 2013;62:e147-239. 23. Yancy CW, Jessup M, Bozkurt B, Butler J, Casey DE, Jr., Colvin MM, Drazner MH, Filippatos GS, Fonarow GC, Givertz MM, Hollenberg SM, Lindenfeld J, Masoudi FA, McBride PE, Peterson PN, Stevenson LW and Westlake C. 2017 ACC/AHA/HFSA Focused Update of the 2013 ACCF/AHA Guideline for the Management of Heart Failure: A Report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines and the Heart Failure Society of America. J Am Coll Cardiol. 2017. 24. Alraies MC and Eckman P. Adult heart transplant: indications and outcomes. Journal of Thoracic Disease. 2014;6:1120-1128. 25. Ponikowski P, Voors AA, Anker SD, Bueno H, Cleland JGF, Coats AJS, Falk V, González- Juanatey JR, Harjola V-P, Jankowska EA, Jessup M, Linde C, Nihoyannopoulos P, Parissis JT, Pieske B, Riley JP, Rosano GMC, Ruilope LM, Ruschitzka F, Rutten FH, van der Meer P, Filippatos G, McMurray JJV, Aboyans V, Achenbach S, Agewall S, Al-Attar N, Atherton JJ, Bauersachs J, John Camm A, Carerj S, Ceconi C, Coca A, Elliott P, Erol Ç, Ezekowitz J, Fernández-Golfín C, Fitzsimons D, Guazzi M, Guenoun M, Hasenfuss G, Hindricks G, Hoes AW, Iung B, Jaarsma T, Kirchhof P, Knuuti J, Kolh P, Konstantinides S, Lainscak M, Lancellotti P, Lip GYH, Maisano F, Mueller C, Petrie MC, Piepoli MF, Priori SG, Torbicki A, Tsutsui H, van Veldhuisen DJ, Windecker S, Yancy C, Zamorano JL, Zamorano JL, Aboyans V, Achenbach S, Agewall S, Badimon L, Barón-Esquivias G, Baumgartner H, Bax JJ, Bueno H, Carerj S, Dean V, Erol Ç, Fitzsimons D, Gaemperli O, Kirchhof P, Kolh P, Lancellotti P, Lip GYH, Nihoyannopoulos P, Piepoli MF, Ponikowski P, Roffi M, Torbicki A, Vaz Carneiro A, Windecker S, Sisakian HS, Isayev E, Kurlianskaya A, Mullens W, Tokmakova M, Agathangelou P, Melenovsky V, Wiggers H, Hassanein M, Uuetoa T, Lommi J, Kostovska ES, Juillière Y, Aladashvili A, Luchner A, Chrysohoou C, Nyolczas N, Thorgeirsson G, Marc Weinstein J, Di Lenarda A, Aidargaliyeva N, Bajraktari G, Beishenkulov M, Kamzola G, Abdel-Massih T, Čelutkienė J, Noppe S, Cassar A, Vataman E, Abir-Khalil S, van Pol P, Mo R, Straburzyńska-Migaj E, Fonseca C, Chioncel O, Shlyakhto E, Otasevic P, Goncalvesová E, Lainscak M, Díaz Molina B, Schaufelberger M, Suter T, Yılmaz MB, Voronkov L and Davies C. 2016 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failureThe Task Force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC)Developed with the special contribution of the Heart Failure Association (HFA) of the ESC. European Heart Journal. 2016;37:2129-2200. 26. Jessup M, Abraham WT, Casey DE, Feldman AM, Francis GS, Ganiats TG, Konstam MA, Mancini DM, Rahko PS, Silver MA, Stevenson LW and Yancy CW. 2009 Focused Update: ACCF/AHA Guidelines for the Diagnosis and Management of Heart Failure in Adults. A Report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines: Developed in Collaboration With the International Society for Heart and Lung Transplantation. 2009;119:1977-2016. 27. Lund LH, Edwards LB, Dipchand AI, Goldfarb S, Kucheryavaya AY, Levvey BJ, Meiser B, Rossano JW, Yusen RD and Stehlik J. The Registry of the International Society for Heart and Lung Transplantation: Thirty-third Adult Heart Transplantation Report—2016; Focus Theme: Primary Diagnostic Indications for Transplant. The Journal of Heart and Lung Transplantation. 35:1158-1169. 28. Chew HC, Kumarasinghe G, Iyer A, Hicks M, Gao L, Doyle A, Jabbour A, Dhital K, Granger E, Jansz P, Hayward C, Keogh A, Kotlyar E, Spratt P and Macdonald P. Primary Graft Dysfunction After Heart Transplantation. Current Transplantation Reports. 2014;1:257-265.

29. Dronavalli VB, Rogers CA and Banner NR. Primary Cardiac Allograft Dysfunction—Validation of a Clinical Definition. Transplantation. 2015;99:1919-1925. 30. Kobashigawa J, Zuckermann A, Macdonald P, Leprince P, Esmailian F, Luu M, Mancini D, Patel J, Razi R, Reichenspurner H, Russell S, Segovia J, Smedira N, Stehlik J and Wagner F. Report from a consensus conference on primary graft dysfunction after cardiac transplantation. The Journal of Heart and Lung Transplantation. 33:327-340. 31. Thomas HL, Dronavalli VB, Parameshwar J, Bonser RS and Banner NR. Incidence and outcome of Levitronix CentriMag support as rescue therapy for early cardiac allograft failure: A United Kingdom national study. European Journal of Cardio-thoracic Surgery. 2011;40:1348-1354. 32. Taghavi S, Zuckermann A, Ankersmit J, Wieselthaler G, Rajek A, Laufer G, Wolner E and Grimm M. Extracorporeal Membrane Oxygenation is Superior to Right Ventricular Assist Device for Acute Right Ventricular Failure After Heart Transplantation. The Annals of Thoracic Surgery. 2004;78:1644-1649. 33. D’Alessandro C, Aubert S, Golmard JL, Praschker BL, Luyt CE, Pavie A, Gandjbakhch I and Leprince P. Extra-corporeal membrane oxygenation temporary support for early graft failure after cardiac transplantation☆. European Journal of Cardio-Thoracic Surgery. 2010;37:343-349. 34. D’Alessandro C, Golmard J-L, Barreda E, Laali M, Makris R, Luyt C-E, Leprince P and Pavie A. Predictive risk factors for primary graft failure requiring temporary extra-corporeal membrane oxygenation support after cardiac transplantation in adults☆. European Journal of Cardio-Thoracic Surgery. 2011;40:962-970. 35. D'Alessandro C, Aubert S, Golmard JL, Praschker BL, Luyt CE, Pavie A, Gandjbakhch I and Leprince P. Extra-corporeal membrane oxygenation temporary support for early graft failure after cardiac transplantation. European journal of cardio-thoracic surgery : official journal of the European Association for Cardio-thoracic Surgery. 2010;37:343-9. 36. Chou N-K, Chi N-H, Ko W-J, Yu H-Y, Huang S-C, Wang S-S, Lin F-Y, Chu S-H and Chen Y-S. Extracorporeal Membrane Oxygenation for Perioperative Cardiac Allograft Failure. ASAIO Journal. 2006;52:100-103. 37. Khush KK, Zaroff JG, Nguyen J, Menza R and Goldstein BA. National decline in donor heart utilization with regional variability: 1995-2010. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 2015;15:642-649. 38. Samsky M, Patel CB, Owen A, Schulte PJ, Jentzer J, Rosenberg PB, Felker GM, Milano CA, Hernandez AF and Rogers JG. Ten Year Experience with Extended Criteria Cardiac Transplantation. Circulation Heart failure. 2013;6:1230-1238. 39. Forni A, Mazzucco A, Chiominto B, Faggian G, Pilati M and Luciani GB. Impact of donor quality on outcome of heart transplantation☆, ☆☆. European Journal of Cardio-Thoracic Surgery. 2010;38:788-794. 40. Khush KKJAoCS. Donor selection in the modern era. 2017. 2017;7:126-134. 41. Nilsson J, Jernryd V, Qin G, Nozohoor S, Goncalves DC, Ragnarsson S, Paskevicius A, Johansson M, Warheim J, Hoglund P, Sjoberg T and Steen S. Non Ischemic Heart Preservation. J Heart Lung Transpl. 2018;37:S13-S13. 42. Ardehali A, Esmailian F, Deng M, Soltesz E, Hsich E, Naka Y, Mancini D, Camacho M, Zucker M, Leprince P, Padera R and Kobashigawa J. Ex-vivo perfusion of donor hearts for human heart transplantation (PROCEED II): a prospective, open-label, multicentre, randomised non-inferiority trial. The Lancet. 385:2577-2584. 43. Dhital KK, Iyer A, Connellan M, Chew HC, Gao L, Doyle A, Hicks M, Kumarasinghe G, Soto C, Dinale A, Cartwright B, Nair P, Granger E, Jansz P, Jabbour A, Kotlyar E, Keogh A, Hayward C, Graham R, Spratt P and Macdonald P. Adult heart transplantation with distant procurement and ex-vivo preservation of donor hearts after circulatory death: a case series. Lancet. 2015;385:2585-91. 44. Gibson H, Seldon WA and Windsor HM. A heart transplantation. 1. Clinical features. Med J Aust. 1969;1:663-5.

45. Windsor HM. Cardiac transplantation. Postgrad Med J. 1970;46:237-42. 46. Chang VP. Reintroduction of cardiac transplantation in Australia. Med J Aust. 1984;140:692- 3. 47. Alexander PM, Swager A, Lee KJ, Shipp A, Konstantinov IE, Wilkinson JL, d'Udekem Y, Brizard C and Weintraub RG. Paediatric heart transplantation in Australia comes of age: 21 years of experience in a national centre. Intern Med J. 2014;44:1223-31. 48. A. I, L. G, A. D, P. R, D. J, B. W, G. K, A. J, M. H, C. JP, P. FM, P. HR, M. GR, K. DK and S. MP. Increasing the Tolerance of DCD Hearts to Warm Ischemia by Pharmacological Postconditioning. American Journal of Transplantation. 2014;14:1744-1752. 49. Iyer A, Chew HC, Gao L, Villanueva J, Hicks M, Doyle A, Kumarasinghe G, Jabbour A, Jansz PC, Feneley MP, Harvey RP, Graham RM, Dhital KK and Macdonald PS. Pathophysiological Trends During Withdrawal of Life Support: Implications for Organ Donation After Circulatory Death. Transplantation. 2016;100:2621-2629.

Chapter 2-7: Publications

While the first heart transplant in 1967 was from a DCD donor, DCD was quickly abandoned after the introduction of the ‘brain-dead rule’. The advantage of a beating heart donor, without exposure to warm ischaemia, assured viability of the donor heart even after 4-6 hours of static cold storage. However, as heart transplantation is increasingly performed for the treatment of end-stage heart failure, the field has been largely limited by donor shortage and as a result led to the expansion of current acceptable donor criterias to include ‘extended’ or marginal donors. This include older donors; donors with borderline left ventricular function; donors with high inotropic requirement; where the anticipated ischaemic time is more than 4-6hours; and DCD donors.

One of the biggest development in heart transplantation which is leading the increasing use of marginal organs is organ perfusion technology. One of the biggest concerns associated with the use of marginal organs is the risk of primary graft dysfunction/failure after transplantation. This is discussed in detail in the review titled Primary Graft Dysfunction after Heart Transplantation which includes the current classification and treatment. In an effort to address the issue of graft failure, there is growing evidence to suggest machine perfusion as an alternate to cold static storage in the management of marginal and DCD organs. The EXPAND trial, by Transmedics OCS™, utilises rejected donor hearts and recovers these organs using normothermic machine perfusion (NMP) with subsequent transplantation. The study, which is ongoing, has reported favourable outcomes to date. Furthermore, our experience in pioneering the DCD heart transplantation program which has achieved excellent outcomes as discussed in the publication Donation after Circulatory Death (DCD) Heart Transplantation: Update of Current Experience and Outcomes in Australia. The

current state of machine perfusion technology in heart transplant, including upcoming new technologies, is also discussed in Donor Heart and Organ Perfusion Technology.

Today, our DCD heart transplant program accounts for up to 20% of all heart transplants performed in our unit. Beginning from early works to understand the ins and outs of DCD and its impact on the heart which helped shape our current DCD protocol, and is presented in Pathophysiological trends during withdrawal of life support: implications for organ donation after circulatory death (DCD).

In our clinical experience with DCD organ procurement and management, our main considerations are techniques to improve and enhance organ recovery during NMP. While supplementation of the cardioplegia solution with GTN and EPO has been shown to improve cardiac allograft recovery, we also considered the effect of heparin in preserving the microcirculation of the organ after death. Furthermore, as antemortem heparin is not currently a widely accepted practise, we designed a set of experiments to investigate the role of antemortem heparin in DCD heart procurement – Ischaemia-Reperfusion Injury and Cardiac Preservation in a Donation after Circulatory Death (DCD) Rodent Model: Impact of Ante-mortem Heparin.

In addition, in an effort to reduce warm ischaemic time and to address the issue of anaemia in the donor, we investigated the role of banked blood for perfusion of the donor heart during NMP. A series of porcine experiments and two clinical attempts to recover hearts using banked blood are presented in Ex-Vivo Perfusion of Donor Hearts: The Feasibility of Banked Blood for Normothermic Machine Perfusion and Banked Blood for Normothermic Machine Perfusion of the Donor Heart: A Clinical Perspective.

Finally, as our DCD clinical outcomes has been excellent, we have recently raised our donor age limit from 45 years to 55 years. Due to concerns that older hearts may not tolerate and recover from the period of warm ischaemia during the withdrawal process, we are reluctant to increase the current age criteria. In order to address the issue of ageing and its effect on the donor heart, a series of experiments using aged rats were performed and the results presented in Ischaemic Tolerance: The Effect of Ageing on Myocardial Susceptibility to Ischaemia in a Donation after Circulatory Death Rodent Model.

Chapter 2 – Publication 1 and 2

Title 1: Primary Graft Dysfunction After Heart Transplantation

Journal: Current Transplantation Reports

Publication date: December, 2014

Title 2: Donor Heart and Organ Perfusion Technology

Journal: Journal of Thoracic Disease (JTD)

Publication status: Accepted for publication 15 March 2019

Declaration: I certify that this publication was a direct result of my research towards this PhD, and that reproduction in this thesis does not breach copyright relations.

Foreword

Primary graft dysfunction/failure (PGD/PGF) is probably the most dreaded complication after heart transplantation and is often associated with a higher mortality. One of the key issues with the diagnosis of PGD is the lack of a clear definition, which accounts for the variability of reported incidence worldwide.

In response, the International Society of Heart and Lung Transplant (ISHLT) has developed a consensus definition at the 2014 ISHLT meeting to address this issue.

The aetiology of PGD is postulated as the accumulative ‘injury’ sustained by the donor heart during the event of brain death and its sequelae, the process from explantation to implantation, and subsequent recipient milieu during reperfusion. Whilst there is no specific biomarker for the PGD, current diagnosis is based on clinical assessment with echocardiography and the necessity for high dose inotropy or mechanical circulatory support in the immediate post- operative period.

Among the key aspects of current research are ways to attenuate injurious pathways and potentially recover donor hearts using organ perfusion technology. Whilst pharmacological supplementation has been shown to improve cardiac recovery post-cold storage and increase donor heart ischaemic tolerance, the use of pharmacological supplementation of the cardioplegic

solution is not universally practiced. Furthermore, pre-clinical research has shown that the protective effects of pharmacological supplementation also applies to donor hearts subjected to circulatory arrest as the mode of death.

Organ perfusion technology to improve recipient outcomes is another area of active research. Currently, there are two clinically trialled systems using different perfusion techniques. The first system is the Transmedics OCS Heart™ which utilises normothermic machine perfusion (NMP) and involves the reanimation and reperfusion of the donor heart ex-situ using a blood-based perfusate. While multi-centre trials have shown no difference in outcome when compared to cold-stored brain-dead donor (DBD) hearts, our group has successfully used the device to recover and transplant circulatory death donor

(DCD) hearts which were previously discarded due to concerns of irreparable injury. To date, the device is being used off-label for DCD heart transplantation, and further multicentre studies are in process to investigate the benefit of NMP in marginal (or extended criteria) brain dead (MBD) hearts.

The second organ system that is currently undergoing clinical trial is Steen’s

Heart Preservation System which utilises hypothermic machine perfusion (HMP).

There is currently no published outcome with this system, however pre-clinical trials using porcine brain death model have shown good results after 24hours of

HMP followed by transplantation.

Primary Graft Dysfunction after Heart Transplantation was published in

Current Transplantation Reports in December 2014, and the Donor Heart and

Organ Perfusion Technology was accepted for publication in January 2019 in the

Journal of Thoracic Disease.

Title 1: Primary Graft Dysfunction After Heart Transplantation

Title 2: Donor Heart and Organ Perfusion Technology

Chapter 3 – Publication 3

Title:

Pathophysiological trends during withdrawal of life support: implications for organ donation after circulatory death (DCD)

Journal:

Transplantation. 2016 Dec;100(12):2621-2629.

Publication Date:

2016 (Co-author)

Author 1 performed experiments for series 1 including data analysis, and author 2 performed experiments for series 2 including data analysis.

Declaration: I certify that this publication was a direct result of my research towards this PhD, and that reproduction in this thesis does not breach copyright relations.

Foreword

Following the first clinical DCD heart transplantation in our unit in 2014, we decided to investigate the events surrounding DCD withdrawal and the physiological changes that occur during this time. Previous DCD porcine experiments focussed on the ischaemic tolerance of the donor heart, which allowed the establishment of the 30minute warm ischaemic time (WIT) that is observed in our clinical practice. However, we are conscious of the physiological differences in brain death versus circulatory death, which led to the following experiments.

While the events surrounding brain-death and their effect on the heart is well understood, the same could not be said in the DCD setting. For instance, the process of allowing the heart to fully arrest exposes the heart to a variable period of warm ischaemia which causes cellular injury to the heart, and indirectly through hypoxic and metabolic changes in the composition of the blood that further impacts the heart. This blood is later collected and used to reperfuse the donor heart with potential deleterious effect.

Furthermore, in our clinical experience, during DCD retrieval, the ventricles appear severely distended, and this poses concerns for ventricular recovery in the post-implantation period.

This study provides an insight into circulatory death and allows for a more tailored approach to cardio-protection, organ resuscitation and recipient selection for improved outcome in the DCD cohort.

Title: Pathophysiological trends during withdrawal of life support: implications for organ donation after circulatory death (DCD)

Chapter 4 – Publication 4

Title:

Outcomes of Donation After Circulatory Death Heart Transplantation in Australia

Journal:

Journal of American College of Cardiology (JACC)

Publication Status:

Accepted for publication Feb 2019

Declaration: I certify that this publication was a direct result of my research towards this PhD, and that reproduction in this thesis does not breach copyright relations.

Foreword

Through many years of DCD research, we performed our first DCD heart transplantation in July 2014. The protocol which was based on large animal experiments performed in the cardiac research institute provided the scientific basis for this. The key concern in utilising DCD hearts is the prolonged period of warm ischaemia during the withdrawal process and its effect on the organ.

Through a series of experiments performed by Iyer et al, they were able to identify the critical time period of twenty minutes after which cardiac recovery is significantly reduced. Furthermore, through supplementing the cardiac flush solution, they were able to increase the ischaemic time from twenty to thirty minutes with excellent functional recovery1. The results from this experiment ultimately helped us set the tolerable warm ischaemic time (WIT) of thirty minutes from withdrawal of life support in our clinical practice.

The second crucial development that allowed us to successfully utilise these hearts, was normothermic machine perfusion(NMP), which gave us the opportunity to reanimate and assess the health of the organ before proceeding for transplantation. The Transmedic OCS system was already being used for DBD cardiac procurement in a few centres in the United States and Europe, and data suggested OCS resuscitation of the donor heart was non-inferior to cold static storage2. Our experience of using the OCS system for MBD donor hearts

provided us with the experience to perform experiments in DCD organs. The use of OCS is now routine for all DCD heart retrievals to determine the viability of the donor heart.

Today, DCD hearts are standard of practice in Australia with survival outcomes comparable to DBD heart transplantation in the short to medium term. Since

2014, 30 DCD heart transplants have been performed in our centre, with only one mortality. All survivors demonstrate normal graft function with no increased risk of rejection episodes when compared to our DBD cohort.

Despite our excellent outcomes to date, there remain challenges in the assessment and recovery of these organs during NMP. Based on current practice, the decision to utilise a recovered heart depends heavily on the lactate profile during NMP. An overall trend of declining lactate, and evidence of lactate consumption is generally regarded as favourable towards organ transplantation.

In addition, visual assessment of the individual ventricles provide reassurance that the donor heart is viable. However, this is not a reliable method of differentiating ‘good’ from ‘bad’ hearts due to its subjectivity. Therefore, there is the need for a set of objective assessments to determine the state of these hearts.

The purpose of this clinical study is to identify ‘objective’ measures that will

supplement current assessment techniques in helping the clinicians decide

whether a DCD heart should be transplanted.

References:

1. Iyer A, Gao L, Doyle A, Rao P, Jayewardene D, Wan B, Kumarasinghe G, Jabbour A, Hicks M,

Jansz PC, Feneley MP, Harvey RP, Graham RM, Dhital KK and Macdonald PS. Increasing the Tolerance

of DCD Hearts to Warm Ischemia by Pharmacological Postconditioning. American Journal of

Transplantation. 2014;14:1744-1752.

2. Ardehali A, Esmailian F, Deng M, Soltesz E, Hsich E, Naka Y, Mancini D, Camacho M, Zucker M,

Leprince P, Padera R and Kobashigawa J. Ex-vivo perfusion of donor hearts for human heart transplantation (PROCEED II): a prospective, open-label, multicentre, randomised non-inferiority trial.

The Lancet. 385:2577-2584.

Title: Donation after Circulatory Death (DCD) Heart Transplantation: Update of Current Experience and Outcomes in Australia

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Chapter 5 – Publication 5 and 6

Title 1: Ex-Vivo Perfusion of Donor Hearts: The Feasibility of Banked Blood for Normothermic Machine Perfusion

Journal:

OBM Transplantation

Publication Status:

Submitted in March 2019

Title 2:

Banked Blood for Normothermic Machine Perfusion of the Donor Heart: A Clinical Perspective

Journal:

Journal of Heart and Lung Transplant

Publication Status:

Submitted in March 2019

Declaration I certify that this publication was a direct result of my research towards this PhD, and that reproduction in this thesis does not breach copyright relations.

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Foreword

Normothermic machine perfusion (NMP) remains the mainstay of device recovery and maintenance of the donor heart. Current recommendation requires the use of donor blood in the reperfusion of these hearts. Whilst crystalloid perfusate in combination with artificial oxygen carriers have been successfully used to reanimate these hearts, the outcome has been less than satisfactory due to early onset oedema that continues to worsen with time 1.

The key challenge with preparing the blood perfusate is the requirement that the donor blood is used in the recovery of these hearts, based on the recommendations of the Transmedics OCS Heart 2. While this is not an issue in the brain dead (BD) setting, the additional time required to collect 1.2-1.5L of donor blood in the circulatory death donor (DCD) can result in significant differences in functional outcome due to warm ischaemic injury 3.

Furthermore, limiting donor factors including small donor size, blood loss and haemodilution from judicious fluid replacement can all lead to inadequate blood volume or inadequate perfusate haematocrit which impacts organ recovery on

NMP. Therefore, the use of banked blood to perfuse donor hearts is favourable, especially in DCD.

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The use of BB however is not novel, and has been successfully used in the

retrieval and perfusion of liver, lungs and kidneys. There is however, little

published data in the use of BB in heart reperfusion, which led to the design of

the following study.

References:

1. White CW, Hasanally D, Mundt P, Li Y, Xiang B, Klein J, Müller A, Ambrose E, Ravandi A, Arora RC, Lee TW, Hryshko LV, Large S, Tian G and Freed DH. A whole blood–based perfusate provides superior preservation of myocardial function during ex vivo heart perfusion. The Journal of Heart and Lung Transplantation. 2015;34:113-121.

2. Esmailian F, Kobashigawa JA, Naka Y, Mancini D, Soltesz E, Hsich E, Camacho M, Baran D, Madsen J, LePrince P, Deng M and Ardehali A. The PROCEED II International Heart Transplant Trial with the Organ Care System Technology (OCS). J Heart Lung Transpl. 2013;32:S95-S96.

3. Chew HC, Iyer A, Connellan M, Granger E, Hayward C, Jabbour A, Jabbour A, Jansz P, Keogh A, Kotlyar E, Spratt P, Macdonald P and Dhital K. ECMO use post DCD Heart Transplantation: A Retrospective Study. 2018;102:S66.

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Title 1: Ex-Vivo Perfusion of Donor Hearts: The Feasibility of Banked Blood for Normothermic Machine Perfusion

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Chapter 6 – Publication 7

Title:

Ischaemia-Reperfusion Injury and Cardiac Preservation in a Donation after Circulatory Death

(DCD) Rodent Model: Impact of Ante-mortem Heparin

Journal:

Transplantation Direct

Publication Status:

Submitted in March 2019

Declaration: I certify that this publication was a direct result of my research towards this PhD, and that reproduction in this thesis does not breach copyright relations.

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Foreword Large and small animal work performed in our laboratory using the brain dead

(DBD) model has shown enhanced preservation of the donor heart when the heart is flushed and stored in Celsior solution mixed with a concoction of supplements including glyceryl trinitrate (GTN), Zoniparide (Z) and erythropoietin (EPO). Previous studies by L. Gao, A. Hing and A. Watson, demonstrated that hearts preserved using the triple supplemented celsior solution have shown improved recovery after 6-8 hours of cold storage in rats and pig experiments 1-5. Furthermore, works by A. Iyer investigating the effectiveness of triple supplementation in a porcine DCD model was able to extend the tolerable warm ischaemic time of the donor porcine heart from twenty minutes to thirty minutes with good functional recovery on resting and working mode using the isolated heart perfusion model developed by

Langendorff 6.

The key difference in the porcine experiments described by Iyer et al. and the current DCD protocol is the use of ante-mortem heparin. Currently, the provision of antemortem heparin is not practised universally due to ethical concerns of its effect on the donor. Whilst there are no data to suggest that heparin increases DCD heart retrieval rates, or improves cardiac recovery during normothermic machine perfusion, studies from DCD liver transplantation have shown that heparin offers ‘cytoprotective effects during circulatory arrest,

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reflected in improved flows during regional perfusion and better biochemical,

functional and histological parameters 7. Cao et al. also noted in a meta-analysis that anti-mortem heparin administration significantly reduces the odds of primary nonfunction of the liver 8.

Based on these studies, we decided to investigate the role of antemortem

heparin in a DCD heart model. Our study compared DCD hearts flushed with

celsior alone or supplemented celsior solution, and further tested the effect of

antemortem heparin in each group.

References: 1. Gao L, Tsun J, Sun L, Kwan J, Watson A, Macdonald PS and Hicks M. Critical role of the STAT3 pathway in the cardioprotective efficacy of zoniporide in a model of myocardial preservation – the rat isolated working heart. British Journal of Pharmacology. 2011;162:633-647.

2. Hing AJ, Hicks M, Garlick SR, Gao L, Kesteven SH, Faddy SC, Wilson MK, Feneley MP and Macdonald PS. The Effects of Hormone Resuscitation on Cardiac Function and Hemodynamics in a Porcine Brain-Dead Organ Donor Model. American Journal of Transplantation. 2007;7:809-817.

3. Watson AJ, Gao L, Sun L, Tsun J, Doyle A, Faddy SC, Jabbour A, Orr Y, Dhital K, Hicks M, Jansz PC and Macdonald PS. Enhanced Preservation of Pig Cardiac Allografts by Combining Erythropoietin With Glyceryl Trinitrate and Zoniporide. American Journal of Transplantation. 2013;13:1676-1687.

4. Watson AJ, Gao L, Sun L, Tsun J, Jabbour A, Qiu MR, Jansz PC, Hicks M and Macdonald PS. Enhanced preservation of the rat heart after prolonged hypothermic ischemia with erythropoietin- supplemented Celsior solution. J Heart Lung Transpl. 2013;32:633-640.

5. Watson AJ, Gao L, Tsun J, Kwan JC, Jabbour A, Macdonald PS and Hicks M. Erythropoietin Improves Functional Recovery of the Rat Heart after Prolonged Cold Storage in Celsior Solution. J Heart Lung Transpl. 2009;28:S189-S189.

6. Iyer A, Gao L, Doyle A, Rao P, Jayewardene D, Wan B, Kumarasinghe G, Jabbour A, Hicks M, Jansz PC, Feneley MP, Harvey RP, Graham RM, Dhital KK and Macdonald PS. Increasing the Tolerance

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of DCD Hearts to Warm Ischemia by Pharmacological Postconditioning. American Journal of Transplantation. 2014;14:1744-1752.

7. Hessheimer AJ, Vendrell M, Muñoz J, Ruíz Á, Díaz A, Sigüenza LF, Lanzilotta JR, Delgado Oliver E, Fuster J, Navasa M, García-Valdecasas JC, Taurá P and Fondevila C. Heparin but not tissue plasminogen activator improves outcomes in donation after circulatory death liver transplantation in a porcine model. 2018;24:665-676.

8. Cao Y, Shahrestani S, Chew HC, Crawford M, Macdonald PS, Laurence J, Hawthorne WJ, Dhital K and Pleass H. Donation After Circulatory Death for Liver Transplantation: A Meta-Analysis on the Location of Life Support Withdrawal Affecting Outcomes. Transplantation. 2016;100:1513-24.

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Title: Ischaemia-Reperfusion Injury and Cardiac Preservation in a Donation after Circulatory Death (DCD) Rodent Model: Impact of Ante- mortem Heparin

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Chapter 7 – Publication 8

Title:

Ischaemic Tolerance: The Effect of Ageing on Myocardial Susceptibility to Ischaemia in a Donation after Circulatory Death Rodent Model

Journal:

Journal of Heart and Lung Transplant

Publication Status:

Submitted for Publication in March 2019

Declaration: I certify that this publication was a direct result of my research towards this PhD, and that reproduction in this thesis does not breach copyright relations.

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Foreword

Local and international organ donation trends have shown increase donor age over the last two decades. While the inclusion of marginal donors may account for the overall age rise more recently, the general consensus is that the population is getting older which accounts for the older donor and recipient trend. One of the main concern with this group of donor is the increase risk of primary graft dysfunction/failure due to reduce myocardial tolerance to ischaemia. This is especially pertinent in the DCD cohort.

In order to address the issue of ageing and ischaemic tolerance, we deviced a rodent experiment to investigate the effect of warm ischaemia on the hearts in a variety of age groups. Rodents were chosen for this set of experiments due to

1. their accessibility for research; 2. Well studied age correlation to humans; and

3. Cost of ageing and maintaining rodents for the purposes of our study.

We hypothesised that ischaemic tolerance dwindles with age, but can be recuperated using pharmacological supplementation as seen in similar DBD experiments by Kumarasinghe1. Our results, however, revealed interesting trends. These are presented in the following article.

References:

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1. Kumarasinghe G, Gao L, Hicks M, Villanueva J, Doyle A, Rao P, Ru Qiu M, Jabbour A, Iyer A, Chew

HC, Hayward CS and Macdonald P. Improved heart function from older donors using pharmacologic conditioning strategies. The Journal of Heart and Lung Transplantation. 2016;35:636-646.

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Title: Ischaemic Tolerance: The Effect of Ageing on Myocardial Susceptibility to Ischaemia in a Donation after Circulatory Death Rodent Model

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Chapter 8 – Discussion and Conclusion

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Heart failure is a leading cause of mortality and morbidity in the world. Current treatments include a combination of medical and resynchronisation therapy aimed at preserving cardiac function and managing symptoms of congestive cardiac failure (CCF) with hopes to control disease progression. However, in patients who continue to deteriorate, mechanical support with left ventricular assist device (LVAD) or heart transplant are the only viable treatment options.

To date, heart transplantation remains the gold standard and definitive treatment for end-stage heart failure with over 90% one-year survival. The improvement in survival outcomes since the 1980’s has substantially increased transplant volumes worldwide. Increasing need for transplantation this day and age has led to the expansion of currently accepted donor pool to include MBD and DCD donors.

While with MBD we saw the acceptance of older donors, donor hearts with borderline LV function and longer cold ischaemic time (>4hours), this pales in comparison to DCD where the donor heart has arrested after a variable period of warm ischaemia post withdrawal from circulatory and respiratory support

(WCRS). As a result, the use of NMP is the standard practice for all DCD heart retrievals.

Today, the number of units performing DCD heart transplants are growing, with more than 5 units in Europe (including UK) and our unit in Sydney, Australia.

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Over 100 DCD heart transplants have been performed, with recipient survival and graft function comparable to DBD heart transplants in the short to medium term (follow up just under 5 years). Despite this, there is still reluctance with the use of DCD hearts due to concerns for organ viability and feasibility for transplantation, and the lack of objective criteria to differentiate ‘good’ from

‘bad’ hearts on NMP.

In an effort to address the above issue, a combination of clinical and animal work have been performed to help us understand and address the key issues impacting donor heart function, post-DCD and more importantly post- implantation. Through thorough review of our clinical experience, we were able to identify a few issues that potentially influence clinical outcome (see chapter

4). These will be discussed briefly below along with our current recommendations.

1. Timing

Similar to myocardial ischaemia, ‘time is muscle’ accurately describes the

effect of WIT on the cardiac allograft. Our earlier works suggested a WIT

of 30minutes with the use of supplemented preservation flush solution

was associated with good recovery, through analysing our clinical

experience, we were able to expand this time by changing the WIT

starting point from the beginning of WCRS to when the systolic BP was

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less than 90mmHg. We further identified the crucial time period that determines the risk for ECMO for delayed graft function was the Asystolic

– cardioPlegia (AP) time. Our current data would suggest that an AP time of under 12 minutes was associated with lower EMCO requirement.

The AP time led us to further assess individual time-points, between asystole and preservation flush, to expedite the retrieval process. The key modifiable time-points identified through our analysis were donor transfer time or Knife to Skin (KTS) time and time to cardioplegia, which includes time required for blood collection.

Current recommendations to reduce KTS time include – updating donor withdrawal location and operating theatre staff briefing prior to organ retrieval. Increasingly, donor withdrawal is performed in the anaesthetic bay or co-adjacent operating theatre, and where this is impossible, transfer time is minimised by selecting the closest ICU bed space and operating theatre. Furthermore, pre-withdrawal briefing in the operating theatre with pre-delegation of roles reduces confusion during patient transfer. These recommendations are currently practiced in Australia.

In contrast, in an effort to minimise time to cardioplegia, we investigated the potential use of banked blood for reperfusion of the donor heart during NMP. Unfortunately, base on current pig model and clinical

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vignettes (Chapter 5), we have concluded that banked blood in its current

unmodified state was not suitable as a replacement of donor blood

during NMP. However, we do recommend for DCD retrieval to be

performed by specialised retrieval teams to prevent any surgical delays.

2. Antemortem heparin and pharmacological supplementation for

enhanced preservation of donor heart.

Currently, the administration of antemortem heparin is not universally

practised in Australia due to legislative differences between the different

states. For example, administration of antemortem heparin is illegal in

NSW, but allowed in ACT, SA and QLD. In the state of VIC, the provision

of antemortem heparin is dependent upon the decision of the intensive

care specialist overseeing the donor management. The main objection

for administration of antemortem heparin is a moral concern that the

high dosage (usually 25,000U) may lead to bleeding that can expedite the

donor’s demise, therefore constituting euthanasia which is illegal in

Australia.

On the flip side, in our clinical experience, we have noticed

hypercoagulation of the donor blood in the absence of peri-mortem

heparin, which can impede blood collection, by way of longer collection

time and/or inadequate volume collected, and a theoretical risk of

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microthrombi affecting the delivery of cardioplegia solution. To address

the issue of slow blood flow, we employ suction in the early phase of

blood collection to facilitate venous drainage.

Furthermore, we performed a series of rodent experiments to investigate

the role of antemortem heparin in the DCD setting (see chapter 6). Our

results suggest that heparin plays an important role in maintaining

coronary patency, with improved coronary flow recovery in heparinised

groups during reperfusion. Furthermore, we noticed a synergistic effect

between heparin and pharmacological supplementation of the

cardioplegia flush solution. We postulate that heparin prevents the

formation of microthrombi in the microcirculation therefore facilitates

the delivery of cardioplegia solution. Further experiments are required to

confirm the benefits of antemortem heparin.

In addition to these recommendations, we have recently broadened our clinical criteria to accept heart donors up to the age of 55 years. Whilst current MBD criteria accept donors up to 65 years of age, we acknowledge that DCD hearts undergo severe ischaemic insult prior to reperfusion on NMP, and are therefore reluctant to match the MBD donor age. Furthermore, our rodent model looking at cardiac recovery post DCD in aged animals confirmed our concerns (see chapter 7). We found that ischaemic tolerance was poor in 3 and 24 month old

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rats, which is equivalent to pre-adolescent and over 60 years in human age.

Furthermore, our study showed that cardiac recovery was best in 12 months followed by 18 month old rats (equivalent to 30 years and 45 years respectively).

Based on these results, we hypothesise that ischaemic tolerance is acquired with age, and after adulthood, begins to deteriorate. In addition, we were able to improve cardiac recovery using pharmacological supplementation. However, the benefit of pharmacological supplementation is less prominent in the 24 month rats.

Conclusion:

DCD heart transplantation accounts for 15-20% of all heart transplant in our unit, and a mortality rate of 3.3% with good short and medium term graft outcome.

The key limitation to wider use of DCD hearts in Australia includes: 1. Expertise for DCD retrieval and organ management and 2. Funding.

As discussed earlier, current NMP management of DCD hearts lacks objective criteria to allow for easy adoption in clinical practise. In our unit, the decision to proceed with transplantation is based on the lactate profile of the organ during

NMP, and visual assessment of the heart. Whilst lactate provides a clear cut-off for organ utilisation, increasingly we are accepting donor hearts with high lactates as long as the trend is favourable and shows sign of venous lactate extraction. Moreover, visual assessment is often inaccurate and subjected to

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inter-person variations. Therefore, there is a great need for a more reliable biomarker or assessment tool to distinguish these hearts.

While the use of NMP is associated with a hefty price tag, there is currently no published cost-benefit analysis comparing the cost of a heart transplant utilising machine perfusion, to standard medical therapy while being on the transplant waiting list. Future research in this area will be instrumental in the wider usage of NMP and possible increase funding for machine management of donor organs.

Furthermore, hypothermic machine perfusion (HMP) are showing promising outcomes in large animal transplant experiments with at least one device being trialled clinically. This will be a welcoming alternative to the current NMP system due to its proposed cost advantage.

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