Specificity and Properties of Anti-HLA Antibodies Associated with Renal Allograft Rejection

245 Pages·2010·2.37 MB·English

Specificity and Propertie s of Anti-HLA Antibodies

Associated with Renal Allograft Rejection

Hooi Sian Eng
Bachelor of Biomedical Science (Hon)
Master of Medical Science

A thesis submitted for the degree of PhD

School of Medicine
Faculty of Health Science
University of Adelaide, South Australia

February 2009

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i. Contents
i. Contents ................................................................................................................................................ 2
ii. List of Tables ......................................................................................................................................... 6
iii. List of Figures ........................................................................................................................................ 8
iv. Summary ............................................................................................................................................... 9
v. Dedication ............................................................................................................................................ 11
vi. Acknowledgements ............................................................................................................................. 12
vii. Declaration........................................................................................................................................... 13
viii. Thesis Structure .................................................................................................................................. 14
ix. Abbreviations ....................................................................................................................................... 15
x. Publications ......................................................................................................................................... 17
xi. Presentations ....................................................................................................................................... 19
xii. Awards ................................................................................................................................................. 22

1 Chapter 1 Literature Review ................................................................................................................ 23
1.1 Overview of Human Major Histocompatibility Complex (MHC) – Gene Discovery ......................... 24
1.1.1 Chronology of HLA Discovery ................................................................................................ 25
1.2 Genetics of MHC ............................................................................................................................. 25
1.2.1 Evolution of MHC Genes ........................................................................................................ 28
1.2.2 MHC Gene Polymorphism ...................................................................................................... 29
1.3 Human Leukocyte Antigen (HLA) System ...................................................................................... 31
1.3.1 HLA Gene Inheritance and Frequency ................................................................................... 33
1.3.2 HLA Class I, II and III Regions ............................................................................................... 33
1.4 Nomenclature .................................................................................................................................. 35
1.5 Structure of HLA Molecules ............................................................................................................ 37
1.5.1.1 HLA Peptide-Binding Grooves ....................................................................................... 40
1.5.2 HLA Epitopes .......................................................................................................................... 42
1.5.3 HLA Expression ...................................................................................................................... 43
1.6 Roles of HLA Molecules .................................................................................................................. 43
1.6.1 Antigen Presentation .............................................................................................................. 43
1.6.1.1 Endogenous Presentation Pathway............................................................................... 43
1.6.1.2 Exogenous Presentation Pathway ................................................................................. 45
1.6.2 T-cell Receptor Repertoire Selection ..................................................................................... 47
1.6.3 Regulation of NK Cell Activity ................................................................................................. 50
1.7 Significance of HLA in Renal Transplantation ................................................................................. 52
1.7.1 Mechanism of Renal Allograft Rejection ................................................................................ 52
1.7.1.1 Isolated MHC Class I Disparity ...................................................................................... 53
1.7.1.2 Isolated MHC class II Disparity ...................................................................................... 56
1.7.1.3 MHC Class I and II Disparity .......................................................................................... 58
1.8 Pre-transplantation HLA Antibody Detection and Crossmatching .................................................. 60
1.8.1 Negative T-cells / Positive B-cells Crossmatches (TN/BP) .................................................. 60
1.8.2 Crossmatch: Current Serum Negative / Historic Serum Positive (CN/HP) ............................ 61
1.8.3 Crossmatch: DTT-Reducible Positivity ................................................................................... 63
1.9 Post-transplantation Monitoring – Donor-Specific Antibodies ......................................................... 64
1.10 MHC Class I Chain Related (MIC) Genes: MICA and MICB .......................................................... 65
1.11 HLAMATCHMAKER – Amino Acid Triplet Program ....................................................................... 66
1.12 Antibody Detection Techniques ...................................................................................................... 67
1.12.1 Complement-dependent Lymphocytotoxicity Assay .............................................................. 67
1.12.2 Flow Cytometry ....................................................................................................................... 68
1.12.3 Solid Phase ELISA ................................................................................................................. 69
1.12.4 Solid Phase Luminex® ............................................................................................................ 69
1.12.5 Method Comparison ............................................................................................................... 70

2 Chapter 2 Materials & Methods ........................................................................................................... 72
2.1 HLA Antigen Typing ........................................................................................................................ 73
2.1.1 Dynabead® T-cell Isolation ..................................................................................................... 73
2.1.2 Dynabead® B-cell Isolation ..................................................................................................... 73
2.1.3 Serology HLA Antigen Typing ................................................................................................ 74
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2.1.4 DNA Quantitation .................................................................................................................... 74
2.1.5 Gel Electropheresis ................................................................................................................ 74
2.1.6 Visualization of PCR Product ................................................................................................. 75
2.1.7 Sequence Base Typing .......................................................................................................... 75
2.1.7.1 Prepare PCR Mixture ..................................................................................................... 75
2.1.7.2 PCR Product Visualization ............................................................................................. 77
2.1.7.3 AMPure PCR Purification .............................................................................................. 77
2.1.7.4 ABI Big Dye Terminator Ready Reaction Kit ................................................................. 78
2.1.7.5 CleanSEQ Dye Terminator Removal ............................................................................. 79
2.1.8 Luminex® Single Stranded Oligonucleotide (SSO) HLA Typing ............................................. 80
2.1.8.1 Prepare PCR Mixture ..................................................................................................... 80
2.1.8.2 Visualization of PCR Product ......................................................................................... 81
2.1.8.3 Hybridisation .................................................................................................................. 81
2.2 Crossmatch ..................................................................................................................................... 82
2.2.1 Direct Crossmatch .................................................................................................................. 82
2.2.2 DTT Crossmatch .................................................................................................................... 83
2.3 Anti-HLA Antibodies Screening ....................................................................................................... 84
2.3.1 SeraClean ............................................................................................................................... 84
2.3.2 Luminex® Screen and PRA .................................................................................................... 84
2.3.3 Luminex® Single Antigen Bead ............................................................................................... 85
2.3.4 Luminex® Single Antigen Beads – Anti-HLA antibodies IgG subclass ................................... 85
2.4 Database ......................................................................................................................................... 86
2.4.1 National Organ Matching System (NOMS) ............................................................................ 86
2.5 Statistics .......................................................................................................................................... 88
2.6 Reagent Preparation ....................................................................................................................... 89
2.7 Equipments ..................................................................................................................................... 94
2.8 Kits .................................................................................................................................................. 95
2.9 Chemicals ........................................................................................................................................ 95
2.10 Consumables .................................................................................................................................. 96

3 Chapter 3 Clinical Relevance of A positive B-cell Crossmatch in Renal transplantation .................... 97
3.1 Introduction ...................................................................................................................................... 98
3.2 Objectives ........................................................................................................................................ 99
3.3 Materials and Methods .................................................................................................................. 100
3.3.1 Study Cohort ......................................................................................................................... 100
3.3.2 HLA Typing ........................................................................................................................... 100
3.3.3 Screening for Autoantibodies ............................................................................................... 100
3.3.4 Luminex® .............................................................................................................................. 101
3.3.5 Antibody Specificity .............................................................................................................. 101
3.3.6 Clinical Data ......................................................................................................................... 101
3.3.7 Statistics ............................................................................................................................... 102
3.4 Results .......................................................................................................................................... 103
3.4.1 Characteristics of Patients with T–B+ crossmatch ............................................................... 103
3.4.2 Luminex® Specificity of Anti B-cell Antibodies ...................................................................... 106
3.4.3 Early Graft Rejection ............................................................................................................ 106
3.4.3.1 Correlation of T–B+ and Early Graft Rejection ............................................................ 107
3.4.3.2 Correlation of Early Graft Rejection and Antibody Specificity in T–B+ ........................ 109
3.4.4 Graft Function at 6, 12 and 36 month Post-transplantation ................................................. 114
3.4.5 Graft Survival ........................................................................................................................ 116
3.4.5.1 Significance of T–B+ in Graft Survival ......................................................................... 116
3.4.5.2 Correlation of Antibody Specificity in T–B+ with Graft Survival ................................... 116
3.4.6 Historic Positive B-cell Crossmatch ...................................................................................... 121
3.4.7 BXM as A Predictor of Graft Failure in 5 years Post-transplantation ................................... 127
3.5 Discussion ..................................................................................................................................... 128

4 Chapter 4 Anti-HLA Antibodies and Risk Factors Associated with Transplant Glomerulopathy ...... 132
4.1 Introduction .................................................................................................................................... 133
4.2 Objectives ...................................................................................................................................... 134
4.3 Materials and Methods .................................................................................................................. 134
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4.3.1 Study Cohorts ....................................................................................................................... 134
4.3.2 HLA Typing ........................................................................................................................... 135
4.3.3 Detection of Anti-HLA Antibodies ......................................................................................... 135
4.3.4 Clinical Data – ANZDATA Analysis ...................................................................................... 135
4.3.5 Statistics ............................................................................................................................... 136
4.4 Results .......................................................................................................................................... 137
4.4.1 Univariate Risk Factors ........................................................................................................ 137
4.4.1.1 Demographic Comparison ........................................................................................... 137
4.4.1.2 Immunosuppression..................................................................................................... 140
4.4.2 Early Graft Rejection ............................................................................................................ 142
4.4.3 Significance of Anti-HLA Antibodies in Transplant Glomerulopathy .................................... 144
4.4.3.1 CDC Class I PRA defined Presensitization ................................................................. 144
4.4.3.2 Luminex® defined Presensitization .............................................................................. 144
4.4.3.3 Pre-transplant DSA as A Risk Factor of TG ................................................................ 146
4.4.3.4 De novo DSA Predict TG ............................................................................................. 146
4.4.4 Multivariate Analysis ............................................................................................................. 148
4.4.5 DSA as A Predictor of TG ..................................................................................................... 150
4.4.5.1 Time to Biopsy ............................................................................................................. 150
4.4.5.2 Association of TG and Graft Survival........................................................................... 151
4.4.5.3 Pre-transplant DSA Associated with Early TG Development ...................................... 156
4.4.5.4 Significance of DSA in TG Progression ....................................................................... 157
4.4.6 DSA are Predictive for Graft Loss in TG .............................................................................. 159
4.5 Discussion ..................................................................................................................................... 161

5. Chapter 5 A Retrospective Study of B-cell Crossmatch with Luminex® Technology in Well-Matched
Highly Sensitized Patients from The Australian National Renal Exchange Programme (ANREP) ... 165
5.1 Introduction .................................................................................................................................... 166
5.2 Objectives ...................................................................................................................................... 167
5.3 Materials and Methods .................................................................................................................. 168
5.3.1 Study Cohort ......................................................................................................................... 168
5.3.2 Crossmatch .......................................................................................................................... 169
5.3.3 HLA Typing ........................................................................................................................... 170
5.3.4 Luminex® Antibody Analysis ................................................................................................. 170
5.3.5 Clinical Data ......................................................................................................................... 171
5.3.6 Statistics ............................................................................................................................... 171
5.4 Results .......................................................................................................................................... 171
5.4.1 Characteristics of Study Cohort ............................................................................................ 171
5.4.2 Graft Outcomes of Highly Sensitized Patients ..................................................................... 176
5.4.2.1 Graft Rejection ............................................................................................................. 176
5.4.2.2 Graft Function .............................................................................................................. 178
5.4.2.3 Graft Survival ............................................................................................................... 179
5.4.3 Predictive Value of B-cell Crossmatch in Highly Sensitized Patients .................................. 181
5.4.3.1 Prevalence of Positive B-cell Crossmatch ................................................................... 181
5.4.3.2 Characteristics of Patients with Positive B-cell Crossmatch ....................................... 181
5.4.3.3 Correlation of B-cell Crossmatch and Graft Rejection ................................................. 185
5.4.3.4 Graft function B-cell Crossmatch and Graft Function .................................................. 187
5.4.3.5 Correlation of B-cell Crossmatch and Graft Survival ................................................... 188
5.4.4 Predictive Value of Luminex® defined Donor-specific Antibodies ........................................ 190
5.4.4.1 Presence of Donor-specific Antibodies ........................................................................ 190
5.4.4.2 Specificity of DSA defined by Luminex® ...................................................................... 194
5.4.4.3 Correlation of DSA and Graft Rejection ....................................................................... 196
5.4.4.4 Correlation of DSA and Graft Function ........................................................................ 198
5.4.4.5 Correlation of Luminex® defined DSA and Graft Survival ............................................ 199
5.4.5 Luminex® defined DSA in B-cell Crossmatches ................................................................... 201
5.4.5.1 Correlation of Luminex® defined DSA and BXM with Graft Rejection ......................... 202
5.4.5.2 Multivariate analysis of BXM and Luminex® defined DSA in predicting rejection........ 202
5.5 Discussion ..................................................................................................................................... 204

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6 Chapter 6 Discussion ........................................................................................................................ 209
6.1 Significance of Preformed Anti-HLA Antibodies Detected by B-cell Crossmatch ......................... 210
6.1.1 Antibodies Causing Positive B-cell Crossmatches and Correlation with Graft Outcomes ... 214
6.2 Luminex® Defined Donor-specific Antibodies as A Predictor of Graft Outcomes ......................... 214
6.1.2 Solid Phase Antibody Analysis: Virtual Crossmatch ............................................................ 215
6.2.2.1 Prediction of Flow Crossmatch Outcomes by Luminex® Assays................................. 216
6.2.2.2 Improved Transplant Rate by Defining Acceptable Mismatches ................................. 219
6.2.2.3 Cost-Effectiveness of Solid Phase Assays Virtual Crossmatch .................................. 219
6.2.2.4 Possible Weaknesses of Solid Phase Assays Virtual Crossmatch ............................. 220
6.1.3 Luminex® Antibody Analysis and Luminex® Lysate Crossmatch ......................................... 221
6.1.4 DSA in Transplant Glomerulopathy – Significance of Post-transplant Monitoring ............... 222
6.3 Summary of Conclusions .............................................................................................................. 223

References ................................................................................................................................................. 225

Appendix .................................................................................................................................................... 243

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ii. List of Tables

Chapter 2
Table 2.1.1: Preparation of PCR Mixture for Sequence Base Typing ......................................................... 75
Table 2.1.2: PCR conditions for Sequence Base Typing ............................................................................. 76
Table 2.1.3: Conditions of sequencing amplification .................................................................................... 79
Table 2.1.5: Preparation of PCR mixtures for Luminex® SSO ..................................................................... 80
Table 2.1.6: Condition of PCR amplification for Luminex® SSO .................................................................. 81
Table 2.1.7: Hybridisation steps for Luminex® SSO .................................................................................... 82

Chapter 3
Table 3.4.1: Descriptive statistics of cohorts by the result of B-cell crossmatching .................................. 104
Table 3.4.2: Immunosuppresion in T–B– and T–B+ groups ...................................................................... 105
Table 3.4.3: Prevalence of cellular, vascular and glomerular rejection in All grafts, T–B– and T–B+ groups
.................................................................................................................................................................... 108
Table 3.4.4: Graft rejection at 6 months according to the antibody specificity in T–B+ ............................. 110
Table 3.4.5: Risk of cellular rejection according to the antibody specificity in T–B+ ................................. 111
Table 3.4.6: Risk of vascular rejection according to the antibody specificity in T–B+ ............................... 112
Table 3.4.7: Risk of glomerular rejection according to the antibody specificity in T–B+ ............................ 113
Table 3.4.8: eGFR at 6, 12 and 36 months by B-cell crossmatch and antibody specificity, ................. 115
Table 3.4.9: Graft survival rates and hazard ratios for graft loss by antibody specificity ........................... 118
Table 3.4.10: Prevalence of cellular, vascular and glomerular rejection in T–B+ subgroups .................... 122
Table 3.4.11: Odds ratios of graft rejection at 6 months in T–B+ subgroups ............................................ 123
Table 3.4.12: eGFR at 6, 12 and 36 months post-transplant in T–B+ subgroups ..................................... 124
Table 3.4.13: Graft survival rates and hazard ratios for graft loss in T–B+ subgroups.............................. 125
Table 3.4.14: Graft outcomes at 5 years post-transplant ........................................................................... 127

Chapter 4
Table 4.4.1: Descriptive statistics of cohort and univariate analyses of risk factor of TG .......................... 138
Table 4.4.2: Immunosuppressants at induction and prior to diagnosis of TG............................................ 141
Table 4.4.3: Cellular, vascular and glomerular rejection as a risk factor of TG ......................................... 143
Table 4.4.4: Univariate effects of HLA presensitization, defined by CDC or Luminex® techniques, in the
development of TG ..................................................................................................................................... 145
Table 4.4.5: Univariate effects of anti-HLA antibodies in the development of TG ..................................... 147
Table 4.4.6: In multiple regression analysis, DSA was the only independent predictor of TG. ................. 149
Table 4.4.7: Graft survival rates in patients with TG, according to antibody specificity ............................. 152
Table 4.4.8: Graft survival and risk of graft loss in TG patients, according to antibody specificity, in 20
years of follow up ....................................................................................................................................... 153

Chapter 5
Table 5.3.1: Patient recruitment ................................................................................................................. 168
Table 5.4.1: Descriptive statistics of study cohort, compared to all other grafts transplanted within the
same period in participating centres .......................................................................................................... 173
Table 5.4.2: Immunosuppressants at induction ......................................................................................... 175
Table 5.4.3: Risk of cellular, vascular and glomerular rejection in highly sensitized patients ................... 177
Table 5.4.4: Graft function, eGFR, of highly sensitized patients ................................................................ 178
Table 5.4.5: Total graft survival rates in highly sensitized patients, compared to all grafts ....................... 180
Table 5.4.6: Demographics of T–B– and T–B+ groups ............................................................................. 182
Table 5.4.7: Immunosuppressants at induction for T–B– and T–B+ groups ............................................. 184
Table 5.4.8: Correlations of T–B+ with cellular, vascular and glomerular rejection ................................... 186
Table 5.4.9: Comparison of graft function (MDRD eGFR) between T–B– and T–B+ groups. ................... 187
Table 5.4.10: Correlation of T–B+ and total graft survival ......................................................................... 189
Table 5.4.11: Demographics of DSA and Non-DSA groups ...................................................................... 191
Table 5.4.12: Immunosuppressants at induction for DSA and Non-DSA groups ...................................... 193
Table 5.4.13: Correlations of DSA with cellular, vascular and glomerular rejection .................................. 197
Table 5.4.14: Comparison of graft function (MDRD eGFR) between Non-DSA and DSA groups ............ 198
Table 5.4.15: Correlation of DSA and total graft survival ........................................................................... 200
Table 5.4.16: BXM results and specificity of DSA in highly sensitized patients ........................................ 201
Table 5.4.17: Independent predictors of graft rejection ............................................................................. 203
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Chapter 6
Table 6.1.1: A summary of studies investigated clinical significance of positive BXM in renal
transplantation ............................................................................................................................................ 212
Table 6.2.1: A review of clinical significance of low titer DSA detected by Luminex® or Flow crossmatch in
sera with negative CDC / AHG-CDC .......................................................................................................... 218

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iii. List of Figures

Chapter 1
Figure 1.2.1: Human MHC gene map .......................................................................................................... 27
Figure 1.2.2: Timescale of MHC evolution ................................................................................................... 29
Figure 1.3.1: The number of antigens and alleles from 1968 to September 2008. ..................................... 32
Figure 1.3.2: HLA gene map illustrates clustering of immune system ......................................................... 34
Figure 1.5.1: Structure of HLA class I molecules ......................................................................................... 38
Figure 1.5.2: Ribbon structure of class I peptide binding groove ................................................................. 38
Figure 1.5.3: Structure of HLA class II molecule .......................................................................................... 39
Figure 1.5.4: Peptide in HLA peptide-binding groove .................................................................................. 40
Figure 1.5.5: Amino acid pockets in the floor of binding groove determine the binding of peptide ............. 41
Figure 1.5.6: HLA molecule epitopes ........................................................................................................... 42
Figure 1.6.1: Roles of peptide loading complex in MHC class I assembly and peptide loading. ................ 44
Figure 1.6.2: Endogenous peptide presentation pathway............................................................................ 45
Figure 1.6.3: Exogenous antigen presentation pathway .............................................................................. 46
Figure 1.6.4: Positive and negative selection of T-cell receptor repertoire .................................................. 49
Figure 1.6.5: Regulation of NK activity by HLA class I molecules ............................................................... 51
Figure 1.7.1: Effector mechanisms of rejection in graft carrying mismatched HLA class I antigen. ............ 55
Figure 1.7.2: Effector mechanism of rejection in graft carrying mismatched HLA class II antigens. ........... 57
Figure 1.7.3: Effector mechanisms of rejection in graft carrying mismatched HLA class I and class II
antigens ........................................................................................................................................................ 59

Chapter 3
Figure 3.4.1: T–B+ as a group was not associated with inferior graft survival .......................................... 119
Figure 3.4.2: Graft survival according to antibody specificity in T–B+ ....................................................... 120
Figure 3.4.3: Significance of DSA class in graft survival ............................................................................ 121
Figure 3.4.4: 5 years graft survival in T–B+ subgroups stratified by peak/current status .......................... 126

Chapter 4
Figure 4.4.1: Cumulative graph for biopsy events in relation to time post-transplantation ........................ 150
Figure 4.4.2: Graft survival in the controls and patients. ............................................................................ 154
Figure 4.4.3: Graft survival before and after diagnosis of TG .................................................................... 155
Figure 4.4.4: Cumulative TG diagnosis events in relation to time post-transplant, according to presence of
DSA ............................................................................................................................................................ 156
Figure 4.4.5: Graft survival in patients with TG according to presence of DSA ......................................... 158
Figure 4.4.6: Graft survival in TG subgroups, compared to the control group ........................................... 160

Chapter 5
Figure 5.4.1: Graft function of highly sensitized patients, compared to all grafts ...................................... 178
Figure 5.4.2: Total graft survival of highly sensitized patients, compared to all grafts .............................. 179
Figure 5.4.3: Correlation of graft function and BXM ................................................................................... 187
Figure 5.4.4: Significance of positive BXM in relation to graft survival ...................................................... 188
Figure 5.4.5: Specificity of HLA class I donor-specific antibodies in highly sensitized patients ................ 195
Figure 5.4.6: Specificity of HLA class II donor-specific antibodies in highly sensitized patients ............. 195
Figure 5.4.7: A trend of poor graft function in DSA group within 1 year post-transplant ........................... 198
Figure 5.4.8: Graft survival in the presence of DSA ................................................................................... 199

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iv. Summary

Identification of the complement C4d fragment in peritubular capillaries as a
specific marker for antibody mediated rejection in renal transplantation revealed the
critical role of antibodies in graft survival. In this thesis, I document the design and
findings of studies performed to investigate the clinical impact of anti-HLA antibodies
present before and/or after transplantation.
Over time, the detection techniques for anti-HLA antibodies has evolved from
the less sensitive complement-dependent lymphocytotoxicity (CDC) crossmatching (XM)
to more sensitive solid phase assays such as Luminex®. Studies have been conducted
to compare the predictive value of different antibody detection techniques.
The first result chapter presents antibody specificity in positive CDC B-cell
crossmatch (BXM), analysed with highly specific Luminex® assays. The study also
investigates the predictive value of BXM in the general transplant population. I found
that donor-specific anti-HLA antibodies (DSA) are only present in one third of positive
BXM and are associated with poor outcomes. The novel finding is that >80% of the DSA
detected by BXM are complement-fixing IgG and IgG subclasses.
1 3
Transplant glomerulopathy (TG) is type of chronic renal graft rejection. The
pathogenesis of TG is unclear. In the second result chapter, I report risk factors and
involvement of anti-HLA antibodies in the development of TG. This study shows that
glomerular rejection, delayed graft function, HLA presensitization and DSA have a
univariate effect on TG development. Multivariate analysis revealed that DSA are an
independent predictor of TG, after adjustment for other risk factors.
I have further investigated the role of BXM in a unique, well-matched, highly
sensitized patient group transplanted under the national renal exchange programme. I
compared Luminex® antibody analysis with BXM in predicting transplant outcomes. In
highly sensitized patients, DSA are found in two thirds of positive BXM. In univariate
analyses, BXM is associated with humoral rejection whereas DSA defined by Luminex®
are associated with total and all rejection types. The major finding is that, by
multivariate analysis, DSA defined by Luminex® are an independent predictor of total
and humoral rejection, but BXM are not. These interesting findings are reported in the
third result chapter.
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Studies reported in this thesis define the clinical significance of anti-HLA
antibodies in renal transplant outcomes. Method comparison studies provide useful
information on antibody specificity and their impact on graft survival. Collectively, a
better understanding of alloantibodies associated with graft rejection and limitation of
antibody detection methods may facilitate donor selection and choice of
immunosuppressants, and consequently improve transplant outcomes.

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