Textbook of Kidney Transplantation Vinant Bhargava, Manisha Sahay, Vinay Sakhuja, Priti Meena, Amit Gupta
Page numbers followed by b refer to box, f refer to figure, fc refer to flowchart, and t refer to table
ABO antigens 164
ABO-compatible 381, 383
ABO-incompatible 102, 103
desensitization protocol 381f
graft 164
kidney transplantation 370, 376, 378, 380f, 382, 384
renal transplantation, outcome of 382t
perinephric 361
renal 362f
Absorption 115, 121
Acanthamoeba 230
Acarbose 296
Acellular pertussis 436
Achilles heel 34
Acid-fast bacillus 250, 289
Acidosis 117t
Acquired immunodeficiency syndrome 207, 227, 238
Actinomyces 256
Activated partial thromboplastin time 56
Active antibody-mediated rejection 163, 164
Acute allograft dysfunction 96
causes of 96
Acute allograft rejection 142
Acute antibody mediated rejection 145, 389
Acute coronary syndrome 86, 87
Acute graft dysfunction 95, 96, 97fc, 98fc
clinical presentation of 96
evaluation of 97, 98
Acute respiratory distress syndrome 223, 397f, 398
Acyclovir 207
Adefovir dipivoxil 205
deaminase 277
monophosphate 41f
triphosphate 41f, 115, 292
Adenoviral nephritis 171
Adenovirus infection 170, 189
Adiponectin 292
Adipose tissue derived mesenchymal stem cells 42f44f
Advisory committee 433, 459
Aggressive fluid therapy 337
Ahmedabad tolerance induction protocol 42f
Air crescent sign 219
aminotransferase 269
glyoxylate aminotransferase 334
Albumin excretion rate 45, 51
Albuminuria 51
Alemtuzumab 102, 103
Alloantibody direct against polymorphic antigens 35
Allograft biopsy interpretation 160
Allograft compartment syndrome 73
Allograft dysfunction 87
causes of 413f
Allograft endothelium 150f
Allograft function 427
Allograft injury 150f
Allograft loss, risk factors of 347t
Allograft nephrectomy
advantages of 352t
disadvantages of 352t
indications of 352t
Allograft parenchymal complications 363
Allograft rejection 142, 143
Alloimmune injury 148
Allopurinol 131, 133, 316
Allorecognition, direct pathway of 26
Alpha-glucosidase inhibitors 296
Alport syndrome 52
Alveolar spaces 239f
Ambulatory blood pressure monitoring 49, 281, 282
American Association for Study of Liver Diseases 197
American College of Cardiology 261
American College of Endocrinology 290
American Diabetes Association 264, 295
American Heart Association 261
American Society of Transplantation 187, 189, 427
Infectious Diseases Community of Practice 432
Amikacin 258
Aminoglycosides 207
Aminosalicylates 133
Aminosalicylic acid 133
Amoebic dysentery 231
Amoxicillin 258, 270
Amphotericin 207
B deoxycholate 229
Amsterdam 111
Amyloid 160
light-chain 57
Amyloidosis 57
Ancillary tests 160, 396, 409
Anemia 86, 139, 315
causes of 315t
Aneurysms, intracranial 58
Angioplasty, percutaneous transluminal 73, 358
Angioregression 345
Angiotensin-converting enzyme 284, 317f
inhibitors 128, 133, 260, 282, 283, 296, 314, 315, 319, 325
Angiotensin-receptor blocker 36, 128, 260, 282, 296, 315, 316, 319, 323
therapy 283
Anisakiasis 235
Anterior rectus sheath flap, tension-free closure of 74f
prophylaxis 246
susceptibility testing 257
Antibody 22f
against nonhuman leukocyte antigens, detection of 35
dependent cellular
cytotoxicity 7, 27
toxicity 150
donor-specific 3, 10, 22f, 23, 26, 29, 30, 30f, 31, 31t, 34, 41, 83, 84, 95, 98, 103, 104, 129, 144, 145, 155, 163, 344, 353, 372, 374, 388
generation 27
immunoglobulin G 398
mediated rejection 7, 26, 32, 34, 83t, 95, 137, 144, 146, 151f, 153, 155, 160, 163, 191, 282, 344, 351, 373, 382, 383
chronic 138, 154t, 163, 164
management of 144t
pathogenesis of 149
testing 20f
Anti-CD20 monoclonal antibody 156
Anticholinergics 91
Antidiabetic drugs 296t
Antiendothelial cell antibodies 28
Antiepileptic drugs, pharmacokinetics of 276t
Antigen presenting cell 4f, 6f, 7f, 36, 136, 143, 148
Antiglomerular basement membrane 60, 329
Anti-hepatitis C virus antibody 412
Anti-human globulin 16f, 17, 43
Anti-human leukocyte antigen 22f, 88, 136, 378, 440
antibody testing, evolution of 16f
Antihypertensive medications 284t
Antilymphocyte globulin 100
Antimetabolites, effect of 293
Antineutrophil cytoplasmic antibody 329
Antiphospholipase A2 receptor antibody 325
Antiphospholipid 329
antibody 59, 75
Antiretroviral therapy 208
combined 207
Antithymocyte globulin 21, 42f, 84, 101103, 121, 133, 134, 155, 173, 178, 181, 191, 207, 213, 238, 350, 371, 373, 374, 381f, 429
Antitubercular therapy 249, 252
Anti-tumor necrosis factor 326
Antivirals 204
prophylaxis 204
therapy 205
timing of 197
Anxiety disorder 452
Apex swap transplant registry 389
Apnea test 395, 409
Apolipoprotein 52, 261
Arrhythmias 262
Arterial anastomosis 65
Arterial blood gas 409
Arterial intimal fibrosis 151
Arterial thrombosis 86
Arteriovenous fistula 282, 358, 404
Artery 67f
catheter, pulmonary 411
hypogastric 66
systolic pressure, pulmonary 263
Artificial urinary sphincter 90
Aspartate aminotransferase 204, 269
Aspergillosis, invasive 219, 220t
Aspergillus 219
fumigatus complex 219
infections 219
Aspiration 234, 235
Aspirin 260
Association of British Clinical Diabetologist and Renal Association 294
Athena trial 133
Atovaquone 241
Atrial fibrillation 58
Atypical hemolytic uremic syndrome 439
Augmentation cystoplasty 91, 92
Australia New Zealand Dialysis Transplant Data System 308, 324
Autoantibody 34
Autosomal recessive congenital nephrotic syndrome 168
Azathioprine 109, 120, 121, 131, 133, 134, 134t, 135, 191, 263, 309, 316, 352
drug interactions of 133t
metabolism 132fc
Azithromycin 118, 231
Babesia microti 230
Babesiosis 230, 235
Bacillus calmette-guérin 435
Bacteria 182
Bacteriuria, asymptomatic 245
Balamuthia 230
Banff categories 162t
Banff classification
scheme 34
system 143
Banff schema 160
Banff scores 160
Barrier methods 430
Barry technique 68
Basal cell carcinoma 310
Basic kidney disease 60
Basiliximab 101103
B-cell 7
lymphoma, diffuse large 172, 308
neoplasms 172
receptor 27, 149
role of 7, 46
Behcet's disease 11
Belatacept 136138, 140, 157, 348, 429
De novo use of 137
mechanism of action of 136f
resistant rejections 139
Beta-cell apoptosis 292
Betaine 338
Bibliotherapy 455
Biochemical abnormality 87, 302
proven acute rejection 111, 121, 124, 127, 133, 134, 348
technique 355
BK nephropathy 188, 191fc, 346
BK viremia 104, 191fc
BK virus 98, 118, 127, 128, 177, 179, 186, 187, 188t, 190, 190t
diagnostic of 170
disease 189
infection 123, 177, 186t
nephropathy 96, 129, 170, 186, 187, 189, 190, 190t, 191, 348
polymerase chain reaction 188
viremia, absence of 191
Bladder 89
abnormality 89, 90fc
assessment 60
augmentation 92, 92f
dysfunction, management of 90fc
incision 92f
neurogenic 89
normal 89
overactive 91
pretransplant workup of 89
Blastocystis hominis 231
renal 117
Yin-yang pattern of 367f
group 398, 402, 412
antibodies, role of 379
antigens 379
compatibilities 379, 379f
oxygenation level dependent 364
pressure 49, 85, 136, 282, 283, 284, 295, 297, 428
diastolic 49
elevated 118
monitoring, self 282
systolic 49, 283
product 57
protozoan infections 227
Bloodstream infection, catheter related 177
B-lymphocytes 100
Boceprevir 199f
fluids 379
mass index 4951, 269, 289, 296, 303f, 443, 445
surface area 49
weight 44f
biopsy 303
disease 304
post-transplantation 109
disorders 358
spectrum of 301f
loss, post-transplantation 303f
marrow 39
edema 278
examination 314
suppression 123, 132
mass 303
metabolism 303f
density 109, 305
disease 301
disorder 261
specific alkaline phosphatase 305
Bortezomib 21, 42f, 43f, 156, 278
Botulinum toxin
A 91
substrate 132
abscess, magnetic resonance image of 219
death 394, 395t, 404
diagnosis 408
donation pathway 408
infections, focal 277
injury, traumatic 409
activity 395
death 395, 408
assessment of 395t
pathophysiology of 397, 397f
prerequisites for 409
reflexes 395, 396
Breast cancer 308
Breastfeeding 135, 430
Brincidofovir 184
British Transplantation Society Guideline 352
Bronchoalveolar lavage 227, 238, 256, 277
Bulldog clamps 65
Burkitt lymphoma 172
Cadaveric transplant 83
Calcineurin inhibitors 38, 85, 97, 98, 100, 114, 117t, 118, 119, 127, 128, 136, 153, 155, 162, 164, 179, 190, 191, 198, 208, 220, 249, 263, 276, 278, 282, 290, 292, 293, 306, 309, 318, 326, 344, 347, 350, 371, 373, 374, 428
levels 117t
mechanism of action of 114fc
nephrotoxicity 117
acute 9698
neurological side effects of 278t
nonrenal side effects of 118
toxicity 97, 137, 164, 346
spectrum of 166f
Calcium 304
channel blockers 118, 282, 284
oxalate crystal deposition, Birefringent fan-shaped 337f
Calcofluor white stain 224f
Cancer 187, 255, 308
donor-related 309, 310
screening 57, 312, 448
Candida pyelonephritis 171
Candidemia 221
Candidiasis 220
invasive 220, 221
Carbamazepine 133, 276
hypersensitivity 11
Carbon dioxide 79
Carcinoma, hepatocellular 271, 316
Cardiac death, donation after 407
Cardiac failure, congestive 260
Cardiopulmonary resuscitation 410f
Cardiovascular disease 57, 260, 265, 446
advanced untreatable 412
screening for 261
spectrum of 262
Cataracts 108
Catastrophic brain injury 395
care 86
directed thrombolysis 75
Cauda equina syndrome 89
Cefotaxime 258
Ceftriaxone 258
Cell death, activation-induced 6
Cellular rejection, acute 96, 114, 134, 281
Centers for Disease Control and Prevention 84
Central nervous system 212, 219, 227, 256, 275, 409, 410f, 446
dysfunction 275
malignancy 279
tumors 310
Central venous
catheter 85
pressure 85
Cerebral angiography 395
Cerebrospinal fluid 276, 277
Cerebrovascular accident 275
irreversible 412
Cerebrovascular disease 279
Cesarean section, lower segment 26
Cestodes 234
Chagas disease 228, 235
Chagasic cardiomyopathy 228
Chemokine, urinary 190
Chemotherapy 311
computed tomography scan of 53, 238
radiographs 251f
X-ray 53, 57, 238
Chickenpox 132
Chimeric tolerance 40
Chloroquine 230
Cholecystitis 58
Cholelithiasis 58
Cholera 434
Chromic silver methenamine stain 172
Chronic allograft dysfunction 344
Chronic allograft injury 343, 344, 344fc, 346, 348
early detection of 347
mechanism of 345
Chronic allograft nephropathy 129, 134, 344
Chronic obstructive pulmonary disease 316
Cincinnati transplant tumor registry 308
Circulatory death, donation after 137, 404, 409
Cirrhosis 438
decompensated 57
Citrate 338
Clavien-Dindo system, Kocak-modified 54
Clazakizumab 157
Clindamycin 241
Clonal anergy 40
Clonal deletion 40
Clozapine 133
Cognitive disorder 452
Cold ischemic time 63
Colony-forming units 244
Column agglutination technique 379
Combined kidney pancreas transplantation 443
Combined liver 440
kidney transplantation 438
Common central nervous system infections postrenal transplantation 277t
cytotoxicity test 100
microlymphocytotoxicity test 10
Complete blood count 131, 398
Computed tomography 60, 73, 75f, 76f, 244, 250, 276, 289, 311, 356, 364, 395
angiogram 77f
contrast-enhanced 356
scan 251f
high-resolution 240, 248
urography 95
Conception, timing of 427
Connective tissue growth factor 345
Constipation 79f, 139
Continuous ambulatory peritoneal dialysis 84, 413
Continuous positive airway pressure 409
Contraception 430, 430t
Contraceptive method 430
Coronary artery disease 260, 262
screening 57
Coronavirus 215
Cortex 356f
Cortical-rim sign 359f
Corticosteroids 116, 242, 278, 292
therapy 255
Costimulatory molecules 7f
Costovertebral angle 136
Cotrimoxazole 133, 241
Cough 139
reflexes, absence of 396
COVID-19 59, 215, 434
infection 164, 191, 215, 222, 450
concomitant 191
messenger ribonucleic acid vaccines 123
pandemic 215, 222
pneumonia 182
moderate-severe 350
vaccination 433
vaccine 59, 138, 434
Cranial nerve response, absence of 396
C-reactive protein 178, 261, 315
Creatinine 337, 337t
Cross-reactive epitope group 32
Cryptococcosis 221, 222t
Cryptogenic cirrhosis 440
Cryptosporidiosis 231
Cryptosporidium infections 231
Crystalline nephropathy 160
Cuboidal metaplasia 239f
Cultured donor bone marrow 42f44f
Cushingoid appearance 108
Cyclic adenosine monophosphate 293
Cyclosporine 131, 134, 189, 259, 263, 273, 278, 293, 327
intravenous 327
Cystinosis 52
Cystogram 79f
Cystoisospora belli 231
Cystoscopy 90
Cysts, renal 52
Cytochrome P450 system 284t
Cytomegalic cells 182
Cytomegalovirus 50, 53, 56, 78, 84, 85, 102, 109, 118, 123, 128, 134, 168, 172f, 176178, 181, 183f, 184t, 189, 208, 212, 238, 261, 270, 271, 291, 315, 316, 347, 364, 383, 388, 398, 428
antigens 183f
disease, laboratory features of 182t
infection 170, 182, 185, 223, 255, 294
nephritis 170
vaccines 185
after kidney transplant, etiology of 317f
post-transplantation 314
Cytotoxic T lymphocyte 4f, 327
antigen 5, 7f
Cytotoxicity, complement-dependent 10, 16, 17t, 18t, 22f, 23, 28, 83, 150, 289, 350, 370, 374, 408
Daclatasvir 199, 199f
Dapsone 241
Daratumumab 157
Dasabuvir 199f
De novo
disease 160
donor-specific antibodies 29, 344
glomerulonephritis 166, 323
malignancies 309
membranous nephropathy 168
primary infection 249
purine synthesis, inhibition of 122fc
Death, clinical determination of 410f
Deep venous thrombosis 86
Deglycerate dehydrogenase 335
Delirium 452
Dendritic cells 5, 8
Dense deposit disease 60, 325
Deoxyribonucleic acid 3, 6f, 10, 11, 12f, 13f, 15, 41, 53, 122, 131, 132, 140, 152, 171, 181, 186, 190, 202, 229, 271, 277, 278, 309, 337, 347, 428
Depot medroxyprogesterone acetate 430
Dequalinium chloride 339
Desensitization 31, 380
Desmopressin 399
Diabetes 89, 264
control 264
and complications trial 291
insipidus 397f
central 397, 398, 399t
mellitus 10, 49, 51, 264, 282, 289, 295, 350
post-transplantation 87, 106, 108, 114, 115, 118, 197, 198, 244, 289291, 291fc, 293, 294, 296, 297, 344
Dialysis 339
after graft failure 351, 353
Diarrhea 132, 139
Dietary oxalate, restriction of 337
Diethylenetriaminepentaacetic acid 86, 357
Digital rectal examination 57
Digital subtraction angiography 73, 358, 359
Dilated renal pelvicalyceal system 362f
Dipeptidyl peptidase 264
inhibitors 296, 297
Diphtheria 434, 436
Diphyllobothriasis 235
Direct-acting antiviral
agents 271
therapy 56
Direct-antiviral agents 195, 199f
Direct-endothelial injury 150
Disseminated intravascular coagulation 397f, 399
Distal aorta 70f
Dithiothreitol 17, 32, 379
Diverticulitis 58
Domino paired kidney exchange 387
assessment of 451, 452b
care 393, 453b
hematopoietic stem cells 45
nephrectomy 62
quality 345
service areas 402
specific antibody 3, 10, 22f, 23, 26, 29, 30, 30f, 31, 31t, 34, 41, 83, 84, 95, 98, 103, 104, 129, 144, 145, 155, 163, 344, 353, 372, 374, 388
assessment of 30
attributes of 32t
monitoring of 30
testing for 152
specific transfusion 43, 44f, 45
role of 40
types of 461
Double filtration
plasma exchange 383
plasmapheresis 373, 378
Double kidney transplants 410
Drainage, percutaneous 361
development 120
drug interaction, clinical implications of 122
interactions 132, 198, 259, 284
level monitoring 127
resistance 184
toxicity 278
Dual-energy X-ray absorptiometry 304, 305, 358
Dyselectrolytemia 117, 117t
Dyslipidemia 108, 265
Dysplasia 89
Echinococcosis 234, 235
granulosus 234
multilocularis 234
Echocardiography 57
Eculizumab 21, 136, 156, 318, 329
Edema, peripheral 139
Ejaculatory dysfunction 425
Elastography, transient 195
Elbasvir 198, 199, 199f
Electrocardiogram 57, 84, 305, 410f
Electroencephalogram 410f
Electrolyte 411
disturbances 275
imbalance 275
Electron microscopy 160, 162, 329
Encephalitis 277
Encephalitozoon intestinalis 231
Encephalopathy, hypertensive 276
Endarteritis 163
Endocrinopathy management 398
Endophthalmitis 219
Endoplasmic reticulum 3
Endothelial cells 5, 26
Entamoeba histolytica 231, 235
Entecavir 205, 271
Enterocytozoon bieneusi 231
Enterohepatic recirculation 122
Enzyme-linked immunosorbent assay 15, 16f, 18, 227, 234
techniques 127
Epithelial mesenchymal transition 345
Epithelial sodium channel 282
Epstein-Barr virus 18, 53, 173, 176, 177, 213, 277, 308, 310, 310f, 315, 317f, 318, 348, 364, 388
Erythrocytosis, post-transplantation 314, 316, 319
Erythropoietin stimulating agents 314, 319
Esophageal candidiasis 208, 221
Esterase inhibitor 155, 156
Estimated glomerular filtration rate 4951, 129, 133, 155, 296, 302
Estrogen 430
Ethylenediaminetetraacetic acid 21, 23, 32, 116
European Society of Cardiology 261
Everolimus 199, 292, 310
Excretion 126
Exophytic heteroechoic mass lesion 365f
Expanded criteria donor 54, 282
External iliac artery 73f, 355, 360f
dissection 72
Extracellular vesicles 36
Extracorporeal machine oxygenation 407
Extracorporeal shock wave lithotripsy 339
Extracorporeal therapy 380f
Familial non-neuropathic systemic amyloid 439
Fasting glucose, impaired 294
Fatty liver disease
metabolic associated 272
nonalcoholic 270, 272, 438
Febuxostat 133
Fertility 425
Fibronectin 37
Fibrosis, interstitial 43, 89, 129, 164, 344
Field stains 230
Fistula, lymphocutaneous 77f
Florid follicular hyperplasia 172, 318
Flow crossmatch 42f, 83
reactivity 18t
Flow cytometry 379
crossmatch 17, 18f, 23t, 373
Flowpra screening 19f
balance 86
challenge 97
therapy, intraoperative 85
Fluorescein isothiocyanate 19
Fluorodeoxyglucose positron emission tomography 259, 357
Folic acid 260, 262
Follicle-stimulating hormone 425, 426f
Foreign antigens, recognition of 4
Foscarnet 207
Fournier's gangrene 264
Fracture 303f
risk assessment tool 303
Framingham risk score 262
culture 224f
hyphae 173f
infections 170, 171, 179, 219, 240
Fungi 182
Galactose 327
Gamma enzyme-linked immunospot assay 144
Gamma-glutamyl transferase 269
Ganciclovir 183, 184
Gastroenteritis 179
Gastrointestinal adverse reactions 123
Gastrointestinal disease 58
Gastrointestinal motility disorders 118
GeneXpert 250
Genomic pathways 107
Giardia duodenalis 231
Giardiasis 231, 235
Giemsa stains 230
Glaucoma 108
Gleason score 59, 310
Glecaprevir 198, 199, 199f
Glibenclamide 296
Gliclazide 296
Glimepiride 296
Glipizide 296
Glomerular basement membrane 162, 168, 169f, 196
Glomerular diseases 324
Glomerular filtration rate 50, 51, 51t, 118, 129, 134, 137, 205, 241, 246, 261, 302, 315, 335, 347, 445
Glomeruli 160
ischemic necrosis of 173f
Glomerulitis 165f
segmental 161f
Glomerulonephritis 60, 164, 166, 198, 323326, 345
after kidney transplantation
classification of 323
types of 324t
chronic 100, 142, 172, 202
membranoproliferative 167, 168, 196, 324, 326
post-transplantation 324
recurrent 166, 325t
secondary 329
Glomerulopathy, transplantation 147, 155, 165f
Glomerulosclerosis focal segmental 41, 60, 96, 114, 167, 324, 325, 346
Glomerulus 161f, 169f
Glucagon-like peptide 264
agonists 289
receptor 296
agonists 295, 297
Glucocorticoids 107, 241, 281, 282, 303, 304
direct effects of 302f
indirect effects of 302f
induced leucine zipper 107
receptor 107
responsive element 106, 107
6-phosphate dehydrogenase 229, 315
perioperative impaired 293
test 50, 291
Glucuronide mycophenolic acid glucuronide 122
Glutamine phosphoribosylpyrophosphate amidotransferase 131
Glycolate oxidase 338
Gomori's methenamine silver 221
dysfunction 78f, 95, 139
assessment 413
function, delayed 85, 134, 137, 282, 346, 353, 363
hydroureteronephrosis 77f
intolerance syndrome 352
biopsy 366
hydronephrosis of 363f
hydroureteronephrosis of 362f
parenchyma of 357f
pyonephrosis of 362f
nephrectomy 77, 172, 352
obstruction 77f
outcome 93
preparation 63
pyelonephritis 96, 98
rejection, effector phase of 5
renal artery 73f, 360f
types of 1f
Gram staining 257
Granular inclusions 170
Granulocyte macrophage colony stimulating factor 184, 317, 433
Granulomatous disease, chronic 255
Granulomatous tubulointerstitial nephritis 244
Grazoprevir 198, 199, 199f
Groningen 111
Ground-glass amorphous appearance 170
Grow urothelial cells 92
factor, transforming 182, 309
retardation 108
monophosphate synthetase 132
triphosphate 132
Haemophilus influenzae 433
Halo sign 219
Hand-assisted laparoscopic nephrectomy 62
Headache 139
disease, structural 260, 262
failure 262
congestive 262
Heat shock protein 107
Heavy chain deposition disease 57
Helicobacter pylori 310f
Hematologic disease 59
Hematological disorders 314
management plan for 319t
Hematoma, perinephric 360, 360f
Hematopoietic stem cells 40, 45
transplantation 255
Hematuria 86, 329, 367
microscopic 51
persistent 49, 51
Hemodialysis 84, 85, 261
Hemoglobin 49, 50, 56, 246, 314, 319
Hemolytic-uremic syndrome 60
Hemophagocytic syndrome 314, 318, 319, 319b
postoperative 86
subarachnoid 409
Hepatic dysfunction 269
Hepatic venous pressure gradient 196
Hepatitis 84
A 434
virus 434
acute 57
B 56, 59, 84, 177, 398, 412, 432, 435
antibody 53
core antibody 53, 59, 204, 398
immunoglobulin 204, 435
surface antibody 59, 398, 436
surface antigen 53, 59, 203, 412
vaccination against 59
virus 53, 59, 177, 178, 202, 205, 208, 270, 310f, 433, 440
B E-antigen 270
C 56, 177, 312, 398, 412
infection 56, 59
virus 53, 59, 177, 195198, 199f, 199t, 207, 209, 271, 289, 291, 294, 310f
chronic 59, 197
D virus 203
Hepatorenal syndrome 438
Hepcidin 314
Hernia 79
incisional 79, 79f
paratransplant 79
post-transplantation 79
Herpes simplex virus 177, 212, 270
treatment of 212t
Herpes zoster virus 212, 213, 213t
Herpesvirus infection 212
Highly active antiretroviral therapy 207, 208, 238
High-performance liquid chromatography 116, 127
Hinman-Allen syndrome 89
Histidine 85
rich protein 230
Histoplasmosis 53
Hodgkin's lymphoma 311, 351
classical 173, 318
Hoeppli-Splendore reaction 256
Hormonal contraception, combined 430
Hormone, parathyroid 56, 301, 303f, 304, 305
Horseradish peroxidase 12
Horseshoe kidney 70
Human cytomegalovirus 181
Human herpesvirus 310f, 311, 315, 317
Human immunodeficiency virus 50, 53, 56, 84, 202, 207209, 227, 238, 241, 255, 275, 289, 310f, 317f, 398, 412, 448
associated nephropathy 207
infection 187
Human leukocyte antigen 2, 2f, 3, 10, 11, 14f, 18, 19f, 20f, 22f, 23, 26, 29, 31, 32, 32t, 34, 40, 45, 46, 50, 76, 83, 102, 104, 147, 148, 164, 181, 186, 291, 325, 344, 347, 352, 370, 371f, 372, 373, 374t, 375, 376f, 387, 411, 450
advantages of 15t
antibody detection assays 16
compatibility 372
complex 10
disadvantages of 15t
matching 402
molecule, structure of 2f, 372f
nomenclature 2f
outcome of 375
sequenced-based 13f
structure 2
typing technologies 10
Human papillomavirus 214, 310f, 434
Human T-cell leukemia virus 215
Hydrocortisone 399
Hydronephrosis 283
Hydroureteronephrosis 78f, 362f
Hydroxy-3-methyl-glutaryl-coenzyme A 108
Hydroxypyruvate reductase 335
Hyperacute rejection 143
Hyperaldosteronism, primary 283
Hyperdense collection 360f
Hyperglycemia 397
management of 294
pathogenic mechanisms for 293t
preoperative care of 85
severe 85
transient post-transplant 290
Hyperinfection syndrome 232
Hyperkalemia 117, 139
transient 119
Hyperlipidemia 118
Hypernatremia 399
Hyperoxaluria 52, 335, 336f, 337f, 439
acquired 335
dietary 336
enteric 335
secondary 335
systemic manifestations of 336
Hyperparathyroidism 301
myointimal 166
plasmacytic 172, 318
Hypersensitivity, delayed-type 46
Hypertension 10, 49, 52, 87, 108, 117, 118, 136, 139, 263, 281, 282, 285, 296
gestational 53
mechanism of 117t
post-transplantation 282, 346
pulmonary 58, 263
resistant 285
secondary 282
treatment of 283
Hypokalemia 139
Hypomagnesemia 117, 294
Hypotension 75f, 87
Hypothalamic-pituitary-gonadal axis 425
Hypovolemia 98
intravascular 74
Hypoxanthine guanine phosphoribosyltransferase 122, 132
Hypoxia 316
Iliac artery, common 66f
Iliac fossa 355, 362f
Iliac vessel
dimension 60
Doppler of 57
Imipenem 258
kidney disease 207
mediated membranoproliferative glomerulonephritis 325
memory, assessment of 29
monitoring assays 183
system 3
Immunity, cellular arm of 38
Immunization, passive 435
Immunobiology, transplantation 1
Immunofluorescence 162, 169f, 329
A 110, 164, 269, 281
nephropathy 167, 324, 325
G 19f, 31, 32, 83, 150, 164, 181, 372, 381f
endopeptidase 157
intravenous 21, 142, 144, 145, 155, 156, 184, 319, 373, 374, 380, 381f, 383, 435
M 17, 18, 164, 183, 227, 270, 398
nephropathy 325
Immunohistochemistry 160, 162, 171f, 172f, 183f
Immunological memory 28
Immunology, transplantation 4
Immunopathogenesis 227
Immunosuppression 83, 136, 309, 350, 380f
chronic 212
effect of 425
management of 351, 428
modification of 260, 264, 296
post-transplantation 205, 209
related factors 281
role of 309fc
therapy 198
withdrawal of 351
Immunosuppressive drugs 100, 293t, 296, 316
Immunosuppressive medications 199t
role of 303
Immunosuppressive therapy 205
Impaired glucose tolerance test 289
Incision 64
Indian Society of Nephrology 432
Indiana approach 440
Induction antithymocyte globulin 260
Induction immunosuppression therapy 104fc
classification of 101t
Induction therapy 100, 103t, 104
Infections 53, 59, 88, 123, 248, 276
malarial 230
primary 181
severity of 241t
chronic 275
granulomatous 160
interstitial 165f, 167f
microvascular 150, 165f
Inflammatory cytokines 345
Influenza 219, 433
ischemic 346, 74fc
nonimmune causes of 347
procurement 345
Inosine monophosphate dehydrogenase 121, 122, 132
Inotropes 411
Insulin 260, 399, 411, 445
Intensive care unit 138, 404, 408, 450, 460
Interferon 4f, 32, 46, 205
gamma release assays 251
regulatory factor 107
Interleukin 6f, 41f, 107, 114, 261, 315
blockade 157
receptor 381f
antagonists 101, 103
Internal iliac artery 64
International Kidney Paired Donation Programs 388
International Pancreas Transplant Registry 443
International Registry in Organ Donation and Transplantation Registry 406, 406t
International Society of Nephrology Global Kidney Health Atlas Methodology 406
Interstitial lymphocytic infiltrate, mild 172
Interstitium 160
Intestinal nematodes 232
Intracranial pressure 397f
Intraglomerular thrombus 167f
Intranuclear inclusion 172f
Intrauterine devices 430
Intrauterine growth retardation 428
Intravesical leadbetter-politano technique 68
Isavuconazole 220
cortical 345
reperfusion injury 29, 381f
Isoniazid 252, 270
Isotonic saline 398
Israel Penn Transplant Tumor Registry 308
Japanese encephalitis 434
John Cunningham
polyomavirus 214
virus 277
Kaposi sarcoma 311
Ketoglutarate 85
Kidney 98, 379, 439, 443
allocation 404t
principles 403b
allograft 355
biopsy, composite picture of 151f
failure 343
rejection, acute 144, 145fc
biopsy 56
frozen section of 413
disease, management of 305
disorders 304t
congenital anomalies of 93
disease 283, 323
chronic 52, 202, 255, 261, 273, 282, 303f, 323, 337, 343, 370, 406, 425, 426f, 428, 432, 438
diabetic 120
end-stage 34, 50, 56, 136, 142, 147, 195, 244, 261, 301, 324t, 343, 352, 387, 425, 438, 443
genetic 56
improving global outcomes 109, 144, 188, 196, 204, 242, 262, 282, 303, 304, 310, 314, 426, 429
polycystic 269
pre-chronic 260
donor profile index 136, 347, 401
function 412
stage 304
glomerular disease 324t
graft implantation 63t
injury, acute 86, 328, 336, 438
insufficiency, chronic 439
paired donation 23, 387, 388f, 390t
preparation 65
replacement therapy 147, 339
solid organ response test 144, 153
stones 52
transplantation 26, 34, 38, 46t, 58, 61, 61f, 72, 88, 100, 101f, 101t103t, 106, 108t, 121, 124, 128t, 129, 142, 147, 176, 178, 208, 261, 279, 289, 301f, 305fc, 314, 318, 319t, 334, 352, 370, 371f, 378, 425, 439441
histopathology of 160
incompatible 373, 374t, 375, 376f, 387
infections in 176
isolated 339, 340
post-transplant outcome of 205
recipient 4f, 56, 83, 111, 129, 133, 176, 181, 191fc, 202, 219, 239b, 244, 246, 246b, 260, 262, 269, 275, 276, 281, 283b, 289, 294, 297, 301, 303t, 308, 314, 426, 428, 428t, 430t, 432, 434, 435, 435t, 450
surgical techniques of 61
total 394f
ultrasound of 57
Killer cell immunoglobulin-like receptors 151
Klebsiella pneumoniae 178
Lacosamide 276
Lactate dehydrogenase 240, 317, 328, 334
Lactation 119
Lamivudine 204
Lamotrigine 276
Laparoscopic right donor nephrectomy 62
Late allograft failure, causes of 351
Ledipasvir 198, 199
Leflunomide 189
Left ventricular hypertrophy 260
donovani 228
infection 228
Leishmaniasis 228
cutaneous 235
mucocutaneous 235
Leptin 292
Lesch-Nyhan syndrome 120
Letermovir 184
Leukocytes 56
Leukocytosis 361f
Leukoencephalopathy, progressive multifocal 177, 214, 277
Leukopenia 86, 139, 314
drug-induced 316
post-transplantation 316
Levetiracetam 276
Levonorgestrel 430
Leydig cell dysfunction 425
Light chain deposition disease 57
Linagliptin 296
Linezolid 258
Lipase 398
high-density 269
low-density 118, 265, 269, 296
Liposomal amphotericin B 229
Live attenuated vaccines 123
Live donor kidney transplant 387
Live vaccines 432
Liver 334, 439, 446
disease 58, 273
causes of 270t
chronic 438
polycystic 273
post-transplantation 196
staging of 195
enzymes 56
abnormal 269
function 412
test 56, 86, 131, 398
injury, drug-induced 272
stiffness measurement 269
transplantation 338
Living donor 461
kidney 65
transplantation 104fc, 370
liver transplantation 340
Living kidney donation
contraindications for 50b
risk factors for 49t
Living kidney donors 54, 402
evaluation of 49, 50t
Lomentospora prolificans 223
Loop-mediated isothermal amplification 240
Low ionic strength solution 379
Lumasiran 338
assay 19
based reverse sequence-specific oligonucleotide method, principle of 13f
crossmatch 21, 22f
platform-based assays 19
sequence-specific oligonucleotide 12
Lung 446
alveoli, histopathology of 239f
injury, acute 397f
lesions 256
Lupus nephritis 10
Luteinizing hormone 426f
Lymphatics, ligation of 64
Lymphocele 76, 77f, 86, 96, 361
posterolateral 77f
assay, modifications of 16, 17t
crossmatch 42f
tests 2
Lymphoid tissue 36
Lymphoma 215
Lymphopenia 316
Maastricht classification 404t
Macrophage colony-stimulating factor 302f
Magnesium concentration 338
Magnetic resonance
angiography 73
imaging 234, 256, 276, 277, 306, 355, 357, 395
Major depressive disorder 452
Major histocompatibility complex 2, 2f, 4f, 7f, 20, 26, 34, 36, 39, 41f, 370
Malaria 229, 235
Malignancy 52, 59, 118, 209, 308, 312, 316
recurrent 309
Malnutrition 275
Mammalian target of rapamycin 38, 75, 118, 126, 127, 153, 155, 212, 259, 265, 291, 293, 315, 317f, 319, 326
complexes 126
inhibitor 101f, 126, 127, 127tt, 128, 128t, 292, 293, 429
mechanism of action of 126f
Maribavir 184
Mean arterial pressure 398, 411
Mean fluorescence intensity 30, 32, 83, 137, 155, 156, 373, 374
Measles, mumps, and rubella 132
vaccine 435
Medicolegal cases 407, 463
Medulla 356f
Meglitinide 296
Membranoproliferative glomerulonephritis, complement-mediated 325
Meningitis 277
Mental status 454
Mercaptoacetyltriglycine 357
Mesangial granular immunoglobulin A 169f
Mesenchymal stem cells 40
role of 40
Messenger ribonucleic acid 126, 188, 229
Metabolism 115, 122, 126, 131
Metformin 296
Methicillin-resistant Staphylococcus aureus 364
Methylmalonic aciduria 439
Methylmercaptopurine 132
nucleotide 132
Methylprednisolone 191, 399
Microbiology 240, 245
Microparticle enzyme immunoassays 116
Micro-ribonucleic acids, quantification of 145
Microsporidiosis 231, 235
Miltefosine 229
plus paromomycin 229
Mineral bone disease 301
Minimum inhibitory concentration 257
Mini-Pfannenstiel incision 62
Minocycline 258
Minor histocompatibility antigens 3, 34
Mitogen-activated protein kinases 6f, 106, 107
Molecular blood biomarkers 144
Molecular microscope diagnostic system 153
Molecular mismatch analysis 32t
Monoclonal deposition disease 160
Monocyte chemoattractant protein 345
Mononucleosis, infectious 172, 318
Monophosphate 122
Motivation enhancement therapy 456
Motor cranial nerve 395
Mucormycosis 222, 222t
risk factors for 289
dysfunction syndrome 235
transplants 139
Multiple inflammatory mediators, steroids reduce expression of 108
Multivitamins 260
Mumps 436
Muscle relaxants 91
Musculoskeletal manifestations 301
Mycobacteria, nontuberculous 253
Mycophenolate 344
mofetil 41, 87, 108, 111, 114116, 118, 120, 121t, 122124, 131, 134, 155, 166, 178, 190, 191, 199, 207, 209, 219, 231, 238, 244, 255, 260, 263, 281, 289, 293, 309, 323, 350, 371, 374, 380f, 381f, 426, 429
clinical pharmacokinetics of 122f
mechanism of action of 122fc
sodium 120
steroids sparing study 133, 134
Mycophenolic acid 103, 120122, 122f, 124, 129, 139, 189, 191, 316, 426
drug level monitoring 124
glucuronide 122f
Myelodysplastic syndromes 314
Myeloid neoplasms 319
Myeloma, multiple 57
Myocardial perfusion 58
Myocytes, vacuolation of 166f
fowleri 230
infections 231
Naïve B cells 27
Nateglinide 296
National Glycohemoglobin Standardization Program 291
National Institutes of Health 16, 16f, 17
National Organ and Tissue Transplant Organization 403, 403b, 408, 412, 464
Natural killer cells 7, 29, 36, 46, 150
Natural orifice 62
transluminal endoscopic surgery 62
Nausea 132, 139
Necrosis, ischemic 173f
Necrotic mediastinal lymph nodes, enlarged 251f
Nedosiran 338
Negative predictive value 153, 52
Neoplastic diseases 123
Nephrectomy 62
pretransplant 60
retroperitoneal laparoscopic 62
Nephrocalcinosis 334
bilateral 335f
Nephrolithiasis, recurrent 334
Nephron loss 167
Nephropathy 106
diabetic 207
ischemic 283
membranous 60, 196, 324, 325, 328
Nephrostogram, antegrade 78f
Nephrostomy, percutaneous 78f
chronic 119
drug-related 364
Neurological disease 58, 275
evaluation in 58
Neurological disorder 215, 275
Neuromodulation 91
Neuroschistosomiasis 233
Neurotoxicity, mechanism of 278
infiltrate of 161f
suppression 46
New-onset diabetes after transplantation 137, 168, 197, 264, 290, 351
Next-generation sequencing 10, 14, 15, 144
Nippostrongylus brasiliensis 256
Nocardia 255, 256
farcinica 255
nova 256
otitidiscaviarum 256
Nocardiosis 255, 256, 259
cutaneous 257
invasive 256
lymphatic cutaneous 256
pulmonary 257
Nomenclature 2, 289
Nondestructive post-transplant lymphoproliferative disorder 172
Nondihydropyridine calcium channel blockers 263
Nondirected anonymous donors 387
Nonheart transplant 228
Non-Hodgkin's lymphoma 308
Nonhuman leukocyte antigen 18f, 34, 36, 36t
antibodies 29, 35, 152, 164
Nonmelanoma skin cancer 310
Nonnucleoside reverse transcriptase inhibitors 208
Nonsteroidal anti-inflammatory drugs 270
Nontransplant organ retrieval centers 407
Normal transplant kidney 357f
Doppler features of 356f
Nuclear factor kappa-beta, receptor activator of 302f
Nuclear medicine 357
Nucleic acid amplification testing 53, 183, 195, 250
Obesity 51, 60, 265
central 293
prevalence of 265
Obstruction 89
Obstructive sleep apnea 282, 283
Ofatumumab 327
Oligoanuria, postoperative 87
assay, sequence-specific 12f
probe, sequence-specific 10, 11
Ombitasvir 199
Oncogenic viruses 310
Oral antibiotic regimens 258
Oral contraceptive pills 270
Oral glucose tolerance test 289, 294
Oral hypoglycemic agents 260
Oral polio vaccine 435
Oral therapy 258
allocation 401, 412, 414, 464
systems 401
donation of 463
maintenance 410
specific management of 398
procurement 102, 209
retrieval 411
transplantation 102, 187, 209, 413
awareness 226
evolution of 393
recipients 234fc, 235t
solid 255, 451
Osteomalacia 301
Osteonecrosis 301, 305
Osteopenia 303
Osteoporosis 108, 301, 303
prevalence of 304
Osteoprotegerin 302f
Owl's eye appearance 182
Oxabact 338
Oxalate 334, 337, 337t
metabolism 334
reduction strategies 340
Oxalobacter formigenes 334, 338
Oxalosis, systemic 336
Oxazyme 338
Oxybutynin 91
Packed cell volume 319
Packed red cells 57
Pain syndrome 278, 306
Pancreas 64, 379, 443
transplants 443
Pancreatitis, chronic 58
Pancytopenia 314, 316, 317
Panel reactive antibody 10, 23, 148, 352
Papanicolaou test 215
Parasitic diseases 226
Parasitic infection 226, 235t
post-transplantation 235
Parathyroidectomy 60
total 305
Parenchymal renal disease 438
Paritaprevir 199
Paromomycin 229, 231
Partial thromboplastin time 411
Parvus et tardus 281
Passenger lymphocyte syndrome 318
Peak systolic velocity 73, 356
Pediatric donor
en bloc kidneys 69
kidneys, preparation of 71f
Pediatric kidneys, transplantation of 69, 69f, 285
Penicillium brevicompactum 120
Pentamidine 241
Peptic ulcer disease 58
Peptide major histocompatibility complex 149f
Perigraft hematoma 74, 75f
Perinephric collection 88, 360
Periodic acid-Schiff 162, 169f
Peripheral blood 145
mononuclear cell 46, 347
stem cells 43
Peripheral nervous system 275
dysfunction 276
Peripheral vascular disease, evaluation of 447
Peritoneal cavity 70f
Peritoneal dialysis 84, 85, 339, 352
Peritubular capillaries 151, 155, 345
Perlecan 37
Peroxidase, streptavidin-horseradish 12f
Peroxisome proliferator-activated receptor gamma 302f
Pertussis 434
Pfizer vaccine 138
Pharmacokinetics 106, 131, 276t
clinical 115, 126
Pharmacotherapy 264
Phenobarbital 276
Phenotype panels 20
Phenytoin 276
Pheohyphomycosis 223
Phosphatase, dual-specificity 106
disorders 301
urinary 338
Phospholipase 107
Phycoerythrin, streptavidin-conjugated 12
Pibrentasvir 198, 199
Pioglitazone 296
cells 100
myeloma 172
exchange 144, 327, 383
glucose, fasting 290
Plasmacytoma 172
Plasmapheresis 156, 318, 327, 374, 381f
Plasminogen activating factor-1 345
Plasmodium falciparum 229
Platelets 56, 57
derived growth factor 345
ratio index 204
Pneumococcal conjugate vaccine 433, 436
Pneumococcal polysaccharide 436
vaccine 436
Pneumocystis 239, 240
carinii 238
jirovecii 179, 223, 238, 249
pneumonia 84, 123, 250
pneumonia 223, 241, 241t
diagnosis of 240t
infection 208
prophylaxis of 242t
risk factors for 239b
treatment of 241t
Pneumonia, community acquired 255
Polio 132
Polyclonal globulins 102t
Polycystic kidney disease 269
dominant 52, 58, 63, 207, 270, 273, 291, 316
recessive 270
Polycystic liver disease 273
Polymerase chain reaction 10, 11, 13f, 14f, 15, 187, 188, 191, 203, 212, 220, 227, 228, 234, 240, 250, 257, 277, 278
Polyomavirus nephropathy 160, 164, 344
Posaconazole 220
Positive end-expiratory pressure 411
Positive predictive value 153
Postchemotherapy 89
Posterior reversible encephalopathy syndrome 276, 278
Post-kidney transplantation course 204
Postradiation 89
Postrenal transplantation 266fc
alterations 301
recipients 261t
Post-transplantation anemia 314, 315, 315fc, 319
causes of 314
prevalence of 314
treatment of 315
Post-transplantation diabetes mellitus 197, 198, 289, 291, 291fc, 293, 294, 296, 297
diagnosis of 290, 291t
impact of 297
incidence of 290
management of 294, 295b
pathogenesis of 290
prevalence of 290
screening of 290
Post-transplantation hypertension 282, 346
etiology of 281
mechanism of 282fc
Post-transplantation lymphoproliferative disease 102, 118, 129, 137, 145, 172, 173, 177, 197, 214, 277, 308, 311, 357, 365, 376
risk of 197
Post-transplantation lymphoproliferative disorder 308, 309, 314, 318
Post-transplantation malignancy 129, 308, 310f, 312
classification of 309t
etiopathogenesis of 309fc
Post-transplantation tuberculosis 250t, 251f, 253
recurrence of 253
Post-traumatic stress disorder 452, 454
Post-tuberculosis 89
Postvoid residual urine 90
Praziquantel 233
Prednisolone 255, 311
Preeclampsia 53
Pre-existing neurological disease 275
Pregnancy 57, 119, 123, 135, 187, 426, 427
management of 427
risks 53
Pretransplant hemolytic uremic syndrome 318
Primaquine 241
Probiotics 339
Progestin 430
only pills 430
Propantheline bromide 91
Propè tolerance 38
Prostate cancer 308
Prostatic enlargement 89
Proteasome inhibitors 156, 373
creatinine ratio 126
serum 56
Proteinuria 329
Prothrombin time 56
Proton pump inhibitor 84
Proximal tubules 166f, 297
Prune belly syndrome 89
Pseudoaneurysm 76, 77f
juxta-anastomotic 76
Pseudomonas 240
aeruginosa 245
Psoas muscle 70f
Psychiatric disease 59
Psychiatric disorders 279
Psychotic disorder 452
Pyelonephritis 89
acute 170, 171, 245
Pyperoxaluria, primary 96, 334336, 338t, 339, 341, 441
Pyrexia 139, 250
Pyridoxal 5'-phosphate 338
Pyridoxine 338
high-dose 338
Pyrimethamine 241
Pyuria 49, 51
Quantitative nucleic acid testing 182
Quantitative polymerase chain reaction 182, 188
benefits of 188
Rabbit antithymocyte globulin 100, 101, 101f, 110, 111, 142, 145, 316
Rabies 435
Ramsay Hunt syndrome 213
Randomized controlled trials 103, 108, 121, 124, 138, 144, 155, 157, 220, 398, 399
Rapamycin, mammalian target of 38, 75, 118, 126, 127, 153, 155, 212, 259, 265, 291, 293, 315, 317f, 319, 326
Real-time polymerase chain reaction 12
Recurrent urinary tract infection 79f, 89, 245
evaluation of 246b
Red blood cells 166, 379
Regional Organ and Tissue Transplant Organization 404, 412, 464
Regulatory T-cell 40, 45
suppression, mechanism of 41f
Reloxaliase 338
Renal allograft 350fc
biopsy, histology of 188
compartment syndrome 73, 74fc, 358
fungal infections of 170
global infarction of 359f
post-transplant infections of 169
rejection of 363
related factors 281
Renal artery 66f
anastomosis of 66, 66f
inferior 77f
kink 72
multiple 62, 69
stenosis 78
diagnosis of 356
thrombosis 75, 359
Renal biopsy 160, 170
summary 168, 169, 171, 172
Renal cell carcinoma 311, 316, 365f
Renal disease 187
end-stage 10, 51, 61, 120, 136, 167, 195, 207, 248, 269, 275, 301, 312, 370, 378, 401, 404, 406
hereditary 52
Renal failure
drug-induced 438
hypovolemia-induced 438
Renal function 350
tests 56, 98, 398
Renal replacement therapy 406, 439, 440
Renal sinus 356f
Renal thrombotic microangiopathy 196
Renal transplantation 3, 26, 29t, 38, 40, 57, 70f, 114, 131, 133, 134t, 166, 248, 253, 262, 310
candidates 261
evaluation of 57
complications 358
fertility after 425
pathology 160
recipients 248, 250, 269, 270, 270t, 315t, 362f, 430
workup for 57
Renal tubular acidosis 117
Renal vein thrombosis 75, 86, 359
Renin-angiotensin-aldosterone system 153, 196, 281, 282
Repaglinide 296
Residual renal function, loss of 352
Resistive index 87
Respiratory syncytial virus 208
goals of 411
hormonal 411
Retina, inflammation of 189
Retinitis 189
Retransplantation 192, 352
Revascularization 65
Reverse transcription polymerase chain reaction 46, 56, 189
quantitative 144
Ribavirin 133
Ribonucleic acid 122, 163, 195, 202, 318, 323, 448
interference 338
agents 338
Rifampicin 249, 250
Ringer's lactate 398
Ritonavir 199
Rituximab 102, 136, 155, 156, 327, 380f, 381f, 383, 429
dosing of 383
monotherapy 311
Robot-assisted laparoscopic nephrectomy 62
Rosiglitazone 296
Rotavirus 132
vaccine 435
Rubella 436
Sabouraud's agar 221
Salivary gland 379
Saxagliptin 296
Schistosoma 233
mansoni 233
infection 233
Schistosomiasis 89, 233, 235
Scientific Renal Transplant Registry 102
Secondary hypertension 282
causes of 283b
Seizure 276
disorder 58
Semidirect pathway 5, 26
Sensitization 31, 352, 402
Serology 240
advantages of 11
disadvantages of 11
Seroma, subcutaneous 77
Sertoli cell atrophy 425
Severe acute respiratory syndrome coronavirus 2 138, 215, 219
Simeprevir 199
Single antigen bead 10, 23, 83, 95
Single nucleotide polymorphism 116
Single-stitch Taguchi technique 68
Sinusoidal obstruction syndrome 272
Sirolimus 127, 129, 199, 292, 310, 315
Sitagliptin 296
cancers 310
management of 310
closure 69
Smooth muscle cells 92, 93f
Sodium 282
chloride cotransporter 282
glucose cotransporter inhibitors 264, 297
Sofosbuvir 198, 199
Soluble urokinase-type plasminogen activator receptor 325
Spectrum 301
Spinal cord injury 89
Spondylitis, ankylosing 11
Sponge theory 353
Spousal donor transplants 462
Squamous cell carcinomas 308
Standard complement-dependent cytotoxicity test 16f
aureus 178
saprophyticus 245
State Organ and Tissue Transplant Organization 404, 408, 411, 464
Steatohepatitis, nonalcoholic 440
Stem cell
factor, serum-soluble 316
therapy 40, 43f, 44f, 45
transplantation 40
Stenosis 78
Steroids 106109, 120, 260, 263, 276
effect 107
free maintenance 110, 111t
mechanism of action of 107f
therapy 108, 110
upregulate anti-inflammatory cytokine interleukin 106
Stiripentol 338
Stomach 120
Streptococcus pyogenes 157, 373
Strongyloides 232, 232f, 233
stercoralis 232
Strongyloidiasis 235
Subcutaneous fat, ischemic necrosis of 78f
Substance misuse, assessment for 454
Substance use disorder 452
Substrate reduction therapy 338
Sulfadiazine 241
Sulfamethoxazole 178, 207, 224, 227, 228, 235, 241, 241t, 255, 258, 270
Sulfasalazine 133
Sulfonamide 258
Sulfonylureas 296
Surgery, steps of 63
Surgical site infection 78, 176
Swap donation 462
Systemic lupus erythematosus 59, 317f
Tachycardia 75f
Tacrolimus 103, 111, 115, 115b, 116, 118, 120, 121, 126, 129, 131, 147, 155, 166, 176, 191, 219, 244, 255, 259, 260, 263, 278, 289, 293, 297, 316, 344, 380f, 429
reducing metabolism of 131
toxicity 78f
activation of 5, 136
cytotoxicity 149f
leukemia, adult 215
hepatosplenic 173
peripheral 172
mediated rejection 142, 147, 148, 153, 162, 350
acute 145, 162, 164
chronic 162, 164
pathogenesis of 148
neoplasms 172
receptor 3, 4f, 7f, 39, 149f
Technetium-99m dimercaptosuccinic acid 50
Tenofovir 205
alafenamide 271
disoproxil fumarate 205
Testicular pathology 425
Testosterone 425
Tetanus 434436
Tetracycline 259
Therapeutic drug monitoring 107, 124
Therapeutic plasma exchange 154156
Thiazolidinediones 296
Thick-walled cavitary disease 251f
Thioguanine nucleotide 132
Thioinosine monophosphate 132
Thiopurine S-methyltransferase 131, 132
Thiouric acid 132
Thrombocytopenia 86, 314
post-transplantation 317
Thromboembolism, pulmonary 86
Thrombotic microangiopathy 78f, 96, 117, 127, 146, 151, 314, 317, 317f, 328, 383
after renal transplantation 169
complement-mediated 327
diagnosis of 87b
Thromboxane 282
Thymoglobulin 102, 136
Thyroid stimulating hormone 397
Thyroxine 399
Tissue 227
injury, pathogenesis of 29
transplantation 459
organization 414
T-lymphocytes 100
Tocilizumab 157
Tolerance induction protocol 43, 44f
Topiramate 276
Total leukocyte count 316
Total lymphoid irradiation 41, 45
acute 166
central 278
chronic 166
peripheral 278
gondii 227
infection 228
Toxoplasmosis 227, 235
Transfusion 57
donor-specific 43, 44f, 45
Transient ischemic attack 58
Transplantation 1, 176, 255
kidney 85
arteriovenous fistula 366f
pseudoaneurysm 367f
nephrectomy, effect of 353
of Human Organ Act 393, 394, 403, 404, 406, 459, 460, 461t
recipients, scientific registry of 207, 448
renal artery stenosis 96, 282, 283, 359, 360f
types of 196
ureteric stricture 363f
Transversus abdominus muscle 74f
Transvesical ureteroneocystostomy, steps of 68f
Tree-in-bud nodules 251f
Tricontinental study 133
Triiodothyronine 399
Trimethoprim 178, 207, 224, 227, 235, 241, 241t, 255, 258, 270
Trypanosoma cruzi 53, 228
Tryptophan 85
Tube method 379
Tuberculin 251
skin testing 251
Tuberculosis 53, 59, 177, 179, 182, 248250, 253, 412
disseminated 244
extrapulmonary 256
global prevalence of 248
multidrug-resistant 252
mycobacterium 208, 242, 249
pathogenesis of 249
prevalence of 248, 248t
reactivation of 177
Tubular atrophy 43, 129, 164, 344
Tubular epithelial cells 171f, 172, 172f, 183f
Tubular necrosis, acute 74, 86, 96, 114, 358, 364, 413f
Tubular oxalate crystal deposition 336f
Tubules 160
renal 337f
Tubulitis, moderate 165f
Tubulointerstitial nephritis 187
Tubulopathy 117
growth factor-beta 41f
necrosis factor 4f, 41f, 107, 149, 149f
Typhoid 132, 434
Ultrasonography 90, 95, 97, 98, 244, 246, 272, 355
contrast-enhanced 355, 356, 364
United States Renal Data System 109, 178, 226, 351
Ureteral leak 75
Ureteric complications 88
Ureteric obstruction 72, 78
ultrasonography assessment for 362
Ureteroneocystostomy 67
extravesical 68, 68f
Urethral stricture 89
Urethral valves, posterior 89
Uridine diphosphoglucuronosyltransferase 122f
Urinary bladder 98
Urinary oxalate measurements 337
Urinary tract 93
infection 84, 139, 177, 178, 209, 220, 221, 244, 245247, 255, 289, 297
impact of 246
recurrent 79f, 89, 245
types of 245
obstruction 97
reconstruction of 67
Urine culture 56
Urine electron microscopy 188
Urinoma 361
Urothelial cells 93f
Vaccination 59, 123, 132, 178, 203
Vaccines 434
Valganciclovir 181, 316
Valproate 276
Varicella 59, 132, 433
vaccine 435
diagnosis of 213
virus 177, 212, 213, 213t, 270, 272
Vascular anastomosis 65
Vascular basement membrane 37
Vascular clamps, application of 65
Vascular endothelial
cells 35
growth factor 128, 310
Vascular obstruction 96
Vascular outcome reduction 262
Vascular thrombosis 74, 96, 97
Vasopressors 411
Vein anastomosis 67f
Velpatasvir 198, 199
Vena cava, inferior 398
Venereal disease research laboratory 412
Venous outflow obstruction 73
Ventilation 411
Ventricular assist device 28, 31
Vertebral osteosclerosis 335f
Vesicoureteral reflux 85, 244
junction 78f
reflux 79
Vesicular nuclei 170
Vessels 160
Vestibulo-ocular reflex, absence of 396
Vildagliptin 296
Vimentin 37
Viral infections 138, 181, 188, 212, 376
nonhepatotropic 271
Virchow's triad 74
Viremia 187
Virological response, early 199
Visceral leishmaniasis 235
deficiency 301
receptor activator 305
supplements 304
K antagonist 59
Voiding dysfunction 89
Vomiting 132, 139
Voriconazole 220
Voxilaprevir 199
West Nile virus 53
White blood cell 101, 132
World Health Organization 264, 291, 295
classification 172
Fracture Risk Assessment Tool 304f
complications 77
dehiscence 77
infection 77
Wright stains 230
Xanthine oxidase 132
inhibitors 133
X-ray absorptiometry 358
Yellow fever 132, 434
Yin-yang pattern 367f
Y-tube system 68f
Ziehl-Neelsen stain, modified 257f
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Immunobiology and Evaluation TechniquesCHAPTER 1

1.1 Transplant Immunobiology
Shruti Tapiawala, Suchita Jogale
Transplantation is the preferred option of treatment for patients suffering from end-stage organ diseases. The immune system is directed to distinguish self from nonself and destroy the nonself. Rejection of a transplanted organ, tissue, or cell is a process in which the immune system identifies the transplanted organ, tissue, or cells as nonself/foreign and initiates a response to destroy it. The challenge to maintain the function of the transplanted organ and prevent rejection is the Achilles heel of transplantation medicine. Understanding the immune responses to the new organ is the basis of evolution of drugs and strategies to prevent rejection of the transplanted organ.
This chapter will explain the terminologies used in immunobiology of transplantation, concepts of transplant immunobiology, key cells and chemicals which are important in the immune response to a transplanted organ, and the mechanisms of rejection and tolerance.
The transplanted organ/tissue is called a graft and based on the origin of the graft, different terms are used (Fig. 1). When an organ or tissue is transplanted from one site to another in the same individual, the graft is termed autograft. If the graft is transplanted into an identical individual, it is termed an isograft. If the graft is transplanted into a nonidentical individual from the same species, the graft is termed an allograft. If a transplanted organ is taken from different species, it is termed xenograft.1
The immune response to an allograft is termed alloimmune response which is initiated by T-cell recognition termed allorecognition of transplantation antigens or alloantigens which are unidentical in recipient and donor and may be responsible for graft rejection.
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Fig. 1: Types of grafts.
The major histocompatibility complex (MHC) is a gene family that produces membrane proteins and is found on the short arm of chromosome 6 (6p21.3). It is the most variable gene cluster in the human genome. In 1958, Jean Dausset (1916–2009) discovered the first human leukocyte antigen (HLA), which he named “MAC” after the three letters of three of his volunteers’ first names. HLA-A2 was the name given to it later. HLA class I and II antigens were discovered via lymphocytotoxicity tests (LCT) and mixed lymphocyte culture (MLC). MHC antigens relevant to clinical transplantation were broadly divided into two classes—classes I and II. Each class is encoded by several loci.2,3
The MHC class I molecules are expressed on almost all nucleated cells and platelets. The MHC class I proteins are subdivided into classical loci HLA-A, HLA-B, and HLA-C. HLA-E, HLA-F, HLA-G, MHC class I polypeptide-related sequence A (MICA) and B (MICB), etc., are classified as nonclassical MHC class I genes. MHC class II molecules are expressed on antigen-presenting cells (APCs) such as B cells, dendritic cells, and macrophages. The clinically relevant HLA class II loci are HLA-DR, -DP, and -DQ.1,4
Human Leukocyte Antigen Class I
Class I molecules consist of glycosylated heavy chains encoded by the HLA class I genes and noncovalently bound to extracellular β2-microglobulin (β2m). Heavy chain (45,000 kD) consists of three α domains, namely α1, α2, and α3. The HLA specificity is conferred due to the polymorphic regions in α1 and α2 domains. The light chain, β2m molecule (12,000 kD), is associated with a heavy chain, not covalently bound to it and not attached to the cell membrane.5
Human Leukocyte Antigen Class II
Human leukocyte antigen class II (cell surface glycopeptides) has a molecular weight of approximately 63,000 kD and consists of two structurally similar α and β transmembrane glycoprotein chains.
These chains have two amino acid domains, the outermost domain containing the variable region of class II alleles. The peptide-binding groove in class I and class II molecules is critical for functional aspects of HLA molecules.6
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Fig. 2: Major histocompatibility complex. (HLA: human leukocyte antigen)
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Fig. 3: Human leukocyte antigen (HLA) molecular structure. (β2m: β2-microglobulin)
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Fig. 4: Human leukocyte antigen (HLA) nomenclature.
All alleles begin with the letters “HLA,” indicating that they are part of the human MHC genes. The following part (HLA-A) tells which gene the allele is a variant of. The first two numbers (HLA-A*24) indicate the antigen type of that specific allele, which usually indicates the serological antigen present. The next two numbers (HLA-A*24:02) 3indicate the allele code of protein, and these are numbered in the order in which they were discovered. The third digit (HLA-A*24:02:01) indicates an allele variant that has a different deoxyribonucleic acid (DNA) sequence than the normal gene but generates the same protein. The fourth digit (HLA-A*24:02:01:01) indicates single or multiple nucleotide polymorphisms in a noncoding region. Finally, the suffix (HLA-A*24:02:01:01N) denotes changes in expression.7,8
Although HLA molecules are best known as transplantation antigens, their primary biological function is to regulate immune response.
Human Leukocyte Antigen Class I
The function of class I MHC is to present endogenous antigens to T cells. Peptide antigens derived from proteins that may be self-protein, altered self-proteins such as those found in cancer cells, and viral proteins found in viral infected cells fit into peptide-binding groove of HLA molecules. These antigens are degraded by large multifunctional proteases (LMPs) and transported to the endoplasmic reticulum (ER) by a transporter associated with antigen processing (TAP) inside the cell. Then, they are transported to cell surface. They interact with CD8+ T cells. There is an inflammatory response if the T-cell receptor (TCR) of a T lymphocyte can bind antigenic peptide.4
Human Leukocyte Antigen Class II
In contrast to class I, class II molecules are synthesized in the ER, but peptide antigens are not placed in the peptide-binding groove. Instead, an invariant chain (Ii) is placed as a stopper combined class II–invariant chain is carried to the endosome. In endosome, another molecule called DM first removes the invariant chain, followed by insertion of class II antigenic peptide. Exogenous proteins, which can be normal self-proteins or microorganisms, are used to make these peptides. Class II molecules are delivered to the cell surface, where they interact with CD4+ T cells, causing antibodies to be produced.4
Table 1 shows the difference between HLA class I and class II.
Foreign peptides are presented by the mismatched HLA present on the allograft to the immune system of the recipient. Once presented to the immune system, donor-specific antibodies (DSAs) are produced. A higher number of HLA mismatches between recipient and donor have consistently been noted to increase rates of early rejections and inferior allograft survival in multiple studies.9
Donor-specific antibodies have been proven to be causative of immune events in the allograft and can cause allograft loss. Preformed DSAs and de novo DSAs formed post-transplant are risk factors for graft loss in organ transplants. With advancing technology, HLA antibodies can be identified by means of sensitive HLA crossmatches and virtual crossmatch, which take into account the donor HLA and HLA antibody specificities identified by solid-phase assays in the patient's serum.10,11
HLA-A, -B, and DR have the most polymorphism, and their mismatching has been seen to be associated with a higher risk of HLA sensitization. Recently, studies have shown that HLA-DQ antibodies commonly develop de novo DSA among post-transplants, and a strong positive correlation exists between the development of donor-specific HLA-DQ antibodies and a higher risk of allograft rejection.4 (See Chapter 1.4 “Donor-specific Antibodies in Kidney Transplantation.)
The minor histocompatibility antigens (MiHA) are proteins which are expressed in some individuals in the population. This creates potential antigenic differences between donors and recipients.
TABLE 1   Differentiating features between HLA class I and class II.
HLA class I
HLA class II
Contains three α domains, namely α1, α2, and α3
One β2-microglobulin
Consists of two structurally similar α and β transmembrane glycoprotein chains
Peptide (number of AA)
Present on
All nucleated cells
Antigen-presenting cells such as B cells, dendritic cells, and macrophages
Peptide-binding region
α1 and α2
α1 and β1
Antigen source
Present endogenous antigens to T cells
Present exogenous antigens to T cells
Peptide generation
By cytosolic proteasome
By endosomal and lysosomal proteases
Interact with
CD8+ T cells
CD4+ T cells
(HLA: human leukocyte antigen)
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Fig. 5: Evolution of immune response after kidney transplant recipients (KTR). (CTL: cytotoxic T lymphocyte; IFN: interferon; MHC: major histocompatibility complex; TNF: tumor necrosis factor)
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Figs. 6A and B: (A) Direct allorecognition; and (B) Indirect allorecognition. (APC: antigen-presenting cell; MHC: major histocompatibility complex; TCR: T-cell receptor)
In theory, a polymorphism of any protein between donor and recipient, e.g., certain cell surface receptors and enzymes that can be processed and presented, can elicit a rejection response. These antigens are recognized by the CD8+ cytotoxic T cells, leading to rejection.1,10
Transplant immunology is an evolving field, and antigenic stimuli have been identified in various proteins over the last five decades. In case of bone marrow transplants, where a well-matched graft has been transplanted, the MiHA play an important role in causing graft versus host disease. Another MiHA noted is the Y-MiHA, produced when a male allograft is transplanted into a female. Here, protein coded by Y-chromosome elicits an immune response. Other non-HLA antibodies that can cause rejection are antiangiotensin-2 receptor, antiglutathione S-transferase T1, antiendothelial antibodies, and antibodies to MHC class I-related chains A and B (MICA and MICB). Some antigens may come from mitochondrial proteins and enzymes.1,4 (See Chapter 1.4 “Donor-specific Antibodies in Kidney Transplantation.)
The rejection process has two parts: (1) An afferent/initiation or sensitization limb where recognition of foreign antigens takes place and (2) an effector limb which destroys the allograft (Fig. 5).12,13
Recognition of Foreign Antigens
Allorecognition (recognition of allograft MHC antigen) is the trigger for allograft rejection. T cells recognize alloantigens via direct and indirect pathways (Figs. 6A and B), which are mutually exclusive.
Direct recognition occurs when recipient T cells recognize intact donor HLAs expressed by donor cells.
In contrast, indirect recognition occurs when the recipient APC processes the donor-HLA antigens prior to presentation to the recipient's T cells.
The direct pathway plays an important role in the immediate post-transplant period and can put forth a strong alloresponse, which occurs primarily due to recipient T cells recognizing the mismatched/nonself-graft antigens. This can cause acute cellular rejection. The indirect pathway may also participate in early acute rejections, but this mechanism predominantly causes late-onset acute and 5chronic rejections. Due to the constant presence of an allograft which is in itself a self-antigen, the APC itself picks up alloantigens which are shed from the allograft and this initiates an alloimmune response.
Semidirect pathway: Dendritic cells (DCs) acquire intact MHC–peptide complexes from other DCs and endothelial cells (ECs) and present them to alloreactive T cells. This pathway involves the uptake and surface expression of intact foreign MHC+ peptide complexes by the recipient APC.
Activation of T Cells
T-cell activation is central to the immune response to an allograft. Tissue destruction occurs due to direct T cell-mediated lysis of graft cells, activation of accessory cells, and alloantibody production with or without complement activation. Three distinct signals are involved from T-cell activation to production of effector T cells and alloantibodies, which ultimately result in damage of the allograft. These include (1) antigen recognition, (2) costimulation, and (3) cytokine-mediated differentiation and expansion of pool of alloreactive effector cells that participate in graft injury and destruction (Figs. 7A and B).1,10,13
Signal 1
The interaction between the antigen-specific T cells and APCs acts as the first signal for T-cell activation. The interaction leads to phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR/CD3 complex.1,10
Signal 2—Costimulatory Signal (Fig. 8)
In addition to the formation of MHC–peptide complex, a costimulatory signal is necessary. In the absence of the costimulatory signal, the T cells become anergic or unresponsive.14,15 Although there are many costimulatory molecules on T cells, the focus of clinical transplantation has been on CD28 and CD154 pathways.
The CD28/B7 costimulatory pathway is involved in allo T-cell activation. CD28 is expressed on the resting T cells, and its ligands are expressed on the activated APCs, the ligands being B7.1 (CD80) and B7.2 (CD86). CD28 signaling reduces the threshold of TCR signaling, which further promotes T-cell proliferation, cytokine production, and differentiation.
CD4 and CD8 T cells play a major role in rejections of grafts mismatched for MiHA. CD4 T cells facilitate CD8 T cells differentiation by direct cell-to-cell contact or by producing cytokines such as interleukin-2 (IL-2) and interferon-gamma (IFN-γ). These, in turn, will help CD8 T-cell proliferation and killing of graft tissue.16,17
The CD154/CD40 costimulatory pathway has also been researched in animal transplantation models. CD154 is expressed on activated T cells, while CD40 is expressed on APCs and B cells.
Blockade of these signals leads to inactivation/anergy of the antigen-specific T cells. T cells also possess cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), a protein that is a negative regulator of the immune response that can compete for binding with CD80 and CD86. Belatacept (LEA29Y), a second-generation humanized fusion protein that binds CTLA4, has been used with a promise to suppress rejection and improve long-term graft survival. Blocking CD28/B7 and CD154/CD40 therapeutically works synergistically to prevent acute and chronic rejections.18
Signal 3 and Effector Phase of Graft Rejection
The costimulatory response after T-cell interaction (signal 1) activates three signal transduction pathways: calcineurin pathway, activated protein kinase pathway, and IκB kinase (IKK)—nuclear factor κB (NK-κB). This initiates the effector phase of rejection. The effector phase of the graft rejection involves the entry of activated T cells into the allograft and the production of cytokines.1,10
CD8+ T cells require cytokine signals produced by macrophages and/or DCs for the generation of effector and memory cells population and to survive to take the cascade further. The CD8+ T cells that do not receive the cytokine signals after signals 1 and 2 are devoid of cytolytic function and become unresponsive.
Once signals 1 and 2 are received, generation of cytokines including CD25, CD154, IL-2, and IL-15 is induced. These interleukins then release growth signals (signal 3) through the mammalian target of rapamycin pathway and phosphoinositide-3-kinase pathway. These subsequently trigger the T-cell growth cycle and proliferation. The completely activated T cells undergo clonal expansion and release cytokines and effector T cells. These eventually produce CD8+ cytotoxic T cells, which help macrophage-induced delayed-type hypersensitivity response with the help of CD4-T helper cells 1. They further help B cells to produce antibodies with CD4+ T helper cells 2.
Complement system also plays a major role in the effector mechanism of rejection. Signals 1 and 2 augment the intracellular cleavage of complement C3. The fragments are transported to the cell membrane, which bind to their corresponding receptors and further amplify the activation signal of T cells. Complement-dependent cytotoxicity remains a crucial step in the lysis of donor cells.19
The effector elements, as mentioned above, are responsible for the clinical manifestations of allograft rejection. A subset of the activated T cells further goes on to become alloantigen-specific memory T cells.1,10,20236
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Figs. 7A and B: (A) The three-signal model; (B) The three-signal model with effectors. (APC: antigen-presenting cell; CDK: cyclin-dependent kinase; DNA: deoxyribonucleic acid; IKK: IκB kinase; IL: interleukin; MAP: mitogen-activated protein; MHC: major histocompatibility complex; mRNA: messenger ribonucleic acid; mTOR: mammalian target of rapamycin; NF-κB: nuclear factor kappa B; NFAT: nuclear factor of activated T cells; TCR: T-cell receptor)
Termination of Immune Response
Transplant rejection is a vicious cycle where release of donor antigens, production of alloreactive T cells, and infiltration of the allograft with effector T cells leads to further shedding of graft antigens and further alloimmune activation till the graft is destroyed.
Effector lymphocytes are generated during an immune response, but most undergo activation-induced cell death (AICD) by apoptosis as the response progresses. Few effector lymphocytes that survive give rise to memory T lymphocytes (Tm). Exhaustion occurs when the effector or memory T lymphocytes repeatedly encounter a persistent antigen as would occur in the case of an allograft. Repeated antigenic stimulation induces the expression of inhibitory molecules that make T cells hypo- or unresponsive.
CD4+ Foxp3+ regulatory T cells (Tregs) are a subset of helper T cells, which help control the response by secreting cytokines (e.g., IL-10 and TGFβ) and inhibitory membrane molecules (e.g., CTLA-4), which inhibit immune responses and prevent the immune response from continuing indefinitely and control inflammation.7
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Fig. 8: Costimulatory molecules. (APC: antigen-presenting cell; MHC: major histocompatibility complex; TCR: T-cell receptor; ICOS: inducible co-stimulator; PDL: programmed death ligand; PD: programmed death; CTLA: cytotoxic T lymphocyte antigen)
These cells also suppress effector cells and modulate dendritic cell maturation.1,23,24
The role of B cells and their progeny, plasma cells, lies in the production of anti-HLA antibodies, which have been implicated in both acute and chronic rejection. B cells express antigen-specific receptors on their surfaces. When these immunoglobulin receptors bind to the respective antigens from the donor with assistance from T helper cells (CD4+TH2), activation occurs. Some activated B cells generate plasma cells, which secrete antibodies and some become memory B cells.2527 B-cell activation is facilitated by membrane contact involving receptors and ligands such as CD40 and CD154 or with the help of secreted cytokines such as IL-4. Plasma cells and CD20+ B cells have been seen infiltrating the allografts. These have been identified to be markers of irreversible acute rejection episodes.28,29
The donor-specific HLA antibodies bind to antigens and cause allograft injury by complement-dependent cytotoxicity or through Fc receptors on natural killer (NK) cells. B cells also participate in the indirect allorecognition pathway where they present allograft-derived antigens to T cells, activate T cells, and further amplify T cell-mediated damage.1,10
Natural killer cells represent a specific population of lymphocytes (5–10% of circulating lymphocytes), which are involved in the innate immune response in an individual, which is involved in antitumor and antiviral defense of an individual. NK cells are naturally cytotoxic and do not require antigen exposure to mediate their effects.30,31
The NK cells exert their lytic effect through direct lysis and antibody-dependent cellular cytotoxicity (ADCC). Recognition of self-HLA molecules by inhibitory receptors, namely the killer cell immunoglobulin-like receptors (KIRs), inhibits the lytic effect of NK cells. Direct lysis happens when the NK cells fail to detect self-HLA molecules on the target cells. ADCC happens when the Fc receptor (FcγRIII) of the NK cell interacts with the Fc fragment of the HLA antibody, recognizing foreign antigen on the target cell and inducing lysis. NK cells, once activated, kill their targets by secreting enzymes such as granzyme, perforin, and IFN-γ. NK cells can prime the adaptive immune system and facilitate the migration of dendritic cells, which can cause an alloimmune response and loss of allograft.3032
The presence of NK cells and donor-specific HLA antibodies (DSAs) has been shown to facilitate rejection. The mere presence of DSAs in the absence of NK cells does not induce rejection, although it can lead to late graft failure. The pathogenic function of NK cells causing antibody-mediated rejection (ABMR) is mediated through the expression of CD16. The pathogenicity can be triggered by the presence of DSAs and can cause graft injury. 8NK cells have been identified in the peritubular capillaries of tissues with ABMR. It is postulated that DSAs bind to the EC where they can interact with the FcγRIII receptors present on NK cells and cause lysis by ADCC. NK cells have also been associated with chronic rejection.1,3234
Specific subsets of NK cells are associated with the induction of tolerance in transplant recipients. Activated NK cells can kill donor-derived DCs through direct lysis, thus dampening the immune response and promoting tolerance of the allograft.1 It is unclear which factors move the NK cells toward donor-derived dendritic cells. It can be thought that NK cells might be able to integrate stimulatory and inhibitory signals, influenced by T-cell behavior, causing the outcome of rejection or tolerance.33
Dendritic cells are APCs found in tissues such as skin and mucosal membranes. Their functions are to process antigens and present them to the immune system and are the bridge between innate and adaptive immunity. Their importance lies in the induction of a protective immune response against pathogens. The key role of DCs in transplantation is in the activation and maintenance of immune responses to allografts. Once the DCs are activated, they move to the lymphatic tissues to further interact with T and B cells to make an adaptive response. Due to their ability to regulate both innate and adaptive immunity, DCs are considered to potentially direct immune responses toward rejection or tolerance. Allograft rejection is initiated at the allograft EC layer and forms an immunogenic barrier for migrating DCs. The DC/EC interactions play an important role in the rejection. Direct and indirect pathways are used by donor DCs and host DCs, respectively, to activate alloreactive T cells and mediate rejection.35
It is thought that clonal deletion, induction of T regulatory cells, and inhibition of memory cell responses help achieve tolerance. These properties have encouraged research to use tolerogenic DC as a therapeutic strategy to promote tolerance in a transplant patient. Infusions of donor/recipient-derived tolerogenic DCs have helped achieve tolerance in murine models.36
An understanding of transplant immunobiology and its orchestra consisting of cells and their effector elements helps us form the basis of transplantation and immunosuppression used to prevent rejection and maintain an allograft. It further helps us work toward therapies and immunomodulation to establish tolerance and accommodations in transplantation.
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1.2 Immunologic Evaluation of Kidney Transplant Candidates
Ajay Kumar Baranwal, Uma Kanga
The human leukocyte antigen (HLA) complex located on the short arm of chromosome 6 is the 4 Mb long, complex genomic region comprising hundreds of genes that are functionally relevant. These genes are highly polymorphic, display linkage disequilibrium, and are inherited en bloc. The antigens encoded by the HLA genes are divided into class I (HLA-A, -B, and -C) and class II (HLA-DR, -DQ, and -DP). According to the latest Immuno Polymorphism Database-ImMunoGeneTics (IPD-IMGT)/HLA database version 3.50 (http://www.ebi.ac.uk/ipd/imgt/hla), approximately 36,016 allelic variants have been reported and this number is growing exponentially. HLA compatibility between recipient and donor is of major clinical relevance in both hematopoietic stem cell and solid organ transplantation.
Human leukocyte antigen laboratory in a setting of organ transplantation is entrusted with two main responsibilities: (1) HLA typing to determine the degree of mismatch between the recipient and donor and (2) detection of HLA antibodies, particularly those directed against donor HLA. Several technologies have been established for accurate HLA typing and detection of anti-HLA antibodies, and these are discussed in detail in subsequent sections.
In 1964, Terasaki and McClelland first introduced the serological HLA typing, which has been the mainstay of HLA typing technology.1 In the 1980s, the deoxyribonucleic acid (DNA)-based methodology was initiated and restriction fragment length polymorphism (RFLP) established for HLA typing. Since the advent of polymerase chain reaction (PCR) in 1986, DNA-based tissue typing techniques have become routine in the histocompatibility laboratories. The PCR-based assays include sequence-specific primers (SSP), sequence-specific oligonucleotide probe (SSOP), reverse SSO, sequence-based typing (SBT), real-time PCR, and next-generation sequencing (NGS).2,3 A brief description of various methods for HLA typing and their advantages and disadvantages are given below.
The serology-based HLA typing or the microlymphocytotoxicity assay is a technique that applies the principle of complement-dependent cytotoxicity (CDC). For this assay, the lymphocytes of an individual are separated and incubated with a set of HLA-specific alloantisera and/or monoclonal antibodies, and later the reaction starts with the addition of rabbit serum, which is used as a source of complement. The method enables identification of HLA 11alleles expressed on lymphocyte surface. Based on this principle, when the antibody binds to HLA antigen on the cell surface, it activates the complement and forms a membrane attack complex that damages the cell membrane and makes it permeable to vital dyes. Results are visualized on addition of the fluorochrome-based dyes (ethidium bromide/acridine orange) or supravital dye (eosin Y). The staining distinguishes live and dead cells, as the live cells look unstained, refractile, and small round shiny structures while the dead cells look large, dark structures. The results can be evaluated by counting the percentage of dead cells on an inverted fluorescence microscope.1,4 The HLA allele is assigned for the particular cell/sample depending on the antibody to which the binding was observed. Several HLA antigens share antigenic determinants (polymorphic epitopes); therefore, one antibody can react with a number of HLA antigens. Extensive quality checks, i.e., good screening of alloantisera or use of monoclonal antibodies, are important for this CDC-based technique.
Advantages and disadvantages of serology: This assay is simple and easy to perform, does not require expensive equipment, and can be performed in a short duration of 3–4 hours. Presently, this technique is not routinely used for HLA typing but is used as an adjunct to molecular methods, i.e., to identify the “N” null alleles or the nonexpressed HLA alleles. However, this conventional method has certain limitations, such as cell viability, need for fresh sample in large volume, dependence on surface expression of HLA alleles, complexity in interpretation due to cross-reactivity of antisera, and cannot be used for high-resolution typing or detection of rare alleles. Therefore, alternative technologies were developed for HLA typing.
Molecular techniques have an advantage over the serological methods as fresh blood/live lymphocytes are not required, and DNA can be extracted from any nucleated cell and stored for long durations. This helps in high throughput testing and also repeat testing if required. All of the DNA-based methods utilize the PCR to identify the genomic region of interest. For most DNA-based methodologies, the PCR process is used only as an amplification step to acquire the required target DNA. The HLA typing process then requires a postamplification step to discriminate between the different alleles. These molecular techniques are employed for low-, intermediate-, and high-resolution typing and are commonly used in laboratories performing clinical testing for diagnostics and/or donor selection for transplantation.
Although HLA typing can be performed by using a single molecular technique, ambiguities can arise depending on the primers and probes used for the region being analyzed. The three possible reasons for the generation of ambiguities are: (1) If the polymorphic sites distinguishing the alleles in question are located outside the amplified region, (2) when multiple allele pairs display identical heterozygous sequences, and (3) when part of the sequence is incomplete within the genomic region being analyzed. The ambiguities can usually be resolved by utilizing multiple molecular techniques. The molecular techniques for HLA typing are described below.
Polymerase Chain Reaction
Sequence-specific Primer
In this technology, SSP pairs selectively amplify target sequences that are specific for a single allele or allele groups present on the target DNA within the locus of interest.5 Several allele-specific primers for the locus of interest are utilized, and multiple individual PCR amplifications are performed.6,7 The synthesis of a new strand is dependent on binding between the genomic DNA and the 3′ end of the primer, as the 3′ end confers specificity to the primers to anneal and thus amplify the target sequence. Furthermore, each reaction is multiplexed with a nonpolymorphic “housekeeping” gene serving as an internal control. Presence of an amplicon, i.e., a positive reaction with one or more allele-specific primer sets, is detected by agarose gel electrophoresis, and the HLA allele is assigned based on the amplification pattern. This technique is unlikely to detect a new unidentified allele unless the primer picks the variation at a specific site and results in an unexpected or unique reaction pattern.
Advantages and disadvantages: Most commercial PCR-SSP assays utilize common thermal cycling conditions for amplification of both HLA class I and II alleles. This is the preferred technology for laboratories requiring results in short duration of 4–5 hours and have lower throughput. This is an effective alternative to serology-based testing. However, this method usually requires a large quantity of DNA, is cumbersome to perform, and generates low-resolution information. Furthermore, intermediate/high-resolution information can be generated by using primers for all alleles within a particular allelic family, but the assay is expensive. This methodology is useful for cadaveric donor typing or for diagnostic and pharmacogenomic screening tests of single alleles such as HLA-B*27 (ankylosing spondylitis), HLA-B*51 (Behcet's disease), B*57:01 (abacavir hypersensitivity), B*15:02 (carbamazepine hypersensitivity), and HLA-DQA/DQB (celiac disease).
Sequence-specific Oligonucleotide Probes
Polymerase Chain Reaction Sequence-specific Oligonucleotide Probes
This method employs locus-specific PCR amplification followed by immobilization of the PCR amplicon on the nylon membrane and hybridization with allele-specific oligonucleotide probes. The HLA typing is obtained 12depending on the binding of each oligo probe with the complementary sequence in the PCR amplicon. Use of radioactive probes was a limitation of PCR-SSOP that was overcome by the development of the option to use biotinylated oligoprobes. Although longer hybridization protocols and the need to employ a large number of probes increased the test duration, SSOP was often used for HLA typing of a large number of samples. Depending on the primers and probes used, PCR-SSOP was employed for both low- and high-resolution HLA typing.8,9 This technology is no longer in regular use as several advanced technologies are now available.
Reverse Line Strip Assay Sequence-specific Oligonucleotide
The above mentioned SSOP technology was further modified with the use of biotinylated PCR primers (specific for exons 2 and 3 for class I and exon 2 for class II) and immobilization of locus- and allele-specific oligos on nylon strips. Therefore, the technique is named reverse line strip (RLS) assay. It is based on three major processes: PCR target amplification of an HLA locus, hybridization of the amplified products to an array of immobilized SSOPs, and detection of the probe-bound amplified product by color formation. Amplicon binding is detected via streptavidin horseradish peroxidase (HRP) and a chromogenic substrate, which generates a blue line at the position of positive probe (Fig. 1). The technology utilizes a combination of probes for polymorphisms that are shared between large numbers of alleles. This leads to significant limitation, i.e., either precise identification of alleles, or generates allelic strings, thus resulting in low- to intermediate-resolution HLA typing and is unlikely to detect a new undefined HLA allele.
Luminex Sequence-specific Oligonucleotide
The SSO assays were further improvised with development of Luminex® xMAP technology.10 The basic principle involves measurement of multiple analytes simultaneously. A series of probes are immobilized on microbeads that are color coded using a blend of fluorescent intensities of two dyes. Target genomic regions of HLA loci (exons 2 and 3 for HLA class I and exon 2 for HLA class II) are amplified and biotinylated PCR amplicons are hybridized with beads carrying locus-specific probes. The binding was detected using streptavidin-conjugated phycoerythrin (SAPE) (Fig. 2). The fluorescent signals are detected using Luminex flow analyzer platform and interpretation software detects the bead reaction pattern assigned to the HLA allele. More than 100 individual beads, i.e., >100 different probes, are available for each assay, which improves the resolution of HLA typing.
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Fig. 1: Reverse line strip blot sequence-specific oligonucleotide (SSO) assay representation. (DNA: deoxyribonucleic acid; SAHRP: streptavidin-horseradish peroxidase)Source: Smith L, Fidler S. HLA typing technologies. In: Mehra NK (Ed). The HLA Complex in Biology and Medicine: A Resource Book. Jaypee Brothers Medical Publishers (P) Ltd; 2010.
Although this technique requires longer execution time, it has selected advantages, namely rapid kinetics reduce incubation times, requires less quantity of DNA, can be applied for high-throughput testing, and provides intermediate- to high-resolution results. Additionally, this technique has been improved for last several years by the addition of a large number of probes; therefore, high-resolution-level HLA typing information can also be obtained using high-definition beads with a larger number of probes. The development of 500 bead system associated with LabScan 3D has helped in reducing allele ambiguities by using powerful software for assessment of reaction patterns and thus improving the reliability of results.11 Therefore, currently, this is the most preferable technique for generic and high-resolution HLA typing of all loci.
Real-time Polymerase Chain Reaction
This DNA-based HLA typing technology was developed for rapid detection of specific HLA alleles with increased sensitivity and utilized the TaqMan assay.12 This approach was applied for typing of single HLA alleles.13 Further advancements in combining the PCR-SSP and SYBR green principles led to the development of the real-time PCR (RT-PCR) assay that can detect PCR products without postamplification manipulation. The PCR is performed with SYBR green in the buffer, and both thermal cycling and melting curve analysis are performed on the quantitative PCR (qPCR) equipment.14 After thermal cycling, PCR amplicons are heated across a temperature range and they melt (become single stranded) at a certain temperature depending on the size, guanine-cytosine content, and sequence motif captured within the DNA fragment. When each amplicon melts, the SYBR green fluorescence decreases rapidly, which is captured by the instrument. This technology helps identify HLA alleles at a low–intermediate resolution in few hours. This approach has considerable advantages for rapid HLA typing required for deceased donor testing in solid organ transplantation.15
Sequencing-based Typing
This technology is based on locus- or group-specific PCR amplification, followed by cycle sequencing using dye terminator technology.1613
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Fig. 2: Principle of Luminex-based reverse sequence-specific oligonucleotide (SSO) method.Source: Smith L, Fidler S. HLA typing technologies. In: Mehra NK (Ed). The HLA Complex in Biology and Medicine: A Resource Book. Jaypee Brothers Medical Publishers (P) Ltd; 2010.
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Fig. 3: Sequenced-based human leukocyte antigen (HLA) typing general workflow. (DNA: deoxyribonucleic acid; PCR: polymerase chain reaction)Source: Smith L, Fidler S. HLA typing technologies. In: Mehra NK (Ed). The HLA Complex in Biology and Medicine: A resource book. Jaypee Brothers Medical Publishers (P) Ltd; 2010.
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Fig. 4: Genomic region sequenced based on primer location. (PCR: polymerase chain reaction)Source: Smith L, Fidler S. HLA typing technologies. In: Mehra NK (Ed). The HLA Complex in Biology and Medicine: A Resource Book. Jaypee Brothers Medical Publishers (P) Ltd; 2010.
Usually, the HLA sequencing is restricted to exons 2 and 3 for HLA class I alleles and exon 2 for HLA class II alleles. Initial amplicons generated are purified to remove unincorporated nucleotides, and product is sequenced using multiple sequencing primers in separate reactions (Fig. 3). The four different 2′,3′-dideoxyribonucleotides, once incorporated in the growing sequence, cause a stop of elongation and emit light at different wavelengths. After PCR, the DNA fragments are sequenced on an automated capillary sequencer and the nucleotides at the end of fragments are identified by the fluorescence emitted.
The sequence generated depends on the primer design for initial PCR and can vary, i.e., (1) primer located within the exon of interest, (2) in flanking introns, and (3) on adjacent exons. Use of primers within introns results in complete exon sequence (Figs. 4 and 5), but can lead to allele dropout due to lack of intron sequences. An alternate approach is the use of multiplexing allele/group-specific primers (Fig. 6).14
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Figs. 5A and B: Polymerase chain reaction (PCR) strategy for sequencing of (A) Human leukocyte antigen (HLA) class I genes (nested sequencing primer); and (B) HLA class II (with universal tags). (UTR: untranslated region)Source: Smith L, Fidler S. HLA typing technologies. In: Mehra NK (Ed). The HLA Complex in Biology and Medicine: A resource book. Jaypee Brothers Medical Publishers (P) Ltd; 2010.
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Fig. 6: Multiplex group/allele-specific polymerase chain reaction (PCR) strategy for human leukocyte antigen (HLA) class II sequencing.Source: Smith L, Fidler S. HLA typing technologies. In: Mehra NK (Ed). The HLA Complex in Biology and Medicine: A Resource Book. Jaypee Brothers Medical Publishers (P) Ltd; 2010.
To resolve ambiguities located outside the amplified regions stated above, additional regions such as exon 4 for class I and exon 3 for class II alleles are also sequenced. HLA typing by SBT was initially employed as a tool to resolve ambiguities arising in other molecular methods; however, despite sequencing, several factors lead to ambiguities. The sequenced region can be shared by several HLA alleles; thus, a pair of common HLA alleles or several less common allelic pairs can be assigned. The ambiguous result due to identical heterozygote sequences can be resolved by group-specific primary amplification and each allele amplified and sequenced.17,18
The advantage of determining the complete nucleotide sequence promoted SBT as the gold standard for high-resolution HLA typing. This technology is not dependent on known polymorphisms and picks up new alleles that are not recognized by existing primers and probes. Additionally, SBT enables evaluation of the synonymous and nonsynonymous mutations. These approaches have enabled identification of thousands of HLA alleles, and these observations have helped in updating the commonly well-defined (CWD) catalog. This technology is expensive and time consuming, but use of capillary-based automated sequencers increased the efficiency and decreased the cost to some extent. However, expertise in interpreting sequenced-based typing results is crucial for final interpretation and allele assignment. Resolving ambiguous allele combinations requires laborious efforts and does not remain cost effective as it has to be combined with other molecular methods and requires many high-resolution workflows.19,20
Next-generation Sequencing
The true innovations, i.e., technological advancements such as NGS, have revolutionized the characterization of polymorphic HLA genes as this technology generates a massive amount of data for a large number of different target DNA sequences in parallel in a single reaction. This technology is based on the monitoring of sequential addition of nucleotides to immobilized DNA templates arranged spatially. NGS provides careful characterization of HLA genes by high-resolution genotyping with relatively simple and cost-effective protocols. The NGS process generally involves few steps: Sample preparation and locus-specific amplification, DNA library preparation, normalization, sequencing, data acquisition, and bioinformatics. Different NGS platforms, such as Ion Torrent, Illumina, Pacific 15Biosciences, and Oxford Nanopore, all differ in terms of sequencing approach and have variable processing time ranging from few hours to days. The manufacturers have now developed varying processes to acquire sequence data from amplified single DNA fragments in their platforms.
As the sequences are generated from single molecules, complications associated with cis/trans ambiguities get rectified at a cost comparable to SBT.21 The benefits and feasibility of NGS approaches as an alternate HLA sequencing methodology have been evaluated using large reference panels previously tested by PCR-SSP/SSO/SBT.22,23 NGS could contribute to expand the knowledge about the biological relevance of both coding and noncoding HLA sequences in solid organ and stem cell transplantation.2426 The clinical efficacy of NGS has been successfully validated, and most histocompatibility testing laboratories define the standards for clinical typing by the different sequencing platforms.27 NGS allows sequencing of multiple HLA loci, i.e., 6–11 loci, and the laboratories can perform high-throughput HLA typing as multiple samples can be barcoded and pooled into one library and sequenced together.
The laboratory's choice of technology to be employed differs and is based on clinical need, throughput, urgency, infrastructure, and staff skills. There are several advantages and disadvantages for each technology (Table 1). Most laboratories employ combinations of methods for perfect assignment of HLA alleles and for resolving ambiguities arising by any single methodology.
The main aim of testing for HLA antibody in allograft recipients is to determine the donor–recipient compatibility and to evaluate the ongoing immune status of the recipient before and after transplantation in order to assess the potential risk for graft loss. This information enables clinicians to make logical decisions regarding donor selection, immunosuppression regimens, and post-transplant patient care. Thus, an accurate, sensitive, and HLA-specific detection of these antibodies becomes very important. In pursuit to achieve this, several methodologies have been developed since the introduction of the original cell-based cytotoxicity assay. Currently, anti-HLA antibodies can be detected by target donor cell-based assays, such as CDC or flow cytometry and HLA protein-based (solid phase) assays, such as an enzyme-linked immunosorbent assay (ELISA) or HLA-antigen-coated fluorescence bead assay system. Importantly, evaluation of assay results depends on understanding the characteristics of each assay, the theoretical principles involved, and the technological or biophysical limitations of the reagents, instrumentation, and analytical methods employed. The methods for detecting HLA antibodies are used in two distinct clinical applications: (1) As a crossmatch method to determine if a patient has antibodies directed against donor and (2) as a screening assay to determine the level of sensitization.
TABLE 1   Advantages and disadvantages of human leukocyte antigen (HLA) typing technologies.
  • Rapid and inexpensive
  • Assesses HLA cell surface expression
  • Time taken 3–4 hours
  • Cross-reactivity
  • Limited polymorphism detection
  • Low resolution
  • Requires viable lymphocytes
  • HLA cell surface expression critical
  • Resolution is better than serology
  • Specific allele can be identified
  • DNA can be stored
  • Low and high resolution possible
  • Time taken 4–5 hours
  • Low throughput
  • Labor intensive
  • High DNA quantity required
  • Several PCR reactions for each locus
  • 24 reactions HLA-A; 48 reactions HLA-B; 24 reactions HLA-DRB1
  • Locus-specific amplification
  • Generic and high resolution possible
  • Low DNA quantity
  • High throughput 96 tests at once
  • Time taken 6–7 hours
  • Sequence of allele must be known
  • Hybridization is critical
  • At times ambiguities issue
  • More reliable and specific
  • Allele sequence detected
  • Low DNA quantity required
  • Novel alleles can be detected
  • Huge infrastructure required
  • Expensive than other techniques
  • Time taken 24–36 hours
  • Most reliable and specific
  • Allele sequence detected
  • Low DNA quantity required
  • Novel alleles can be detected
  • Infrastructure is expensive
  • Cost effective if high throughput
  • Bioinformatics expertise required
  • Time taken 3–4 days
(DNA: deoxyribonucleic acid; NGS: next-generation sequencing; PCR: polymerase chain reaction; PCR-SSO: PCR-sequence-specific oligonucleotide; PCR-SSP: PCR-sequence-specific primers; SBT: sequence-based typing)
16The specific features and limitations of the available techniques for detection of anti-HLA antibodies are discussed in detail in the subsequent paragraphs, and their evolution is depicted in Figure 7.
Cell-based Assays
Complement-dependent Cytotoxicity
The complement-dependent microcytotoxicity (CDC) is a lymphocyte-based biological assay that requires antibody binding, complement activation, and cellular damage to register a positive reaction (Figs. 8A and B). The results are scored using a standardized ranking system introduced by Terasaki and later adopted by the National Institutes of Health (NIH).4 The technology for performing the test and association of positive crossmatch transplant with hyperacute rejection in renal allografts was described by Terasaki et al.1,28 Soon, it became a working paradigm to perform CDC crossmatch (CDCXM) test before proceeding for renal transplantation and positive CDCXM was considered a contraindication to transplantation.
Modified Lymphocytotoxicity Assays
Several modifications to the original method have been introduced in order to increase the sensitivity and specificity.
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Fig. 7: Evolution of antihuman leukocyte antigen (HLA) antibody testing. (AHG: antihuman globulin; ELISA: enzyme-linked immunosorbent assay; NIH: National Institutes of Health)
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Figs. 8A and B: Standard complement-dependent cytotoxicity (CDC) test. (A) Schematic presentation of CDC; and (B) Representative results of CDC. Dead cells are shown as black by eosin dye. Negative reaction (grades 1–2) and positive reaction (grades 4–8). (DSA: donor-specific antibody; HLA: human leukocyte antigen)Source: Nakamura T, Ushigome H, Shirouzu T, Yoshimura N. Donor-specific anti-HLA antibodies in organ transplantation: transition from serum DSA to intra-graft DSA. In: Mahdi BM (Ed). Human Leukocyte Antigen (HLA). London: IntechOpen; 2018. [online] Available from https://www.intechopen.com/chapters/62771. [Last accessed September, 2022].
TABLE 2   Modifications of lymphocytotoxicity assay over standard complement-dependent cytotoxicity (CDC).
Modification over standard CDC (NIH)
Modification detail
One wash prior to complement
To remove anticomplementary factors
Amos (modified)
Three to four washes prior to complement
To remove anticomplementary factors
Antihuman globulin (AHG) enhanced
Addition of AHG prior to complement over Amos (modified) technique
To increase the ability to detect low titer antibodies
Extended incubation
60 minutes serum/cell; 120 minutes complement; no washes
To increase the binding of low avidity antibodies
DTT treatment
Treatment of serum with DTT
To reduce IgM pentamers to monomers, rendering them incapable of fixing complement
(DTT: dithiothreitol; IgM: immunoglobulin M; NIH: National Institutes of Health)
These modifications as given in Table 2 include the addition of wash steps to eliminate anticomplementary factors,29 the use of an antihuman globulin (AHG) to increase the ability to detect low-titer and/or noncomplement fixing antibodies,30 extended incubation to increase the binding of low avidity antibodies,31 and the use of separate T and B lymphocyte target cells.32 Of all the modifications mentioned above, the most widely utilized is the use of AHG-CDC to enhance the sensitivity of complement fixing alloantibody detection.33
Complement-dependent cytotoxicity assays detect both immunoglobulin M (IgM) and IgG classes of antibodies. Clinically, greater emphasis is put on IgG antibodies rather than IgM antibodies. Moreover, most of the autoantibodies are IgM antibodies and can interfere with the detection of IgG antibodies. To negate the effects of IgM antibodies, it is an accepted laboratory practice to pretreat the test serum with heat or reducing agents such as dithiothreitol (DTT).
The CDC assay was further modified as an antibody screening assay by using cell panels composed of randomly selected HLA-typed donor lymphocytes in an effort to mimic the natural distribution of HLA antigens within a population. The results are expressed as %PRA (percent of cells in the panel giving a positive reaction). PRA is a measure of sensitization. However, the %PRA for a given serum sample could vary significantly depending on the antigen composition of the test panel. This means a high %PRA could either be because of serum containing several different specificities or just a single specificity corresponding to an overrepresented antigen on the test panel. Later, commercial cell panels became available, providing a limited degree of consistency in PRA testing. CDC-based PRA testing has several important drawbacks, such as it neither reflects the complex array of antibodies that could be present nor reflects the specificity of the antibody. In addition, non-HLA antibodies could give CDC-positive results, thus further skewing the %PRA result.
Flow Cytometry Crossmatch
The flow crossmatch technique was first introduced in 1983 by Garovoy et al. primarily to address some of the problems inherent to the CDC assay.34 The assay involves incubation of purified donor mononuclear cells with the patient's serum and then adding fluorescein conjugated, secondary antihuman immunoglobulin antibody to detect antibody in the patient's serum bound to the cellular targets (Figs. 9A to D). Subsequently, it was modified to employ two-color analysis of T and B cells in separate tubes and a three-color analysis of T and B cells in a single tube to permit the simultaneous evaluation of T- and B-cell reactivities.35,36 This involved an additional incubation step with anti-CD3 PerCP/CD19-PE to identify the T- and B-cell populations, respectively.37 Moreover, by varying the type of secondary antibody used, one can test for IgG, IgM, or IgA immunoglobulin isotypes. The amount of cell-bound antibody is quantified by fluorescence intensity and the degree of positivity is expressed as channel shift. A 40-channel shift for T cells and an 80-channel shift for B cells are generally considered a positive test.38 However, it is apt for the laboratories to set their own criteria. The possible combination of CDC and flow crossmatch reactivity and their interpretation is shown in Table 3.
Even though flow cytometry crossmatch (FCXM) is much more sensitive to CDCXM, it is not free of limitations. Most notably, nonspecific binding of immunoglobulins to Fc receptors can be mistaken as positive result. Non-HLA antibodies as well as monoclonal antilymphocyte antibodies such as thymoglobulin, rituximab, and alemtuzumab used for immunosuppression can also interfere with the assay giving a false-positive result.39,40 Unlike CDCXM, it is a binding assay and not a functional test because it only provides information on the binding of the DSA to its potential donor target and not on the killing of the target cells. Furthermore, it does not discriminate between complement and noncomplement fixing antibodies. The use of pronase has been utilized to reduce the rate of false-positive B-cell crossmatch due to nonspecific binding of immunoglobulins to Fc receptors as well as reactivity with therapeutic antibodies specific for CD20.18
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Figs. 9A to D: Flow cytometry crossmatch (FCXM). (A) Schematic presentation of FCXM; (B) An example of positive reaction by non-human leukocyte antigen (HLA) antibodies; Representative results of FCXM using (C) T cell or (D) B cell. (AUTO: auto cross match sample; DSA: donor-specific antibody; IgG: immunoglobulin G; MFI: mean fluorescence intensity; NC: negative control; PC: positive control; PE: phycoerythrin; XM: cross match sample)Source: Nakamura T, Ushigome H, Shirouzu T, Yoshimura N. Donor-specific anti-HLA antibodies in organ transplantation: transition from serum DSA to intra-graft DSA. In: Mahdi BM (Ed). Human Leukocyte Antigen (HLA). London: IntechOpen; 2018. [online] Available from https://www.intechopen.com/chapters/62771. [Last accessed September, 2022].
TABLE 3   Different combinations of complement-dependent cytotoxicity (CDC) and flow crossmatch reactivity and its interpretation.
CDC crossmatch
Flow crossmatch
T cell
B cell
T cell
B cell
Scenario 1
Anti-HLA class I (low titer)
Scenario 2
Anti-HLA class I (low titer) ± anti-HLA class II
Scenario 3
Anti-HLA class II ± anti-HLA class I (low titer) or therapeutic antibodies
Scenario 4
Anti-HLA class I (high titer) ± anti-HLA class II
Scenario 5
Autoantibodies (IgM antibody)
Scenario 6
Likely T-cell-specific non-HLA antibody
(HLA: human leukocyte antigen; IgM: immunoglobulin M; Neg: negative; Pos: positive)
Solid-phase Assays
Enzyme-linked Immunosorbent Assay
Kao et al. developed an ELISA-based HLA antibody detection method using affinity-purified HLA class I from platelets as the target antigens for the detection of HLA antibodies in patient sera.41 Subsequently, several ELISA-based assays to detect HLA antibodies were introduced in the 1990s, and all were more sensitive than CDC assay but less sensitive than the FCXM. The viability of this assay as a clear indicator of the amount of antibody detected is questionable, since the test uses quantities of HLA antigens much greater than the normal biologic expression on the immune cell. Undoubtedly, ELISA was a significant step forward, but it was cumbersome to perform and has been superseded by the bead-based assays.
Flow Cytometry-based Panel Reactive Antibody
This technique was first developed by Pei et al. in 1998 to address the issues associated with ELISA such as sensitivity and separate test required to discriminate HLA class I and II antibodies.42 This approach utilized isolation and purification of HLA class I and II proteins from Epstein–Barr virus (EBV) transformed cell lines and their subsequent coupling to microparticles that could easily pass through a flow cytometer. This assay combined 30 individual beads for class I and 30 individual beads for class II with each bead coated with either HLA class I or class II phenotype. Two distinct properties, namely bead size and inherent bead fluorescence, help to discriminate beads expressing HLA class I antigens from class II antigens.19
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Figs. 10A and B: FlowPRA screening. (A) Schematic presentation of FlowPRA screening; (B) Examples of positive FlowPRA screening results about class I (upper) and class II (lower). (HLA: human leukocyte antigen; IgG: immunoglobulin G; MFI: mean fluorescence intensity; NC: negative control; PE: phycoerythrin)Source: Nakamura T, Ushigome H, Shirouzu T, Yoshimura N. Donor-specific anti-HLA antibodies in organ transplantation: transition from serum DSA to intra-graft DSA. In: Mahdi BM (Ed). Human Leukocyte Antigen (HLA). London: IntechOpen; 2018. (online) Available from https://www.intechopen.com/chapters/62771. [Last accessed September, 2022].
The class I beads are smaller and nonfluorescent while the class II beads are slightly larger and are impregnated with a fluorescent dye that has excitation/emission properties similar to phycoerythrin (PE). A positive reaction is determined by the number and fluorescence intensity of beads. Beads that exhibit increased fluorescence [fluorescein isothiocyanate (FITC) antihuman, IgG] compared to background controls are considered positive. These differences permit simultaneous testing for both class I and II HLA antibodies. The %PRA is calculated as the percentage of beads that demonstrate a significant positive fluorescence shift above background (Figs. 10A and B). It has been shown that the FlowPRA can detect HLA antibodies 10–20% more frequently than ELISA or cytotoxicity.43
In addition to increased sensitivity and simultaneous detection of antibodies against HLA class I and class II, the assay has additional advantages of being specific for HLA antigens without significant interference from non-HLA antibodies. It is not affected by subjective microscopic readings or cell viability and gives a semiquantitative assessment of antibody binding but failed to determine individual HLA specificities. Subsequently, it was modified to coat microparticles with individual HLA molecules.44 This assay used panels of 8 or 11 individual beads, each identified by their unique level of red fluorescence signature. Green fluorescence indicates the quantity of HLA antibody that is bound. Hence, a broadly sensitized patient could have all his/her HLA antibodies identified, thereby making it possible to predict compatibility with specific donors. Although extremely useful, the assay was cumbersome requiring up to 15 tubes per patient, with each tube containing 8 or 11 individual, single HLA-antigen-coated beads. This proved to be a major drawback and a significant technical handicap. Thus, the high degree of HLA diversity and the challenges of highly sensitized patients made its use limited.
Luminex Platform-based Assays
This technology has completely transformed the histocompatibility laboratory's approach and ability to detect HLA antibodies. Its usefulness lies in the characterization of HLA antibody specificities in highly reactive sera.45,46 The technique has been successfully applied to monitor patient antibody profiles in relation to particular specificities, such as following desensitization treatment at pretransplant stage and therapeutic monitoring of antibody removal therapies.
The Luminex assay is a semiquantitative bead-based array system that utilizes polystyrene microbeads coated with purified HLA class I or class II antigens. These beads are impregnated with a unique blend of different intensities of two fluorescent dyes that are simultaneously excited by red laser at 635 nm. The emitted light can be detected at wavelengths of 660 nm (red) and 730 nm (infrared) using a dedicated footprint flow cytometer. By measuring the composition of emission intensities for both channels, up to 100 distinct microbead sets can be identified concomitantly (classifier parameters). These microbeads act as molecular carriers that capture samples. The detection of HLA antibody is achieved by using a secondary antibody conjugated with the reporter fluorophore R-PE, which is excited by green laser (532 nm) and detected at 576 nm. The fluidics of the Luminex cytometer aligns the microbeads in a single file as occurs with a flow cytometer.20
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Fig. 11: Schematic presentation of human leukocyte antigen (HLA) antibody testing using single antigen bead (SAB) assay. Recombinant HLA bound to “single antigen” microbeads bind with anti-HLA antibody present in patient serum and are stained by a secondary antihuman immunoglobulin G (IgG) antibody conjugated with phycoerythrin (PE). Data acquisition is done on Luminex platform wherein red laser identifies the bead being examined and green laser measure the assay result on its surface.
The optics then focuses on each bead to generate and collect the fluorescent signals, while the electronics convert and digitize the signals for computer analysis. Figure 11 schematically shows the principle of HLA antibody screening using Luminex technology.
Based on the composition of target antigens, there are three versions of the test, namely pooled antigen, phenotype panels, and SAB in the order of increasing resolution. The “pooled antigen panels” are typically used as a preliminary screening test to detect the presence or absence of class I and II antibodies in a single test. The bead set is composed of 10–17 beads, and each of them is coated with affinity-purified HLA antigens obtained from multiple individual cell lines. Although this is relatively less expensive with a very high negative predictive value, it is unable to specify possible antigen and is purely qualitative. The “phenotype panels” detect antibodies and their specificity against HLA class I and II antigens separately. The class I panel contains approximately 55 beads, while class II contains 35 beads. Each bead is coated with class I or class II HLA antigen derived from a single individual cell line. Since there is more than one HLA specificity present on each bead, greater expertise is required for the interpretation of results as compared to the pooled or single antigen panel.47,48
One of the major advantages of this technique is that through single antigen test panels, it enables detection of epitope-specific antibodies to individual and specific HLA alleles and thus provides information on the virtual crossmatch (VXM).49 Moreover, it permits identification of antibodies to HLA loci that were not amenable to detection by traditional cell-based assays. These include HLA-C antigens that are poorly expressed on the lymphocyte surface, DQ and DP antigens, as well as DRB3, 4, and 5, which were difficult to distinguish from DRB1 antigens coexpressed on the surface of B cells. This technology also enables detection and characterization of antibodies against major histocompatibility complex (MHC) class I-related chain A (MICA). The MFI of the beads is a measure of the degree of saturation of the total antigens present on the beads by antibodies and is used as a surrogate marker for the level of antibody titers. There is no recommended cutoff value for MFI positivity and most laboratories set their own cutoff levels. In our center, MFI > 1,000 is taken as relevant.
This widely accepted assay is not free of limitations, and there are important critical factors that should be considered while analyzing the data. Being highly sensitive, the technology provides false-positive results, leading to unnecessary delays on the waiting list. The current panel of single HLA beads does not cover the whole diversity of HLA alleles. Hence, populations such as Asian Indians that are characterized by many novel alleles and unique HLA haplotypes will be at a disadvantage because antibodies raised against such HLA alleles are likely to be missed. Luminex beads accommodate both intact and denatured HLA molecules. The immobilization procedure of the HLA molecules on beads may alter the tertiary structure of the molecule exposing neoepitopes which may lead to false-negative and/or -positive reactions. Morales-Buenrostro et al. described the presence of naturally occurring antibodies to HLA class I and II molecules, which may be produced as a result of cross-reactivity with epitopes on 21vaccines or with epitopes on environmental proteins such as microbes or ingested foods.50 Epitope analyses have revealed that these natural HLA antibodies react with polymorphic amino acid residues that are usually not exposed to the molecular surface.51
The relative density of a particular antigen, especially HLA-Cw, -DQ, and -DP on SAB, is higher as compared to phenotype beads and human cells. As a consequence, antibodies directed against these antigens run the risk of being overestimated yet may represent only a low immunologic risk for renal transplant rejection.52 Conversely, antibodies against public epitopes such as Bw4 or Bw6 may appear underrepresented because a single antibody may be dispersed across many beads underestimating its actual level (peanut butter effect).53 Disparities in relative antigen quantity exist across the different bead formats and different HLA molecules on the SAB.
The technique remains susceptible to the interference caused by substances inherent to serum or by exogenous substances. This may lead to the potential underestimation of HLA antibodies due to false-negative reactions or may give false-positive results. Similar to the prozone or Hook phenomenon, the serum appears weak or negative when tested neat and becomes more strongly reactive upon dilution, especially in highly sensitized patients. The main reason for this prozone-like effect using Luminex beads is attributed to the presence of IgM antibodies and complement component C1. Dilution or hypotonic dialysis of serum as well as treatment of sera with DTT, ethylenediaminetetraacetic acid (EDTA), or heat activation helps to overcome this phenomenon.54 Therapeutics such as intravenous immunoglobulin (IVIg), antithymocyte globulin (ATG), bortezomib, and eculizumab also cause interferences for detection of HLA antibodies. High-dose IVIg (2 g/kg) interferes with the assays using an antiglobulin reagent. It leads to more than fivefold increase in reactivity with negative control beads. Treatment with ATG results in misleading HLA-specific reactivity. Treatment with bortezomib and eculizumab leads to significant reduction in the strength of antibody. However, hypotonic dialysis of sera prevents any such reduction in antibody strength.55
Parameters that indicate interferences in Luminex-based assays include:48
  • High reactivity with the negative control bead
  • Low reactivity with the positive control bead
  • Sudden change in the pattern of reactivity in sequential sera from a patient in the absence of any specific treatment or event
  • Reactivity that does not reconcile with the results of CDC or FCXM tests
  • Reactivity with the patient's own HLA antigens.
The specific features along with the limitations of the available techniques based on cell and solid phase assays for the detection of anti-HLA antibodies are summarized in Figure 12.
Luminex Crossmatch
Luminex crossmatch (LUXM) detects donor-specific anti-HLA IgG antibodies against class I and/or class II antigens in single well. Donor lymphocytes isolated from peripheral blood are solubilized with a nonionic detergent to obtain a lysate and are used as the source material for HLA. Two of the beads in a single blend of Luminex beads are coated with mouse antibodies with specificity for nonpolymorphic sequence on HLA class I and II molecules. Beads capture HLA molecules of the donor from donor lysate cells. Capture beads are then reacted with recipient sera, and the bound antibody, if present, is detected with fluorescently labeled secondary antibody, and results are read as per the conventional bead assay (Fig. 13). The blend of Luminex beads also includes background and conjugate control beads. The main advantage of the technique is that the donor lysate can be stored frozen for future testing and avoids the cell viability issues. This makes it ideal to use both for pre- and post-transplant testing. Bacterial contamination of the samples as well as the presence of immune complexes or other immunoglobulin aggregates in the sample may cause an increased nonspecific binding and produce erroneous results in this assay. Several studies have demonstrated the failure to detect class II antibodies especially against DQ and DP antigens. LUXM is not recommended to be used as a stand-alone method for transplant decisions.56
Virtual Crossmatch
Luminex SAB assay and DNA-based HLA typing at high resolution have permitted the concept of VXM gain acceptance worldwide. The VXM is defined as “An assessment of immunological compatibility based on the patient's alloantibody profile compared to the donor's histocompatibility antigen.”57 The VXM takes into account the HLA antibody profile of the patient to predict crossmatch negative donors. In other words, it gives an insight into acceptable mismatches. In majority of cases, VXM correlated with FCXM. Disagreement between FCXM and VXM should be considered with much deliberation rather than disregarding the results as false.5861 Table 4 highlights the discordant scenarios of FCXM and VXM, factors responsible for such discordance and further evaluation to confirm or negate such factors.
The degree of sensitization as measured by PRA is imprecise and highly variable as it is dependent on panel cell construction and not true representation of donor pool. In 2007, the United Network of Organ Sharing (UNOS) approved a proposal by the Histocompatibility Committee to use calculated PRA (cPRA). It is based on antibody specificity, not from positive reactions on a panel, and is computed from HLA frequencies in different ethnic populations.22
zoom view
Fig. 12: A summarized view of the cell-based [complement-dependent cytotoxicity (CDC) crossmatch and flow cytometry] and solid phase-based assays (Luminex-based technology) for the detection of antihuman leukocyte antigen (HLA) antibodies, highlighting main advantages and disadvantages. (IgG: immunoglobulin G; IgM: immunoglobulin M; MFI: mean fluorescence intensity; MICA: major histocompatibility complex class I-related chain A)Source: Mehra NK, Baranwal AK. Clinical and immunological relevance of antibodies in solid organ transplantation. Int J Immunogenet. 2016;43(6):351-68. Reproduced with permission from John Wiley & Sons.
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Fig. 13: Schematic presentation of Luminex crossmatch. Donor lysate is prepared from donor lymphocytes using lysis buffer. Lysate is mixed with DSA beads to capture donor class I and II human leukocyte antigen (HLA) on to beads. Donor-specific antibodies present in patient serum binds with HLA on capture beads and is then stained by a secondary antihuman immunoglobulin (IgG) antibody conjugated with phycoerythrin (PE). Data acquisition is done on Luminex platform and the results are analyzed. (Con: control)
TABLE 4   Discordant scenarios based on flow cytometry crossmatch (FCXM) and virtual crossmatch (VXM) results, factors responsible, and further evaluation.
Flow crossmatch (FCXM)
Factors responsible
Further evaluation
Scenario 1
Auto-flow crossmatch
Nonspecific binding
Pronase treatment
Alloantibody to shared epitopes (“peanut butter effect”)
Non-HLA antibody
Test for non-HLA antibody
Donor allele/antigen not covered by SAB
Check for allele representation on bead
Prozone/Hook effect
Pretreat sera with EDTA/heat or dilution of sera with repeat assay
Incomplete/incorrect HLA typing
Ensure complete and correct typing
Scenario 2
Antibody toward denatured HLA
Acid treat SAB
DSA with low affinity/avidity
Low HLA expression
(DSA: donor-specific antibody; EDTA: ethylenediaminetetraacetic acid; HLA: human leukocyte antigen; SAB: single antigen bead)
zoom view
Flowchart 1: Algorithm for candidates with potential living donor.(AD: alternate donor; CDC: complement-dependent cytotoxicity; DSA: donor-specific antibody; KPD: kidney paired donation; PRA: panel reactive antibody; SAB: single antigen bead; XM: crossmatch)
It takes into consideration the frequency of unacceptable HLA in the donor pool. The computer software system uses an established formula and HLA frequencies derived from the HLA types of that population to calculate the cPRA value. Different countries have different calculators suited to their ethnic population. It provides a more representative estimate of sensitization not only because of the use of more accurate HLA frequencies, but also includes both class I and II antigens in the calculation, unlike that of traditional PRA, where class I and II specificities are measured separately.62
Proper history-taking of a sensitizing event is critical for the correct interpretation of the crossmatch and antibody detection assays. Flowchart 1 highlights the algorithm for the candidates with potential living donors. It may differ among different setups based on the preferences and availability of the assays.24
The Luminex-based SAB assay coupled with HLA sequencing data provides a platform to identify epitopes against which HLA antibodies are directed. This in turn leads to a better opportunity for clinical decision-making with accuracy and maximizes the success of transplant. In view of intricacies associated with rapid development of techniques, it is imperative for the transplant physicians to communicate with transplant immunologists on an individual patient basis.
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  1. Amos DB, Bashir H, Boyle W, MacQueen M, Tiilikainen A. A simple micro cytotoxicity test. Transplantation. 1969;7:220-3.
  1. Johnson AH, Rossen RD, Butler WT. Detection of alloantibodies using a sensitive antiglobulin microcytotoxicity test: identification of low levels of pre-formed antibodies in accelerated allograft rejection. Tissue Antigens. 1972;2(3):215-26.

  1. 25 Cross DE, Whittier FC, Weaver P, Foxworth J. A comparison of the antiglobulin versus extended incubation time crossmatch: results in 223 renal transplants. Transplant Proc. 1977;9(4):1803-6.
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  1. Zachary AA, Klingman L, Thorne N, Smerglia AR, Teresi GA. Variations of the lymphocytotoxicity test. An evaluation of sensitivity and specificity. Transplantation. 1995;60(5):498-503.
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  1. Bray RA, Lebeck LK, Gebel HM. The flow cytometric crossmatch. Dual-color analysis of T cell and B cell reactivities. Transplantation. 1989;49:834-40.
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  1. Bray RA, Gebel HM. Clinical utility of flow cytometry in allogeneic transplantation. In: Keren D (Ed). Clinical Flow Cytometry. Chicago, IL: ASCP Press; 2007. pp. 275-92 [Chapter 14].
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  1. Vaidya S, Partlow D, Susskind B, Noor M, Barnes T, Gugliuzza K. Prediction of crossmatch outcome of highly sensitized patients by single and/or multiple antigen bead Luminex assay. Transplantation. 2006;82(11):1524-8.
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  1. El-Awar N, Terasaki PI, Nguyen A, Sasaki N, Morales-Buenrostro LE, Saji H, et al. Epitopes of human leukocyte antigen class I antibodies found in sera of normal healthy males and cord blood. Hum Immunol. 2009;70:844-53.
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  1. Cecka JM. Calculated PRA (CPRA): the new measure of sensitization for transplant candidates. Am J Transplant. 2010;10(1):26-9.26
1.3 Donor-specific Antibodies in Kidney Transplantation
Rahul Grover, Narayan Prasad
The report by Patel and Terasaki in 1969 regarding the significance of positive crossmatch in kidney transplant, “24 of 30 with positive crossmatch versus just 8 of 195 with negative crossmatch failed to function,” made screening for preformed cytotoxic antibodies against donor an integral part of transplant evaluation.1 Since then, the methods for assessing circulating donor-specific antibodies (DSA) have undergone many refinements. DSA either maybe present pretransplant or may develop anytime post-transplant. The presence of DSA predisposes to the development of antibody-mediated rejection (AMR), which is considered the leading cause of graft loss. Pretransplant evaluation for DSA to assess the degree of sensitization as well as post-transplant monitoring of DSA to predict, prevent, and manage rejections and prevent graft loss continues to evolve.2
Human Leukocyte Antigens
Human leukocyte antigens (HLA) are dimeric glycoprotein molecules that are present on nucleated cells and perform the role of antigen presentation through their highly polymorphic peptide-binding groove.3 Class I HLA molecules (HLA A, B, and C) are dimers of alpha-chain and beta 2-microglobulin. They present intracellular peptides to CD8+ T cells. Class II HLA molecules (DR, DP, and DQ) are dimers made of a constant alpha chain (DR) or polymorphic alpha chain (DQ, DP) with polymorphic beta chains (DP, DQ, and DR). They are constitutively expressed on B cells and antigen-presenting cells (APC). Their expression can be induced on endothelial cells (ECs) and activated T cells. They present endocytosed extracellular peptides which get loaded via phagolysosomes to CD4+ T cells.
The genes encoding HLA molecules reside on the short arm of chromosome 6 in the major histocompatibility complex (MHC) region. They show a high degree of polymorphism (>16,000 alleles reported). This polymorphism while being a protective mechanism against infectious organisms acts as a barrier for organ transplantation. Exposure to non-self HLA leads to allorecognition via direct, semidirect, or indirect immunological pathways.4 See Table 1 for terms and definitions.
Direct Pathway of Allorecognition
Direct pathway involves donor APC [passenger cells] presenting nonself [donor] HLA peptide complex to recipient CD4+ T cell. CD4+ cells then provide T cell help for the effector function of CD8+ cells. This occurs in early post-transplantations and lasts till the donor APCs become depleted. This pathway is considered to be responsible for early acute cellular rejections.
Indirect Pathway of Allorecognition
Indirect pathway involves recipient APC phagocytosing and processing donor allopeptide and presenting them on self-MHC class II molecules to CD4 +ve T cells. These CD4 +ve T cells provide help to de novo allospecific CD8 +ve T cells. This leads to allorecognition and initiation of chronic allograft rejection.
Semidirect Pathway of Allorecognition
It is recognized that intact MHC class I and class II molecules are transferred between recipient and donor APC. This is accomplished by either direct cell to cell contact [trogocytosis] or through extracellular vesicles [exosomes] that are engulfed by APCs. In this way, recipient APCs acquire and present intact [unprocessed] donor peptide MHC complex on their surface [referred to as crossdressing].
Recipient APC thus has ability to express simultaneously on their surface ‘self-HLA molecule with processed donor peptides [class II] to CD4 +ve Th cells’ as well as intact [unprocessed] donor peptide expressing donor HLA molecule [class I] to CD8 +ve T cells.27
TABLE 1   Terms and definitions.
Donor-specific antibodies, usually against HLA
DSA to non-HLA
Human leukocyte antigens
Antibody-mediated rejection
Major histocompatibility complex
A highly polymorphic region on short-arm chromosome 6 contributes to the immunological repertoire, autoimmunity, sensitization
Class I HLA molecules
HLA A, B, and C
Present on all nucleated cells
Class II HLA molecules
Present on antigen-presenting cells, B cells
Antigen-presenting cells
CD4+ T cells
T helper cells
CD4+ Tfh
T follicular helper cells
CD8+ T cells
Effector T cells
Complementarity-determining region
Part of the variable region of B-cell receptor, antibodies, T-cell receptor that binds to the antigen
Part of antigen that is recognized by the immune system
Part of the antibody that recognizes epitope
Clusters of two to three amino acid sequences (out of the 15–22 that make the epitope) that are recognized by HLA antibodies
Can be considered as functional epitopes
Cross-reactive epitope groups
Defined by the presence of cross-reactive HLA antibodies due to the presence of shared epitopes
Immunologically naive
No previous exposure to alloantigen
Absence of DSA does not mean immunologically naive status
Immunological sensitization
Previous exposure to alloantigen
DSA maybe present
Panel-reactive antibody
  • A method to assess the degree of sensitization
  • Patients’ serum tested against around 100 representative blood donors
This constitutes the three cell model or linked model of allorecoginition. This model is considered pertinent in development of alloantibodies. (Figs. 1A and B).
Naïve B cells carry a huge preimmune antibody repertoire with complementarity-determining regions (CDR) that help them recognize varied non-self HLA epitopes. On encountering the non-self HLA, the cognate naive B cell undergoes activation, proliferation, and maturation to DSA-specific memory B cells and long-lived plasma cells. The antigen-stimulated rapidly proliferating B cells in the germinal center of lymphoid tissues undergo somatic hypermutations in variable regions of their B-cell receptors (BCR).
zoom view
Figs. 1A and B: (A) Traditional three-cell model T cells cross talk, also called linked model, in which donor APCs activate both CD4 and CD8 T cells; (B) In four-cell models, recipient CD8+ T cells stimulated through the direct pathway by donor APC can receive T-cell help or suppression from CD4+ T cells activated via the indirect pathway by recipient dendritic cells. (APC: antigen-presenting cell)
A subset of allospecific CD4+ T cells that gets activated via the indirect or semidirect pathway moves to the T-cell zones in the lymphoid follicle. These CD4+ Tfh cells help complementary B cells in cross switching from short-lived immunoglobulin M (IgM)-producing B cells to long-lived immunoglobulin G (IgG)-producing B cells as a part of affinity-based maturation—a method of negative selection so that only cells with highest antigen affinity proliferate. DSA are thus generated as a consequence of alloimmune recognition and close interaction of APC, B cells, and T cells.5,6 These are regulated by a host of co-stimulatory or subordinate signaling pathways including cytotoxic T-lymphocyte-associated protein 4 (CTLA4), CD28, CD40L to B7, and CD40 on T and B cells, respectively. Interaction of CD28–B7 promotes interleukin-2 (IL-2) release causing proliferation and differentiation of B cells and CD40L–CD40 interaction promotes class switch.
While these DSA show high specificity to respective HLA molecules, they also show cross-reactivity with groups of non-self HLA molecules (cross-reactive epitope groups or CREGs). This is explained by the presence of shared epitopes (antigenic/immunogenic domains of HLA) or shared eplets [configuration of polymorphic amino acid determinant (usually three to five amino acid long) of epitope]. It is realized that it is the eplets to which the DSA actually bind and epitopes of different HLA molecules may also have shared eplets.7,8
While DSA against the non-self HLA are the chief cause of AMRs, non-HLA DSA are also generated and can be responsible for some of the rejections. These non-HLA antibodies include those against MHC class I chain-related molecule A (MICA), anti-protein kinase C (PKC) zeta, angiotensin II type I receptor (ATR1), antiendothelial cell antibodies (AECA), anti-peroxisomal trans-2-enoyl-CoA reductase (PERC), antiendoglin, MIG, ITAC, etc.
H-Y minor histocompatibility antigen encoded by Y chromosome can be a target for alloimmune response against male kidneys transplanted in female recipients.
Exposure to non-self HLA by sensitizing events such as blood product transfusions, pregnancy, previous transplant, and implants [ventricular assist device (VAD), homografts, etc.] leads to host mounting an immunological response. This response is both cellular, i.e., “generation of donor-reactive memory T and B cells” and humoral, i.e., generation of anti-HLA antibodies. Alloreactive immune T-cell response may also arise due to cross-reactivity of infectious pathogens with donor antigens. Individuals with these exposures are considered to be sensitized and are likely to pose an anamnestic immunological response post solid organ transplant leading to allograft rejection. A person not exposed to the above can be considered to be immunologically naive and is unlikely to host an anamnestic immune response against allograft.
No single laboratory test can determine the sensitization status of a patient. Cellular memory responses are difficult to assess; tests such as interferon-γ enzyme-linked immunosorbent spot (ELISpot) assay do provide some insight.
Donor-specific antibodies are the most readily used laboratory tests to determine sensitization.
Circulating HLA antibodies both donor specific and non-donor specific give an idea about the presence of sensitization. Sensitization is usually measured by detecting the presence of antibodies against a panel of representative HLAs from the general population. These antibodies are called the panel-reactive antibodies. The more the number of antibodies present, the more the sensitization.
There are, however, limitations in defining the specificity of measured DSA. On one hand, complement-dependent cytotoxicity (CDC) is very specific. On the other hand, single antigen bead (SAB) on Luminex platforms is highly sensitive.29
TABLE 2   Immunological workup for renal transplant.
Assessment of immune memory
HLA typing and DSA: Laboratory assessment
Clinical attributes of DSA
Molecular mismatch analysis
(DSA: donor-specific antibodies; HLA: human leukocyte antigen)
SAB assays pick up a lot of noise due to the detection of DSA to cryptic epitopes of HLA (that are not clinically significant) that get exposed due to the limitations in the technical process of synthesizing and loading HLA on the beads. As such, the tests have to be interpreted in terms of clinical history.
Evaluation of sensitization involves documenting the history of HLA-sensitizing events, inflammatory events that may boost alloimmune memory (such as major surgery, infections, and vaccinations), and non-DSA and DSA documented at any time pretransplant. DSA appearing within 2 weeks and up to 3 months post-transplant may still suggest the presence of immunological memory rather than de novo antibody responses. Autoantibody assessment as well as determination of non-HLA antibodies is also mandated.
Key points for Immunological workup for renal transplant are provided in Table 2.
Alloimmune recognition of the engrafted organ leads to the generation of de novo DSA. Risk factors for the development of de novo DSA include a high number of HLA mismatches, inadequacy of immunosuppression, and factors that increase graft immunogenicity such as viral infections, ischemia-reperfusion injury, etc. They often develop against class II antigens, especially against DQ due to its highly polymorphic beta chain. New-onset DSA develop commonly in the first year post-transplant but can develop at any time post-transplantation.
Donor-specific Antibodies
Class I Donor-specific Antibodies
These are formed against the alpha chain of HLA A, B, and C. They are usually of IgG1 and IgG3 subclass and are complement-binding antibodies. Preformed class I DSA may result in positive T-cell crossmatch contraindicating transplant. De novo class I antibodies develop relatively early post-transplant and can cause acute AMR and graft dysfunction. C4d positivity is seen and rejections are often responsive to treatment.
Class II Donor-specific Antibodies
These are formed against the alpha and beta chains of DR, DQ, and DP antigens. They are usually of IgG2 and IgG4 subclass, and complement activation is weak. They result in a positive B-cell crossmatch and are responsible for chronic and subclinical rejections. They are difficult to desensitize and less amenable to treatment.
Nonhuman Leukocyte Antigen Donor-specific Antibodies
These are usually formed by humoral response toward non-HLA on ECs. They came into recognition as causative antibodies for unexpected hyperacute AMR in identical twin donations. A wide variety of antibodies are now recognized and they play a role in acute and chronic allograft rejections. Their routine detection, attributable risk, and pathogenic role on graft outcomes continue to be evaluated.9
The ECs constitutively express class I antigens (with HLA A, B finding greater expression than class C). Class II antigens expression gets induced in the presence of events promoting inflammation (such as ischemia-reperfusion injury, delayed graft function, etc.). DSA bind to expressed HLA depending on the avidity of eplet–antibody interaction that determines their pathogenicity.
The DSAs cause graft injury by one of the following mechanisms—by promoting complement activation, by ADCC, or by inducing modifications in endothelium.10
Occasionally the DSAs bind to endothelial antigen without any inflammation or endothelial injury, a process referred to as accommodation.
Immunoglobulin M, IgG1, and IgG3 classes of antibodies are complement fixing. Their binding activated the classical complement pathway leading to the activation of MAC and lysis of antibody-coated cells. The by-products of the complement pathway additionally participate in the recruitment of inflammatory cells. Tissue deposits of C4d are histological markers for the activation of complement pathway.
Antibody-dependent cell-mediated cytotoxicity is induced by the interaction of Fc-gamma receptors of natural killer (NK) cells (as well as neutrophils and macrophages) with Fc-gamma region of DSA. This results in the formation of synapses and release of granzymes and perforins leading to apoptosis of the target cell. The release of chemokines further enhances HLA expression and inflammation. All classes of DSA including noncomplement activating DSA (IgG2, IgG4) participate in ADCC. Histologically, it is characterized by microvascular inflammation in the absence of C4d deposits.
Additionally, DSA binding to class I HLA on ECs induces intracellular signaling cascades. These lead to EC activation and phenotype modifications enhancing cell proliferation and survival. This contributes to the development of intimal proliferation, fibrosis, transplant glomerulopathy, and vasculopathy (Fig. 2).30
zoom view
Fig. 2: Pathogenesis of complement-binding donor-specific antibodies (DSA) and complement nondependent binding of DSA.
Presence of DSA is tested by one of the following methods: Classical cytotoxicity crossmatch, flow crossmatch, and solid phase assays (beads conjugated with single antigen or multiple HLA). The SAB assays are very sensitive, while cytotoxic crossmatch is very specific.
The DSA are assessed for their strength, complement-fixing ability, and subclass.11
The strength is measured in terms of mean fluoresce intensity (MFI) on Luminex assays. Higher MFI increases the likelihood of complement activation and tissue injury. However, the correlations between MFI and tissue injury are not definitive. There is often a high coefficient of variance (CV%) in MFI value, different DSAs may have similar MFI but may not cause similar tissue injury. The affinity for the beads may not correspond to the in vivo antigen binding on the ECs. DSAs targeting shared epitopes may get diluted on the multiple beads. Inhibitors, prozone effects, and denatured beads may affect interpretation.
C1q-binding DSAs help to improve specificity. C1q-binding DSAs show higher odds in predicting tissue injury compared to DSA that do not bind C1q. C3d- and C4d-binding DSA serve to assess more downstream complement activation. C3d and C4d assays used alone or along with C1q-binding assays serve to be a better predictor of AMR. However, there are limitations in these approaches. Complement fixation strongly correlates with MFI titers and as such often is only a surrogate of high MFIs. Also, it is recognized that c4d staining is seen histologically in AMRs in which DSA were not fixing complement in vitro.
Donor-specific antibody IgG subclasses give an insight into the maturity of immune response. Sequential IgG subclass switching occurs during immune response from IgG3 → IgG1 → IgG2 → IgG4. IgG3 and IgG1 tend to activate complement, while IgG2 does so minimally and IgG4 does not activate the complement cascade. The presence of IgG2 and IgG4 thus represents a more advanced stage of immune response. These DSA recruit effector cells via Fc-gamma receptor. IgG3-dominant AMR occurs earlier and is associated with C1q positivity, microcirculation injury, and C4d positivity. IgG4-dominant AMR maybe more subclinical and is associated with transplant glomerulopathy, interstitial fibrosis, and tubular atrophy.
Donor-specific antibodies are monitored as a part of pretransplant risk stratification, monitoring efficacy of desensitization, and post-transplant to predict AMR.
Risk Stratification
Risk is stratified on the basis of two types of immune responses: (1) The immune memory that leads to 31anamnestic response and (2) HLA mismatch that leads to the development of de novo alloimmune recognition. Features and outcomes of AMR related to memory versus de novo responses may vary.
All HLA immunizing events are not equal. The alloimmune response varies depending on the type of immunizing event. There maybe differences in response between a term pregnancy, miscarriage, blood product transfusion, previous transplant, etc. The antibody titers or MFI can be measured serially to assess strength and trends. DSA measurements have limitations, MFI does not always correlate with titers, the absence of DSA/calculated panel-reactive antibody (cPRA) does not necessarily mean immunologically naive status, acceptable antigens used in the listing may also have alloimmune sensitization but below thresholds used for listing, pretransplant titers do not necessarily correlate with post-transplant antibody responses, and c1q negative DSA can still mount c4d +ve AMR [at least six c1q DSA molecules in close proximity are required to activate complement, lower titers of complement fixing antibody can thus go undetected]. Thus, there remains significant unpredictability in the assessment of memory for risk stratification. Also, measuring memory cell responses remains difficult adding to unpredictability.
Similarly, assessing for de novo alloimmune response is also challenging. An HLA epitope has six CDRs out of that the third region of the variable heavy-chain antibody-binding site (CDR H3) is the eplet. This three to five amino acid region binds most strongly and represents the “functional epitope” for antibody specificity, while all the six CDRs together represent “structural epitope” and determine antibody avidity. HLA serological matches do predict the development of DSA and long-term graft outcomes. Those with the higher number of HLA mismatches have higher chances of development of DSAs and deterioration of graft function over time. However, serological assessment of HLA mismatches has inherent immunogenicity of organ transplanted (kidney vs. liver), adequacy of immunosuppression, and type of induction also need to be considered for risk stratification.2,12
The recommendations from Sensitization in Transplantation: Assessment of Risk (STAR) working group meeting reports (2017 and 2019) regarding DSA assessment and monitoring are summarized in Tables 3 to 7.
Currently, HLA antibody titers are used to assess desensitization protocol. Beyond that assessment of the effect on immunological memory in desensitization protocols like limitations and does not predict de novo alloimmune responses accurately.
TABLE 3   Comparison of class I and II donor-specific antibodies (DSA).
Class I DSA
Class II DSA
A, B, and C
DR, DQ, and DP
Epitopes’ location
α- and β-chains
All nucleated cells
Antigen-presenting cells
Preformed DSA:
Positive crossmatch
T cells
B cells
De novo DSA:
  • Detection
  • IgG subclasses
  • Complement binding
  • Frequency
  • Sooner
  • IgG1, IgG3
  • Strong
  • Fewer
  • Later
  • IgG2, IgG4
  • Weak/no
  • Common, especially DQ
Antibody-mediated rejection:
  • Phenotypes
  • Presentation
  • Graft dysfunction
  • C4d deposit
  • Treatment
  • Graft loss
  • Acute
  • Early
  • Rapidly
  • Positive
  • More responsive
  • Early
  • Chronic/subclinical
  • Later
  • Slowly
  • Negative
  • Less responsive
  • Later
(HLA: human leukocyte antigens; IgG: immunoglobulin G)
TABLE 4   Assessment for immune memory.2,12
Immune memory
HLA-sensitizing events
  • Pregnancy transfusions
  • Previous transplant implants (VAD, homografts, etc.)
Latent potential for alloimmune memory response
  • History of sensitizing event
  • Non-DSA HLA antibody detected at one or more time prior to transplant
  • Non-DSA detected pretransplant
Inflammatory events that boost preexisting alloimmune memory
  • Major surgery
  • Major infection
  • Recent vaccination
Active potential for alloimmune response
DSA present at the time of transplantation or in the historical sample
Alloimmune memory response
DSA response post-transplant or pretransplant DSA or new DSA within 2 weeks post-transplant
Notes: Patients without HLA-sensitizing events to be considered low risk for immunological memory.
Immunological memory status should be used for risk stratification, determining the frequency of DSA testing post-transplant.
(DSA: donor-specific antibodies; HLA: human leukocyte antigens; VAD: ventricular assist device)
TABLE 5   Attributes of donor-specific antibodies (DSA).2,12
  • High MFI
  • High antibody titers
  • Complement-fixing antibodies
Increased risk of AMR
IgG subclass
  • IgG1, IgG3—acute AMR
  • IgG2, IgG4—chronic active AMR
DSA-associated cell signaling
Endothelial cell modifications
Memory cell alloimmunity
T- and B-cell assays (donor HLA-specific IFN gamma-secreting T cells, panel reactive T cells, T follicular helper cells, frequency of HLA-binding memory B cells, etc.)
At research level
(AMR: antibody-mediated rejection; IFN: interferon; HLA: human leukocyte antigens; IgG: immunoglobulin G; MFI: mean fluoresce intensity)
TABLE 6   Human leukocyte antigen (HLA) laboratory assessment.2,12
HLA typing
Should be comprehensive
HLA A, B, C, DRB1, DRB3/4/5, DQA1/DQB1, DPA1/DPB1
HLA antibody assessment
Solid-phase assays
Report up to allele level
Inhibitors should be removed
DTT, EDTA, titration studies, heat inactivation
Epitope sharing to be analyzed
Stacking antibodies against CREGs
MFI cut-off 1,000–1,500
Possible false negative if it belongs to CREG
False positive if against denatured/cryptic epitopes, autoantigen/allele, nonspecific patters (hot beads)
Differences in MFI <25% not significant
High coefficient of variance in MFI
(CREG: cross-reactive epitope group; DTT: dithiothreitol; EDTA: ethylenediaminetetraacetic acid; HTA: human leukocyte antigens; MFI: mean fluoresce intensity)
TABLE 7   Molecular mismatch analysis.2,12
HLA matchmaker
Donor–recipient amino acid sequences to determine continuous and discontinuous eplets that maybe recognized by antibody CDRs
  • Focus on polymorphic regions
  • All mismatched eplets are given same value
  • Generates an eplet mismatch score
  • Verified and unverified eplet terms used to define detected eplets not serologically confirmed
  • Immune response does not work on total loads
Electrostatic mismatch score
Electrostatic surface value of amino acids on HLA molecules
Tries to add physiochemical properties over and above the number of mismatches
  • Predicted indirectly recognizable HLA
  • Epitopes presented by recipient HLA-class II
  • Antigens = PIRCHE-II
Focus on HLA DR presentable antigens
T-cell help for B-cell maturation is needed
Amino acid sequence comparison
Looks at number of amino acid mismatches (rather than eplet mismatch)
No prior assumptions regarding the relevance of mismatched amino acids make it less biased
(CDR: complementarity-determining region; HLA: human leukocyte antigens)
For a given HLA serotype, there are differences at epitope and eplet levels. Also, there may exist immunodominant HLA epitopes. A good matching at HLA, especially at epitope or eplet levels, remains a desirable goal. Computerized algorithms for molecular mismatch load analysis, eplet matching are being evaluated for risk stratification for de novo alloimmune response and clinical outcomes.
Risk stratification also depends on factors other than preexisting immune memory and epitope/eplet mismatches. Factors such as age of the recipient (young vs. old), 33effects on memory B or T cells remains difficult to assess.
The impact of attributes of DSA such as complement-binding ability, antibody class, and isotype switch remains undefined.
Post-transplant Monitoring
The frequency of screening for DSA post-transplant should be based on immunological risk (immunological memory, HLA serology/epitope/eplet mismatches, and factors such as age and type of organ transplanted). However, the cost–benefit for these approaches needs to be evaluated.
  1. Patel R, Terasaki PI. Significance of the positive crossmatch test in kidney transplantation. N Engl J Med. 1969;280(14):735-9.
  1. Tambur AR, Campbell P, Chong AS, Feng S, Ford ML, Gebel H, et al. Sensitization in transplantation: assessment of risk (STAR) 2019 Working Group Meeting Report. Am J Transplant. 2020;20(10):2652-68.
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  1. Bezstarosti S, Bakker KH, Kramer CSM, de Fijter JW, Reinders MEJ, Mulder A, et al. A comprehensive evaluation of the antibody-verified status of eplets listed in the HLA Epitope Registry. Front Immunol. 2022;12:800946.
  1. Matsuda Y, Sarwal MM. Unraveling the role of allo-antibodies and transplant injury. Front Immunol. 2016;7:432.
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1.4 Role of non-HLA Antigen in Kidney Transplantation
Jasmine Sethi, Vivek Kumar
Kidney transplantation is a modern scientific achievement that has transformed the lives of patients with end-stage kidney disease (ESKD). Over the last few decades, remarkable progress has been made to prevent acute rejection of the kidney allograft. However, preventing immunologic allograft loss in the long term is still considered the “Achilles’ heel” in management of kidney transplant recipients. Despite best efforts, most kidney transplant recipients would develop varying degrees of chronic allograft rejection in the long term.
Traditionally, the concept of “self versus other” has centered around the human leukocyte antigens (HLAs). These are encoded by the major histocompatibility complex (MHC) on the short arm of chromosome 6. These are considered the most important alloantigens as presence of anti-HLA antibodies directed against donor (also called donor-specific antibodies or DSAs) is strongly associated with acute or chronic rejection and decreased allograft survival. The presence of DSAs is an important characteristic of antibody-mediated rejection (ABMR). However, there has been increasing recognition of the fact that rejection of the kidney allograft can also occur in recipients who do not have demonstrable or clinically significant DSAs. With the advent of molecular typing techniques that allow epitope- and eplet-level resolution of HLA mismatches, the role of non-HLA antibodies has gained more significance. Although adsorption of DSA within the allograft has been proposed to explain the occurrence of ABMR in DSA-negative recipients, mounting evidence point to possible role of non-HLA antibodies in allograft rejection.1 The first indirect evidence for the involvement of non-HLA antibodies in allograft outcome came from reports of allograft rejection in HLA-identical sibling transplants that had a negative crossmatch for anti-HLA antibodies. In the 2017 update on the Banff classification scheme for allograft histology, testing for non-HLA antibodies was strongly advised in cases where ABMR occurred in the absence of detectable or clinically significant DSAs.2
As is suggested by the name, any non-MHC-encoded polymorphic protein that is sufficiently antigenic to induce an immune response against the allograft will qualify as a non-HLA. Such antigens may initiate cascade of immunologic damage to the kidney allograft in the absence of anti-HLA antibodies or DSAs. These are also known as minor histocompatibility antigens (mHA). These are likely to be transmembrane or extracellular or cell surface proteins which are polymorphic and hence could be different between different individuals of the same species. Nonself alloantigens are identified by either direct or indirect allorecognition. It is the indirect allorecognition by the recipient's immune system that is primarily responsible for recognition of non-HLA and development of antibodies against mHA.3
The allograft endothelium is the first interface of contact between recipient immune system and donor tissue antigens. HLAs are constitutively expressed on endothelial cell surface. Therefore, it is not surprising that non-HLA or proteins expressed on allograft endothelium are considered as primary culprits behind DSA-negative allograft rejection. 35However, we must remember that not all proteins that may be different between recipient and donor are likely to be expressed on the endothelial surface. Some of the potential antigens may be cryptic, i.e., their usual location and conformation prevent access by the immune system. Tissue damage due to various reasons may create permissive conditions that may lead to exposure and presentation of such cryptic antigens to the recipient's immune system. This in turn may lead to development of alloantibodies (if the protein is polymorphic) or autoantibodies (if the protein is monomorphic). Binding to these antibodies to antigenic targets may start a cascade of vascular inflammation and allograft injury.1 The specificity of these could be broad and mechanisms are currently not very clear.
Why is Routine Evaluation Negative?
Detection of specific antibodies is dependent on characterization of specific antigens. In the case of DSAs, this is facilitated by decades of extensive research into high-resolution characterization of different types and subtypes of HLA in various populations. The current methods of routine identification and quantitation of DSAs are targeted exclusively against HLA. Therefore, allograft rejections in the setting of negative DSAs represent the clinical phenotype of non-HLA-mediated immunologic injury provided falsely negative interpretations of DSAs are taken care of. Antibodies against non-HLA are not usually detected by methods routinely used for crossmatching since these antigens may not be expressed on the lymphocytes.4
Classifying Nonhuman Leukocyte Antigen Antibodies
There is no formal classification of antibodies against non-HLA in kidney transplantation. However, these can be broadly classified into three different types:5
  1. Alloantibodies direct against polymorphic antigens: These antigens differ between the recipient and donor, e.g., antimajor histocompatibility complex class I-related chain A (MICA) antibody.
  2. Autoantibodies that recognize self-antigens which are usually cryptic: These constitute the majority of currently characterized non-HLA antibodies. A significant proportion of these react with autoantigens expressed by vascular endothelial cells (VECs). These antiendothelial cell antibodies (AECAs) include a spectrum of different antibodies depending on antigenic specificity such as anti-angiotensin-II type 1 receptor (AT1R) antibody, anti-endothelin-1 type A receptor (ETAR) antibody, antiperlecan/three laminin-like globular domains (LG3) antibody, and antiagrin antibody.
  3. Natural antibodies that exist in the absence of exogenous antigen exposure: These are polyreactive, e.g., antiapoptotic cell antibody.
Detection of Antibodies against Nonhuman Leukocyte Antigens
More than 10% of cases with C4d-positive ABMR fail to show circulating anti-HLA antibodies or DSAs. Causes of DSA-negative ABMR could be falsely negative DSAs due to technical issues (variations in assay sensitivity or reporting threshold), rare donor HLA not represented in the assay kits, DSAs’ sequestration within the allograft, or presence of antibodies against non-HLA donor antigens. As of now, there are no commercially available methods for the reliable detection of antibodies against non-HLA donor antigens. Different testing approaches have been used to detect these.6 As VECs are considered the primary interface, extracts or preparations of endothelial cells have been used as substrates for broad or specific antibody screening assays. Detection of antibodies targeted against specified antigens can be done through commercially available assays for antibodies against MICA, AT1R, and ETAR. Proteomic and genomic approaches have also been advocated and tested to ascertain variations between recipients and donors. Genome-wide incompatibility and development of allogenomics mismatch score have been shown to add value to HLA incompatibility with respect to allograft outcomes in small studies. However, lack of extensive data and limited availability of such approaches forbid routine applications at present.
Pathogenic Mechanisms
Mechanisms for production of non-HLA antibodies and associated allograft injury have been described, though these are still not fully understood.5,7,8 The process of pathogenic antibody production starts with allograft injury. Allograft injury by insults such as ischemia reperfusion injury (IRI), chronic inflammation, surgical trauma, infection, nephrotoxicity, or rejection exposes the cryptic non-HLA.9 It induces the production of non-HLA antibodies and their binding to now exposed or expressed cryptic antigens. Some non-HLA antibodies (e.g., antiperlecan) are present in the body even prior to transplant and bind to their targets following tissue injury in the post-transplantation period.10 Vascular access creation and manipulation may also play a role in the production of non-HLA antibodies prior to transplant.36
Allograft injury leads to release of extracellular vesicles (EVs) in circulation that express autoantigens on the surface. These EVs can be exosomes, microparticles, or apoptotic bodies and may contain numerous autoantigens. These are then presented, most likely, by the recipient's antigen presenting cells (APC) to the recipient's autoreactive T cells. This indirect alloreactive pathway is considered to be more important in context of non-HLA histocompatibility antigens. CD4 T cells indirectly stimulate autoreactive B cells and thus lead to autoantibody production. Activation of autoreactive T cells results in production of proinflammatory cytokines and local allograft injury. Tertiary lymphoid tissue (TLT) along with Th17 support the formation and proliferation of autoreactive B cells and autoantibodies. Interleukin (IL)-17 produced by Th17 leads to leukocyte recruitment and allograft damage. Finally, autoreactive B cells escape apoptosis in TLT due to sustained local production of IL-21 which promotes B-cell survival. These autoantibodies act through both complement-dependent and complement-independent cytotoxicity. These can also induce lysis of target cells through activation of natural killer (NK) cells, by a process known as antibody-dependent cell-mediated cytotoxicity (ADCC). These can also induce vascular endothelial call activation/injury and subsequent immune dysfunction.
There may be a complex relationship between tissue injury, anti-HLA, and non-HLA immunity in enhancing allograft damage. Inflammatory response induced by non-HLA antibodies could upregulate HLA expression and increase the risk of developing anti-HLA DSAs.11
Specific Nonhuman Leukocyte Antigens
The important non-HLA against which antibodies have been described are as follows (Table 1):
  • Major histocompatibility complex class I-related chain A (MICA): MICA antigens are expressed on the surface of dendritic cells, fibroblasts, VECs, and monocytes but not on lymphocytes. Since MICA is not expressed on resting lymphocytes, it cannot be detected with classical lymphocyte-based crossmatching techniques. Normally, MICA antigens are not expressed on the surface of quiescent VECs. However, stressful conditions such as infection, inflammation, and hypoxia induce MICA expression on the surface of these cells. MICA gene locus is highly pleomorphic and is located on chromosome 6 in close linkage equilibrium to HLA-B locus. Antibodies against MICA antigens may form due to the cross-reactivity with other antigens such as in infections. MICA sensitization can also occur through mechanisms similar to HLA, such as previous transplants, pregnancy, or transfusion. Anti-MICA antibodies have been associated with both ABMR and acute cellular rejection (ACR).1214
  • Angiotensin-II type 1 receptor (AT1R): AT1R is a transmembrane G-protein coupled receptor (GPCR), which has seven transmembrane domains and is expressed on VECs, podocytes, and other kidney tissues. Aldosterone acts through AT1R and regulates salt and water retention, inflammation, and vascular remodeling. IRI may modulate AT1R and facilitate interaction with AT1R antibodies. AT1R antibodies are agonistic and lead to increased production of proinflammatory cytokines and further stimulate T cells. AT1R antibodies have been associated with severe acute vascular rejection and malignant hypertension.15 Compared to DSA-mediated rejection, AT1R Ab-associated rejection is associated with higher prevalence of hypertension, more vascular rejection, and lack of complement deposition. Angiotensin II receptor blockers represent an interesting therapeutic option in patients with AT1R-mediated rejection in combination with plasmapheresis. However, their clinical utility remains unproven.
    TABLE 1   Common nonhuman leukocyte antigens.8
    Heparin sulfate proteoglycan and a major component of vessel wall
    Intermediate filament protein present in the cells of mesenchymal origin, including endothelial cells
    Endothelin 1 type A receptor (ETAR)
    Receptor for endothelin 1
    Major histocompatibility complex class I-related chain A (MICA)
    Expressed on endothelial cells, keratinocytes, monocytes, dendritic cells, fibroblasts, and epithelial cells
    Angiotensin-II type 1 receptor (AT1R)
    G protein-coupled receptor that mediates the action of angiotensin II
    Type III intermediate filament protein, expressed by lymphocytes and macrophages
    Collagen I, Collagen V, and α-tubulin
    Collagens are extracellular matrix proteins, and tubulin is the major constituent of microtubules
    RSP4Y protein
    This is located on Y chromosome, may induce alloreactivity in gender mismatched transplants
    (LG3: three laminin-like globular domains)
  • 37Endothelin-1 type A receptor (ETAR): It is also a transmembrane GPCR that acts as a receptor for endothelin 1 (ET1). Similar to AT1R antibodies, ETAR antibodies are also agonistic antibodies. Binding of ET1 to ETAR induces vasoconstriction and its mitogenic and proinflammatory effects lead to obliterative vasculopathy and progressive tissue fibrosis. Anti-ETAR antibodies are associated with vasculopathy and arteritis.16
  • Perlecan/LG3: It is a heparin sulfate proteoglycan and a component of the vascular basement membrane (VBM). Its C terminal domain contains LG3. The LG3 domain can be released by caspase-dependent cleavage during apoptosis and might act as neoantigen and promote antibody production. High levels of anti-LG3 antibodies in both pre- and post-transplant periods are associated with increased risk of vascular rejection.17 Bortezomib can block the production of anti-LG3 autoantibodies triggered by exosome-like vesicles and may prove useful for preventing autoantibody production before transplantation.18
  • Collagen-IV, vimentin, and fibronectin: Autoantibodies to collagen-IV and fibronectin have been associated with transplant glomerulopathy (TG).19 Autoantibodies to vimentin have been found to be elevated in patients with interstitial fibrosis and tubular atrophy and those with previously failed renal allografts.20
Immune mechanisms that lead to non-HLA antibody formation are complex and poorly understood. Mounting evidence has shown that various forms of antibodies against non-HLA play an important role in acute or chronic allograft rejection and portend a poor long-term allograft outcome. Identification of these antibodies by advanced molecular techniques, integration of proteomics or genomics data to further refine alloincompatibility beyond HLA mismatch, and standardization of testing platforms for widespread applicability are required to make progress in this field.
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  1. Haas M, Loupy A, Lefaucheur C, Roufosse C, Glotz D, Seron D, et al. The Banff 2017 kidney meeting report: revised diagnostic criteria for chronic active T cell-mediated rejection, antibody-mediated rejection, and prospects for integrative endpoints for next-generation clinical trials. Am J Transplant. 2018;18(2):293-307.
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  1. Tinckam KJ, Chandraker A. Mechanisms and role of HLA and non-HLA alloantibodies. Clin J Am Soc Nephrol. 2006;1(3):404-14.
  1. Zhang X, Reinsmoen NL. Impact of non-human leukocyte antigen-specific antibodies in kidney and heart transplantation. Front Immunol. 2017;8:434.
  1. Dragun D, Catar R, Philippe A. Non-HLA antibodies in solid organ transplantation: recent concepts and clinical relevance. Curr Opin Organ Transplant. 2013;18(4):430-5.
  1. Etta PK, Madhavi T, Parikh N. The multifaceted role of non-human leukocyte antigens’ immune response in renal allograft rejection. Saudi J Kidney Dis Transpl. 2021;32(3):622-36.
  1. Dragun D, Catar R, Philippe A. Non-HLA antibodies against endothelial targets bridging allo- and autoimmunity. Kidney Int. 2016;90(2):280-8.
  1. Zou Y, Stastny P, Susal C, Dohler B, Opelz G. Antibodies against MICA antigens and kidney-transplant rejection. N Engl J Med. 2007;357(13):1293-300.
  1. Chowdhry M, Makroo RN, Singh M, Kumar M, Thakur Y, Sharma V. Role of anti-MICA antibodies in graft survival of renal transplant recipients of India. J Immunol Res. 2018;2018:3434050.
  1. Baranwal AK, Mehra NK. Major histocompatibility complex class I chain-related A (MICA) molecules: relevance in solid organ transplantation. Front Immunol. 2017;8:182.
  1. In JW, Park H, Rho EY, Shin S, Park KU, Park MH, et al. Anti-angiotensin type 1 receptor antibodies associated with antibody-mediated rejection in patients without preformed HLA-donor-specific antibody. Transplant Proc. 2014;46(10):3371-4.
  1. Banasik M, Boratynska M, Koscielska-Kasprzak K, Krajewska M, Mazanowska O, Kaminska D, et al. The impact of non-HLA antibodies directed against endothelin-1 type A receptors (ETAR) on early renal transplant outcomes. Transpl Immunol. 2014;30(1):24-9.
  1. Cardinal H, Dieude M, Brassard N, Qi S, Patey N, Soulez M, et al. Antiperlecan antibodies are novel accelerators of immune-mediated vascular injury. Am J Transplant. 2013;13(4):861-74.
  1. Dieude M, Bell C, Turgeon J, Beillevaire D, Pomerleau L, Yang B, et al. The 20S proteasome core, active within apoptotic exosome-like vesicles, induces autoantibody production and accelerates rejection. Sci Transl Med. 2015;7(318):318ra200.
  1. Angaswamy N, Klein C, Tiriveedhi V, Gaut J, Anwar S, Rossi A, et al. Immune responses to collagen-IV and fibronectin in renal transplant recipients with transplant glomerulopathy. Am J Transplant. 2014;14(3):685-93.
  1. Lopez-Soler RI, Borgia JA, Kanangat S, Fhied CL, Conti DJ, Constantino D, et al. Anti-vimentin antibodies present at the time of transplantation may predict early development of interstitial fibrosis/tubular atrophy. Transplant Proc. 2016;48(6):2023-33.38
1.5 Tolerance and Accommodation in Renal Transplantation
Aruna V Vanikar, Pankaj R Shah
Lord Ganesha with an elephant's head transplanted over a human body is the first example of successful xenotransplantation accepted by the human race. Subsequently, examples in other civilizations involving transplantation of limbs, heart, and the spirit have been recorded. This chapter briefly discusses the immunology of rejection, tolerance, and different modes of inducing and measuring it. It also touches upon accommodation.
Successful kidney transplantation was achieved simultaneously by Merrill and Murray in identical twins in Boston, and by Hamburger who transplanted kidney from sister to brother in Paris. Surgical skills were established by the 17th century ad.1 However, immunological success is still not achieved in the 21st century. A transplanted kidney being a foreign body gets rejected by the immune system of the host. The advent of immunosuppressants/immune modulators beginning from 6-mercaptopurine, calcineurin inhibitors (CNI), or mammalian target of rapamycin (m-TOR) inhibitors supported by mycophenolate, azathioprine, and steroids have helped in controlling acute rejections.25 However, none could usher in immunosuppression-free normal life. Immunosuppressants have toxic side effects of suppressing the general immune response of the recipient making him susceptible to bacterial, viral, and fungal infections, malignancies/diabetes mellitus, and a myriad of other problems in the long run. In spite of optimum immunosuppression, eventually, the graft is lost to chronic attrition after an enormous financial burden to the system/family.4,6,7 Striking a balance between prevention of rejection and drug toxicity is still elusive.
The introduction of an antigen is recognized as a foreign invasion by the host immune system evoking a cellular or humoral immune response involving T-cell/B-cell activation. Immunity protects the human body from infections/malignancy; paradoxically, the same immune system is responsible for the rejection of a graft. Hence, for the survival of transplanted graft, the host immune system needs to be suppressed/downregulated.
Immune tolerance or immunological tolerance is a state of absence of a destructive immune response by the recipient immune system toward a well-functioning donor organ/graft in the absence of maintenance immunosuppression and with a fully intact immune system.8
Clinical/Operational Tolerance
Tolerance has been achieved anecdotally in renal transplant recipients by the successful withdrawal of immunosuppressive medications.9 Although such withdrawal was not planned in most cases, a number of trials were conducted for immunosuppression withdrawal.917 These trials were not without risk; in renal transplant patients, acute rejection episodes correlated strongly with the development of chronic allograft nephropathy and reduced graft function was recognized as a risk factor for cardiovascular death.12,18 Selection of ideal patients before transplantation by immunological and molecular phenotypic analyses, coupled with aggressive immune monitoring post-transplantation, is of paramount importance for successful tolerance induction. Thus, the term “operational tolerance” evolved, which is defined as stable graft function without clinical features of chronic rejection and in the absence of any immunosuppressive drugs, usually for >1 year.19,20 It has been observed in liver transplant patients more than in other allograft recipients. Biopsy was not a requisite for operational tolerance.
Propè Tolerance
After substantial efforts by many groups, immune or even operational tolerance was emerging as a Utopian dream. This led to the addition of one more term to the tools defining transplantation tolerance called “Propè tolerance.” Propè (or almost) tolerance was described as a state in which graft acceptance is maintained by a low, nontoxic dosage of maintenance immunosuppression, which may not be required indefinitely.21
Mechanisms of Tolerance
In 1938, Gorer identified immune rejection of transplanted tissue.22,23 He believed that the humoral arm of immunity was responsible for rejection and if this was arrested, tolerance would be achieved. Billingham et al. established the concept of chimeric tolerance (cellular arm of immunity) 39through Owen's model of natural tolerance induction in bovine twins, which they extrapolated in the neonatal mice model.24,25 This was revived with bone marrow (BM) and liver transplantation in the clinic.26
Tolerance is believed to be induced by two basic pathways: Central or thymic tolerance and peripheral tolerance. In central tolerance, the double positive T cells (CD4+ CD8+) are selected to become either CD4+ if the T-cell receptors (TCR) react with major histocompatibility complex (MHC) class II antigens or CD8+ if the TCR react with MHC class I antigens on antigen-presenting cells (APCs) (dendritic cells, macrophages, thymic medullary epithelium). They may undergo apoptotic deletion if this engagement fails. A negative selection follows in which most self-reactive CD4+ or CD8+ cells undergo TCR-induced death if they interact with MHC molecules on APCs carrying self-peptides. Alloreactive cells can also undergo negative selection in the thymus during the interaction of antigen-specific T cells with “tolerogenic” dendritic cells expressing varying levels of co-stimulatory molecules.
In peripheral tolerance, autoreactive/alloreactive cells that escape thymic deletion may be deleted or controlled in circulation by activation-induced cell death, by programmed cell death, or by the suppressive action of Tregs and cytokines. Alloreactive cells repeatedly stimulated with alloantigen can undergo activation-induced cell death by “death receptors” (Fas) cross-linking Fas ligand, leading to caspase cascade activation and cell apoptosis. This pathway is critical to the inhibition of cell proliferation as well as to the development of tolerance2729 (Fig. 1).
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Fig. 1: Proposed mechanism of tolerance. The figure displays the central and peripheral mechanisms for regulation of autoimmune/alloimmune responses. CD3þ cells that are initially double positive for CD4 and CD8 first undergo positive selection to become either CD4+ [major histocompatibility complex (MHC) class II interaction] or CD8+ (MHC class I interaction) followed by deletion or negative selection if autoreactive/alloreactive. However, some CD4+ and CD8+ cells can escape negative selection and become autoreactive/alloreactive Th1 cells in the periphery (lymph nodes, blood, spleen). Mechanisms to regulate these peripherally reactive T cells that can cause autoimmune diseases and transplant rejection are displayed: (1) Immunoregulation from natural (thymic-derived) or induced (in the context of proregulatory cytokines/dendritic cells) regulatory T cells (Tregs) or other regulatory cells; (2) Activation-induced cell death; and (3) No activation resulting in programmed cell death. (IL-10: interleukin-10; PCD: programmed cell death; TCR: T-cell receptors; TGF-β: tumor growth factor-beta)
40The mechanisms proposed for peripheral tolerance induction include:9,18,3035
  • Chimeric tolerance (simultaneous peaceful existence of two different kinds of cells in one host)
  • Clonal deletion (removal of rejecting cell groups)
  • Clonal anergy
  • Regulation/suppression (of these rejecting clones)
Long-lasting tolerance is believed to involve multiple mechanisms with the goal of profound reduction in clonal T-cell expansion along with active immune regulation.9,10 Multiple receptors, ligands, and signaling intermediates have been identified to serve therapeutic targets for tolerance induction strategies including co-stimulatory blockade, TCR targeting, and profound T-cell depletion.36 Animal and human data suggest that administration of donor antigens concurrent with these immunomodulatory agents may be an important adjuvant therapy for successful tolerance strategies.
Importance of the blood transfusion effect gets diminished with the introduction of cyclosporine.3739 Suppression of alloimmune responses after blood transfusion could be partly attributed to induction of regulatory CD4+ T cells, which appears to be dependent on recognition of an allopeptide shared between blood/organ donor and recipient in the context of self-human leukocyte antigen (HLA) class II.40 Presentation in this manner induces hyporesponsiveness to other alloantigens presented on the same APC bearing the shared class II molecule, a phenomenon called linked suppression.41 These and similar studies provide a better understanding of the mechanisms of beneficial effect, which should also help to prevent one of the most feared complications of this form of therapy, i.e., recipient sensitization.
Mesenchymal stem cells (MSCs) have immunomodulatory, immunosuppressive, and tolerogenic effects, and also enhance the action of hematopoietic stem cells (HSCs). MSCs are a potential “homeostatic niche” for Tregs and play role in Treg recruitment, regulation, and maintenance in vitro.4244 Immunoregulatory functions of MSC are not fixed, but are rather consequences of the microenvironment they encounter in vivo.45
Tregs are a large group of Regulatory T cells (with various markers), known to use a variety of mechanisms to mediate cell-mediated suppression effects. In addition, Tregs can produce copious amounts of immune-suppressive cytokines, including tumor growth factor-beta (TGF-β), interleukin (IL)-10, and IL-35 known for inhibiting a wide spectrum of cellular activities (Fig. 2). CD4+CD25+ Tregs at the time of transplantation were shown to be capable of suppressing rejection initiated by a 100-fold excess of donor-alloantigen-specific CD8+ TCR-transgenic T cells demonstrating the potency of CD4+CD25+ T cells.46,47 Treg pool contains a population of committed Tregs and a small fraction of uncommitted Tregs. Such uncommitted Tregs can lose their expression and become T-effector cells. Ex vivo expanded Tregs exhibit a signature similar to T-effector cells rather than Tregs.48 This may be one of the key reasons for the instability of Tregs and, therefore, there is paucity of Tregs in vivo in transplant recipients, even under tolerizing conditions. Subclinical rejection of renal allografts refers to histological patterns of acute rejection despite stable renal function. For finding out whether the presence of Tregs could help determine the functional importance of tubulointerstitial infiltrates observed in protocol biopsies, cases of subclinical rejection were also evaluated. The presence of Treg discriminated harmless from injurious infiltrates, evidenced by independently predicting better graft function 2 and 3 years after transplantation.49 Furthermore, the Treg/CD3+ T-cell ratio positively correlated with graft function at 2 years post-transplantation, suggesting that an increasing proportion of Treg within the global T-cell infiltrate may facilitate renal engraftment; therefore, immunostaining for Treg in patients with subclinical rejection on protocol biopsies may ultimately be useful to identify patients who may require alterations in their immunosuppressive regimens. In vitro studies have shown that Tregs are capable of suppressing the proliferation of syngeneic alloactivated T cells.50 Treatment of positively selected CD4+CD25+ cells enabled them to prevent acute graft-versus-host disease (GVHD).51
Stanford University and the association between the Universities of Louisville and Northwestern Harvard University in the United States and Trivedi et al. in India have attempted tolerance induction in renal transplant recipients using stem cell (SC) transplantation (Fig. 3).52,5341
zoom view
Fig. 2: Mechanisms of regulatory T cell (Treg) suppression. (AMP: adenosine monophosphate; ATP: adenosine triphosphate; IL: interleukin; MHC: major histocompatibility complex; TGF-β: tumor growth factor-beta; TNF: tumor necrosis factor)
  • Stanford group attempted tolerance in 12 HLA-matched recipients who received donor CD34+ selected cells [5–16 × 106/kg bodyweight (BW)] and CD3+ T cells (1–10 ×106/kg BW), intravenously (IV) on day 11.54 Conditioning regimen was total lymphoid irradiation (TLI) (10 doses, 80–120 cGy) to spleen, lymph nodes, thymus plus anti-T-cell antibodies [five doses of rabbit antithymocyte globulin (r-ATG)] during the first 10 days after kidney transplantation. Immunosuppression of mycophenolate mofetil (MMF) (2 g/day) × 1 month; cyclosporine discontinued 6–17 months after transplantation. Chimerism persisted for at least 6 months according to short-tandem-repeat analysis of deoxyribonucleic acid (DNA) from blood granulocytes and lymphocytes and there was no evidence of GVHD or rejection (clinical/biopsy).55 They succeeded in 8 out of 12 patients and had a mean follow-up of 25 months. Recurrence of focal segmental glomerulosclerosis (FSGS) was observed in one patient. Subsequently, more patients were added. The last report states that 24/29 recipients have been taken off immunosuppression.52
  • The association of the Northwestern group attempted it in eight patients.56 Salient features in their protocol were HLA-mismatched kidneys and infusion of tolerogenic graft facilitating cells (FCs) with HSC. Conditioning was with fludarabine, total body irradiation (TBI; 200 cGy), and cyclophosphamide. Immunosuppression was with tacrolimus and MMF. The absolute neutrophil counts reached a nadir 1 week after transplant, with recovery by 2 weeks. Multilineage chimerism was observed at 1 month (6–100%). Conditioning was well tolerated. Transient chimerism was noted in two out of eight patients who were maintained on low-dose tacrolimus monotherapy. One patient had viral sepsis 2 months post-transplant. Five patients displayed durable chimerism, immunocompetence, and donor-specific tolerance by in vitro proliferative assays and were weaned off all immunosuppression 1 year after transplant. None of the recipients produced donor-specific antibodies (DSA)/GVHD. They concluded that their protocol was a safe, practical, and reproducible means of inducing durable chimerism and donor-specific tolerance in solid-organ transplant recipients.
  • Trivedi et al. attempted tolerance induction in 69 recipients across HLA barriers.57 Renal transplantation was performed from living donors after a TLI-based clonal deletion protocol with no post-transplant maintenance immunosuppression planned. If needed, immunosuppression was started on a patient-specific basis, adding one drug at a time, a strategy named “DAWN” (drugs added when needed).42
    zoom view
    Fig. 3: The Ahmedabad tolerance induction protocol. (ADMSC: adipose tissue-derived mesenchymal stem cells (donor); ATG: antithymocyte globulin; Bort: bortezomib; CBM: cultured donor bone marrow; FCM: flow crossmatch; LCM: lymphocytotoxicity crossmatch; PBSC: donor-derived peripheral blood stem cell infusion cyclophosphamide + ATG)
    zoom view
    Figs. 4A and B: Follow-up of tolerance induction protocol using stem cell therapy (SCT)-representative cartoon. (ADMSC: adipose tissue-derived mesenchymal stem cells (donor); Bort: bortezomib; CBM: cultured donor bone marrow; DST: donor-specific transfusion; FCM: flow crossmatch; IRB: institutional review boards; LCM: lymphocytotoxicity crossmatch; MP: methylprednisone; PBSC: donor-derived peripheral blood stem cell infusion; r-ATG: rabbit antithymocyte globulin; SA: single antigen)
    Following this strategy, at the last follow-up, 40 of the 69 patients (58%) had to be rescued by conventional immunosuppression, 23 (33%) had to be started on daily prednisone, and 6 (9%) remained with no maintenance immunosuppression. The overall rate of de novo DSA produced was 36% (in 25/69 patients), at a mean time of about 4 months. Acute rejection episodes with humoral components were noted in 19 cases (27%), of which 14 were pure antibody-mediated rejection, 5 combined antibody- and T-cell-mediated rejection, and 6 episodes (9%) of pure T-cell-mediated rejection. Although complete clonal deletion was not achieved, 42% (29/69) were on prednisone alone/with no immunosuppression for a total mean follow-up of 13.3 months. The mean serum creatinine of 16 patients was 1.33 ± 0.2 mg/dL, over a mean follow-up of 19.3 months. It was concluded that clonal deletion can be used to transplant patients without maintenance immunosuppression, adding drugs only as needed.
  • The only successful tolerance induction protocol (TIP) starting from zero conventional immunosuppression was performed by the Ahmedabad group at Institute of Kidney Diseases and Research Centre–Institute of Transplantation Sciences (IKDRC–ITS) (Figs. 4A and B).36 They undertook open-labeled two armed clinical trials comparing the role of donor-specific transfusion (DST) with SC infusion pretransplant to induce tolerance in living-related renal transplantation. Both the arms, the SC arm and DST arm, had 10 patients each, across HLA barriers. SC arm included infusion of adipose-derived MSC (ADMSC), BM-HSC, and peripheral blood stem cells (PBSC) in liver. DST arm included DST in the peripheral circulation of recipients. Conditioning was with bortezomib methylprednisone (125 mg IV), one cycle, pre- and post-transplant, and r-ATG at the end of transplantation surgery followed by rituximab on day 1 of transplantation. No conventional immunosuppression was given. Immune monitoring included DSA, T- and B-cell flow crossmatch and antihuman globulin (AHG) crossmatch pretransplantation. Post-transplant monitoring was with DSA weekly for the first 3 months and 3 monthly along with monitoring of Tregs (CD 127low/–/CD4+/25high) (representative Figs. 5A and B). There was 100% patient and graft survival 1-year posttransplant in the SC arm. One graft was lost to chronic graft dysfunction with the biopsy revealing unexplained interstitial fibrosis and tubular atrophy (IF/TA) and absence of DSA. Two patients were lost, one to pneumonia at 33 months post-transplant and one to sudden cardiac arrest at 38 months post-transplant.44
    zoom view
    Figs. 5A and B: Tolerance induction protocols with (A) Stem cell therapy (SCT); and (B) Donor-specific transfusion (DST). (ADMSC: adipose-derived mesenchymal stem cells; BW: bodyweight; CBM: cultured donor bone marrow; Treg: regulatory T cell; PBSC: peripheral blood stem cells)
    At 6 years post-transplant, out of seven patients, five were on no conventional immunosuppression and two on mycophenolate sodium (360 mg BD) and prednisone (5 mg/day).56 In the DST arm, one patient was lost to de novo FSGS and associated complications at 10 months post-transplant, two grafts were lost, one at 17 months to chronic graft dysfunction and the second at 40 months post-transplant to IF/TA. Both had developed DSA. Out of seven patients on follow-up at 6 years, four were on no conventional immunosuppression, two on mycophenolate sodium, and one on triple immunosuppression of tacrolimus, mycophenolate, and prednisone.
  • The other reports are mentioned in Table 1.586645
TABLE 1   Tolerance trials across the globe.
Number of recipients
HLA-matched status (n/6)
Full/mixed chimerism
Transient mixed chimerism
Stanford, USA
Mixed chimerism
Mixed chimerism
DHSC and recipient Tregs (mixed chimerism)
Yet to start
Northwestern, USA
Alemtuzumab and donor HSC infusion
Durable chimerism, FCRx
09-b + 120-d
Treg (TRACT therapeutics)
Johns Hopkins, USA
Full chimerism (FCRx)
The One Study (combined)
Donor-alloantigen-reactive Tregs (UCSF)
Autologous tolerogenic dendritic cells
Donor-derived regulatory macrophage
Tregs (UK)
Tregs (Germany)
Tregs + belatacept (Boston)
IRCCS, Italy
Full chimerism (FCRx)—multicenter
Yet to be started
Sam Sang University, South Korea
Mixed chimerism
Across HLA barriers
TLI-based DAWN protocol with SCT
IKDRCITS, Ahmedabad, India
Combined MSC + HSC administration in thymus, liver + conditioning with bortezomib, r-ATG + rituximab
DST + conditioning with bortezomib, r-ATG + rituximab
(b: completed; c: ongoing; CIRM: California Institute of Regenerative Medicine; d: to be initiated soon; DAWN: drugs added when needed; DHSC: donor hematopoietic stem cells; DST: donor-specific transfusion; FCRx: DHSC plus facilitating cells; HLA: human leukocyte antigen; HSC: hematopoietic stem cells; IKDRC–ITS: Institute of Kidney Diseases and Research Centre–Institute of Transplantation Sciences, Ahmedabad, India; IRCCS: Istituto di Ricovero eCura a Carattere Scientifico; MGH: Massachusetts General Hospital; MSC: mesenchymal stem cells; r-ATG: rabbit antithymocyte globulin; SCT: stem cell therapy; TLI: total lymphoid irradiation; Treg: regulatory T cell; TRACT: Treg adoptive cellular transfer; UCSF: University of California at San Francisco)
The absence of DSA in patients with established tolerance has been noted and their reappearance with rising in serum creatinine or subclinical rejections has been observed by Trivedi et al.57 Thus, measurement of antibodies by the luminex platform can be one of the best-established biomarkers currently that has fulfilled the litmus test of proving the absence of antibodies in subjects with tolerance.46
TABLE 2   Biomarkers in operational tolerance in kidney transplantation.
Technique of measurement
NK cells
Peripheral NK cells
Flow cytometry
T cells
Peripheral T cells
Flow cytometry
T-cell alloreactivity
  • IFN-γ ELISpot
  • Trans-vivo DTH assay
Whole blood gene expression
B cells
Peripheral B-cell phenotype
Flow cytometry
B-cell cytokine production
IC cytokine staining
PBMC/B-cell gene expression
Whole blood gene expression
Urinary gene expression
Serum HLA antibodies
(DTH: delayed-type hypersensitivity; IC: intracellular; HLA: human leukocyte antigen; IFN: interferon; NK: natural killer; PBMC: peripheral blood mononuclear cell; RT-PCR: reverse transcription polymerase chain reaction)
Normal graft biopsy, although invasive can still be considered as the gold standard of tolerance, provided the graft function is normal with maintained immune status in absence of immunosuppression.
Mixed chimerism although being used as markers for tolerance, the studies have established that transient chimerism has been observed in patients with sustained tolerance. Hence, chimerism may not be considered as biomarker for testing tolerance.
Tregs (CD4+/25high/127low) in peripheral blood can be established as both immunomodulators, helping in establishing tolerance as well as biomarkers for tolerance.6769
Some examples of biomarkers in operational tolerance in kidney transplantation are provided in Table 2.70
Previously B cells were only considered to have pathogenic role in kidney transplantation, but now there are growing evidence showing that B cells also have important role in the induction and maintenance of transplant tolerance. Regulatory B cells (Bregs) are characterized by secretion of IL-10 and other cytokines such as TGF-β and IL-35. The two important subsets of Bregs are CD5+CD19+CD1dhi B10 and CD19+CD21hiCD23hi CD24hi transitional-2 (T2) Bregs. The main functions of Bregs are given below:
  • Suppression of CD8+ effector T-cell function
  • T-cell apoptosis induction via binding the FAS and programmed cell death 1 (PD-1) receptors
  • Induction of Tregs
  • Suppression of antigen-presenting and cytokine secretion by dendritic cells and M1 macrophages
  • Natural killer (NK) cells and neutrophils suppression
Accommodation is a state of acquired resistance of a grafted organ to immune-mediated damage.72 It was first observed in ABO-incompatible kidney transplants, which survived and functioned normally in recipients with high titers of anti-blood group antibodies that were directed against the graft antigens.73 The mechanism of development of tolerance is being understood a little better than that of accommodation. Accommodation differs from tolerance in that the immune system retains the capability to reject fresh tissue from the same donor, but accommodated donor tissue remains protected even if retransplanted into a new recipient.74 Moreover, biopsies of normal functioning grafts revealed ABO antigens on their endothelial surfaces.75 Hallmark of these biopsies is CD4 deposition on peritubular capillaries without rejection, unlike intolerance.
Tolerance induction is feasible, and TIP described above is safe. The important arms of sustained tolerance induction are clonal deletion and stem cell therapy (SCT), which may induce chimeric tolerance initially and link suppression with infectious tolerance subsequently. Moreover, financially tolerance induction is proving cost-effective. SCT under safe and effective nonmyeloablative conditioning does not lead to GVHD. If transplantation with zero immunosuppression is not feasible, the initial protocols of immunosuppression minimization can be practiced safely.
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