Antibodies in kidney transplantation

329 Pages·2015·7.87 MB·English

by  Andrew John Bentall

Antibodies in
Kidney Transplantation

Andrew John Bentall

A thesis submitted to the University of Birmingham for the degree of
Doctor of Medicine (MD)

Department of Nephrology
School of Immunity and Infection
College of Medical and Dental Sciences
The University of Birmingham
January 2014
University of Birmingham Research Archive

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Abstract
The aim of this thesis is to examine the effect of anti-donor antibodies in the clinical
management and outcomes of antibody incompatible kidney transplantation. Initial
studies were conducted to improve measurement of anti-ABO specific blood group
antibodies. The specificity of antibody binding to blood group antigens depended upon
the assay platform and the nature of the core structure to which the blood group antigen
was bound. A standardised haemagglutination assay was shown to produce excellent
reproducibility, which was then applied to the analysis of samples derived from the
ABOUT-K study. In this study of ABO incompatible kidney transplantation (ABOiKTx) in
the UK, good clinical outcomes were achieved but there was wide variation in the
method and result reported in local assays quantifying blood group antigen specific
antibodies. This did not seem to alter graft survival within the limitations of a study of
100 patients however may have altered the exposure to treatment.
In a highly sensitised HLA incompatible kidney transplant recipients (HLAiKTx), I
demonstrated long term outcomes were poor compared to a compatible cohort. In
particular outcomes were worse with pre-formed donor specific anti-HLA Class II
antibodies than with HLA class I alone. The histological injury of antibody damage
occurred significantly earlier than with Class I antibodies. I then demonstrated that
despite the activation of complement, anti-donor ABO specific antibodies did not display
the same inflammatory phenotype in allograft biopsies as anti-HLA antibodies. Finally,
the inhibition of terminal complement activation, whilst reducing early antibody-
mediated rejection did not abrogate all inflammation. This resistance to inhibition of
terminal complement activation may be contributed to by IgM DSA which may play a
role in cellular recruitment into the allograft but can be removed easily through plasma
exchange.
Reproducible and standardised assays are needed for antibody assessment in order to
make good clinical decisions to improve patient outcomes. Further studies are needed to
stop production or block mechanisms of ongoing cellular infiltrate to improve patient
outcomes.
Acknowledgements

I am very grateful to my supervisors Dr Simon Ball and Professor Lorraine Harper for
their support and guidance throughout my research appointment. Professor David
Briggs has provided both insightful and practical help into the area of histocompatibility
and Dr David Lowe and David Atkinson have guided me in practical assay development.

Dr Mark Stegall has been enormously helpful mentor and supervisor at Mayo Clinic and I
am very grateful to have had the opportunity to have worked with him and Walter Park
in his transplant research group.

Clinical research is collaborative in nature, so I am very grateful to the investigators and
research nurses who have helped with recruitment and data collection of patients
throughout the UK for the ABOUT-K study. Manjit Braitch performed the single user
haemagglutination assays countless times.

I am grateful to the University of Birmingham Mayo Exchange Programme who funded
my research collaboration with Mayo Clinic, USA and the Queen Elizabeth Hospital
Birmingham Charity who provided funding for ABO assays to be performed.

Finally, my wife and children, Aimée, Esther and Daniel have been very supportive and
encouraging, both emotionally, patiently, practically and willing to move countries to
help me with my research goals. I am particularly grateful to their faith, perseverance
and fun!
Contents
Contents .............................................................................................................................................. 2
Work Arising from this Thesis .................................................................................................. 12
Papers ...................................................................................................................................................................12
Abstracts ..............................................................................................................................................................13
Index of Figures .............................................................................................................................. 16
Index of Tables ............................................................................................................................... 20
Abbreviations ................................................................................................................................. 22
Chapter 1 Introduction ................................................................................................................ 25
1.1 End Stage Renal Failure in the UK .....................................................................................................26
1.2 Live Donor Transplantation as a treatment option for ESRF .................................................26
1.3 Immunological barriers to transplantation ...................................................................................27
1.3.1 ABO Blood Group .............................................................................................................................30
1.3.1.1 Chemistry of A/B antigen formation ........................................................................ 30
1.3.1.2 Development of anti-ABO specific antibodies ....................................................... 30
1.3.1.3 Subtyping of ABO Blood Group Individuals ........................................................... 32
1.3.1.4 ABO antigen and core structures ............................................................................... 32
1.3.1.5 Clinical effect ABO Blood Groups ............................................................................... 34
1.3.2 HLA sensitization .............................................................................................................................35
1.4 Antibodies and the measurement of antibodies ..........................................................................35
1.4.1 Isotype production ..........................................................................................................................35
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1.4.2. Significance of Isotype ..................................................................................................................36
1.4.3. Anti-HLA IgM ....................................................................................................................................37
1.4.4. IgG Subclasses ..................................................................................................................................40
1.4.4. HLA antibody measurement .......................................................................................................41
1.4.3.1 Complement Dependent Cytotoxicity (CDC) ......................................................... 42
1.4.3.2. Flow Cytometric Crossmatch (FXM) ........................................................................ 42
1.4.3.3. Single Antigen Bead Detection (SAB) ...................................................................... 43
1.4.3.4. Combination of techniques .......................................................................................... 43
1.4.5. Anti-ABO specific antibody measurement ............................................................................46
1.4.6. Other anti-donor antibodies .......................................................................................................47
1.5 Preformed antibodies as a barrier to transplantation. ..............................................................48
1.5.1 ABO incompatible kidney transplantation ............................................................................48
1.5.2 HLA incompatible kidney transplantation ............................................................................50
1.5.3 Limitations of pooled exchange programme ........................................................................51
1.6 Desensitization ..........................................................................................................................................51
1.6.1 Antibody Production ......................................................................................................................51
1.6.2 Extracorporeal Antibody Removal Treatment.....................................................................53
1.6.3 Prevention of allograft damage ..................................................................................................54
1.7 Rejection .......................................................................................................................................................55
1.7.1. Defining Antibody-Mediated Rejection ..................................................................................56
1.7.2 Peritubular capillaritis (PTCitis) ...............................................................................................57
1.7.3 Glomerulitis ........................................................................................................................................57
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1.7.4 Microcirculation injury scores ....................................................................................................58
1.7.5 Complement and Antibodies .......................................................................................................60
1.7.5.1 Complement Activation ................................................................................................. 60
1.7.5.2 Complement Activation and Chronic Injury .......................................................... 61
1.7.5.3 Antibody damage without Complement ................................................................. 62
1.7.6 Chronic Antibody-Mediated Rejection ....................................................................................63
1.7.7 Accommodation ................................................................................................................................63
1.8 What happens after an antibody incompatible transplant ......................................................65
1.8.1 Clinical Outcomes in HLAiKTx ....................................................................................................65
1.8.2 Clinical Outcomes in ABOiKTx ....................................................................................................67
1.9 Summary and Scope of this thesis .....................................................................................................69
Chapter 2 Novel Assay Development for anti-ABO blood group antigen specific
antibodies ........................................................................................................................................ 70
2.1 Introduction ................................................................................................................................................71
2.1.1 ABO Titration .....................................................................................................................................71
2.1.2 Immunoglobulin Isotypes.............................................................................................................72
2.1.3 Poor reproducibility of ABO titration ......................................................................................72
2.1.4 Alternative techniques for anti-ABO antibody assessment ............................................74
2.2 Anti-ABO specific antibody assay development ...........................................................................75
2.2.1 Haemagglutination ..........................................................................................................................75
2.2.1.1 Introduction ....................................................................................................................... 75
2.2.1.2 Methods................................................................................................................................ 76
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2.2.1.2.1. Preparation of Red Blood Cell Test Blood Group Sample. ........................... 76
2.2.1.3 Assay variability ............................................................................................................... 77
2.2.1.4. Conclusions ........................................................................................................................ 81
2.2.2 Flow Cytometry Assay ...................................................................................................................81
2.2.2.1 Methods................................................................................................................................ 82
2.2.2.1.1 Isotypes ................................................................................................................................ 83
2.2.2.1.2 Subclasses ............................................................................................................................ 83
2.2.2.1.3 Purification of antibody ................................................................................................. 83
2.2.2.2 Results .................................................................................................................................. 84
2.2.2.2.1 Isotypes ................................................................................................................................ 84
2.2.2.2.2 Subclasses ............................................................................................................................ 88
2.2.2.3 Conclusions ......................................................................................................................... 91
2.2.3 Solid Phase Assay .............................................................................................................................92
2.2.3.1 Microsphere Assay ........................................................................................................... 92
2.2.3.1.1 Introduction ........................................................................................................................ 92
2.2.3.2 Methods and Results ....................................................................................................... 93
2.2.3.2.1 Coupling carbohydrate antigen to microsphere beads ..................................... 93
2.2.3.2.2 Microsphere assay protocol ......................................................................................... 97
2.2.3.2.3 Microsphere assay development strategies .......................................................... 98
2.2.3.2.4 Protocols used for Reducing Non-specific Binding ........................................... 100
2.2.3.2.4.1 Beads from Different Manufacturers ............................................................. 100
2.2.3.2.4.1 Conjugation Testing .............................................................................................. 100
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2.2.3.2.4.3 Pre-incubation protocols .................................................................................... 103
2.2.3.2.4.4 Different Wash Regimes ...................................................................................... 109
2.2.3.2.4.5 Plasma testing ......................................................................................................... 109
2.2.3.3 Conclusions ...................................................................................................................... 110
2.2.4 Surface Plasmon Resonance ..................................................................................................... 110
2.2.4.1 Introduction .................................................................................................................... 110
2.2.4.2 Methods............................................................................................................................. 113
2.2.4.2.1 Antigen coupling ............................................................................................................. 113
2.1.4.2.2 Buffering dilution ........................................................................................................... 113
2.2.4.2.3 Control testing ................................................................................................................. 114
2.2.4.2.4 Binding ................................................................................................................................ 114
2.2.4.2.5 Modelling ........................................................................................................................... 123
2.2.4.5 Conclusions ...................................................................................................................... 124
2.2.5. Statistical Analysis ....................................................................................................................... 125
2.3 Anti-ABO specific Antibody Conclusions ..................................................................................... 126
Chapter 3 The ABOUT-K study ............................................................................................... 130
3.1 Introduction ............................................................................................................................................. 131
3.2 Methods ..................................................................................................................................................... 131
3.2.1 Study Aims ....................................................................................................................................... 132
3.2.2 Treatment Protocols .................................................................................................................... 133
3.2.3 Study Selection and Clinical Assessment............................................................................. 134
3.2.3.1 Informed consent .......................................................................................................... 136
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