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HLA Typing for Blood Bankers Kaaron Benson, M.D. Professor and Senior Member Departments of Oncologic Science, and Pathology and Cell Biology H. Lee Moffitt Cancer Center Slide 2 Introduction to the HLA System HLA Human Leukocyte Antigens Key role in immunologic function Part of the major histocompatibility complex (MHC) HLA antigens encoded for by genes from 6 primary loci on chromosome 6 Slide 3 Structure of HLA Class I and Class II Molecules N Engl J Med 2000;343:702. HLA typing targets Slide 4 HLA Class I Ribbon Structure Slide 5 Slide 6 Nomenclature: Serology vs Molecular Typing Serology Ab reagents DR4 Molecular DNA amplification DRB1*0401 DRB1*0402 DRB1*0403 DRB1*0404 DRB1*0405 DRB1*0424 Slide 7 Nomenclature HLAHLA complex HLA-ALocus HLA-A*02Ag equivalent HLA-A*02:01Allele specificity HLA-A*02:01NNull allele HLA-A*02:01:01Silent mutation (coding region) HLA-A*02:01:01:01Noncoding region mutation HLA-A*02:01:01LLow expression gene Slide 8 Slide 9 New Nomenclature: April 2010 HLA-Cw becomes HLA-C Cw*0202 now C*02:02, represents Cw2 Ag Colons (:) to delimit separate fields A*0301 becomes A*03:01 A*2601 becomes A*26:01 A*02 and A*92 to change A*9201 becomes A*02:101 A*9202 becomes A*02:102 B*15 and B*95 to change B*9501 becomes B*15:101 B*9502 becomes B*15:102 Slide 10 Nomenclature Confusion: One Example HLA-DRB1*03:01 HLA-DRB1*03 HLA-DR17 HLA-DR3 This one allele could be written these four different ways. All four do not refer to the same thing. Slide 11 Class II Class III Class I Chromosome 6 B1 A1 B1 A1 B1 B3/4/5 A C4 TNF DP DQ DR B C A DRB1*0401 DRB1*0402 DRB1*0403 DRB1*0404 DRB1*0405 DRB1*0406 B*0702 B*0801 B*1301 B*1401 B*1501 A*0101 A*0201 A*0301 GENES OF THE HUMAN MHC SYSTEM Slide 12 http://www.ebi.ac.uk/imgt/hla/intro.htmlhttp://www.ebi.ac.uk/imgt/hla/intro.html (1968: 7000) Slide 13 Identification of New HLA Alleles Slide 14 Family Genotype Mother Father Possible combinations in children (4) Slide 15 Chance of Finding an HLA-Matched Sibling 1 (0.75) n n = no. of siblings No. of sibsChance (%) 125 244 358 468 576 682 787 890 992 1094 Slide 16 Crossover Mother Father Slide 17 Slide 18 HLA Typing Methods Serologic assays Microlymphocytotoxicity test Cellular assays Mixed lymphocyte culture Molecular assays Sequence-specific primer (SSP) Sequence-specific oligonucleotide probe (SSOP) Sequence-based testing (SBT) Slide 19 Slide 20 All dead = Red Score =8 All alive = Green Score = 1 Slide 21 HLA Typing by Molecular Methods Pros More accurate and precise than serology e.g. DRB1 >300 alleles but only 17 serotypes Better matching between pt and donor Less sample required, nonviable cells Wider variety of samples can be used Becoming easier + automated Cons Does not account for genes that are present but not expressed: Genotype Phenotype Rare alleles = growing list of ambiguities Slide 22 Slide 23 Slide 24 Sequence-Specific Primer PCR (SSP) Gel Slide 25 Class II DQB1 High Resolution Gel 12341234 ML H G F E D C B A * ** * * Slide 26 * * Slide 27 * DQB1*02:02 Slide 28 Sequence-Specific Oligonucleotide Probe Hybridization (SSO, SSOPH) Multiple Microparticles (Luminex) Slide 29 Slide 30 SSO Pros and Cons Pros Fairly rapid High volume High(er) resolution Reasonable cost/test Cons Single typing is more expensive Requires more DNA Expensive equipment Slide 31 Sequence-Based Typing (SBT) Gold standard for HLA typing Detects novel alleles Expensive Sequencers are costly (but other uses) Requires highly skilled technologists Cis/trans polymorphisms - ambiguities result - require additional testing Slide 32 DNA Typing Resolution Low serologic equivalent DRB1*04, DRB1*13 Intermediate DRB1*04:01 or *04:03 or *04:04 DRB1*04:01/03/04 High allele level DRB1*04:01 DRB1*04:01/36/45/52/56 Slide 33 DNA Typing Resolution Low serologic equivalent DRB1*04, DRB1*13 Intermediate DRB1*04:01 or *04:03 or *04:04 DRB1*04:01/03/04 High allele level DRB1*04:01 DRB1*04:01/36/45/52/56 ambiguities Slide 34 High Resolution Typing Type to a single common allele Rare allele definition: For Class I < 1:50,000 alleles For DRB1 < 1:100,000 alleles B*15:01, 35:01 B*15:01/29/33/34, 35:01/40N/42 Slide 35 Sequence-Based Typing (SBT) Sanger sequencing (chain-termination method) Slide 36 Sequence-Based Typing (SBT) Homozygous sequence Heterozygous sequence Slide 37 A*01:01, 02:01 Slide 38 Confirmatory HLA Typing New sample must be collected Patient: prior to final donor selection Donor: prior to stem cell collection Both MRD and MUD transplants NMDP donor typing counts as one typing Only one typing must be at high resolution Level of typing (LR, IR, HR) decided by program MCC: IR/HR typing for original and repeat Slide 39 Clinical Applications of HLA Typing Population studies Disease associations Pharmacogenomics Platelet transfusion TRALI risk reduction Transplantation hematopoietic stem cell solid organ Slide 40 Population Studies: HLA Antigen Frequencies (%) Slide 41 HLA and Disease Associations Disease HLA RR Ankylosing spondolytis B*27>100 NarcolepsyDRB1*15:01 94 Celiac diseaseDQB1*02:01 11 Rheumatoid arthritisDRB1*04 11 Multiple sclerosisDRB1*15:01 5 HIV slow progressDQB1*06:05 9 HIV fast progressB*07 3 Slide 42 HLA-B27 and Ankylosing Spondylitis A.S. >90% B27+ HLA-B*27 ~8% of population ~2% of B*27+ dev AS Slide 43 Ingelman-Sundberg M. Pharmacogenomic Biomarkers for Prediction of Severe Adverse Drug Reactions NEJM 2008;358:637-639 Slide 44 Platelet Transfusion Platelet refractoriness: non-immune vs immune Non-immune refractoriness infection, splenomeg, BMT, DIC, bleed, meds Immune platelet refractoriness HLA Abs (other Abs: plt-specific, drug-induced, ABO) HLA alloimmunization: due to allogeneic WBC exposure via prior transfusion or pregnancy (WBCs cl. I and II, PLTs cl. I only) Management: more PLTs vs. histocompat. PLTs Prevention: WBC-reduced blood Slide 45 Histocompatible Platelets HLA-matched Crossmatch-compatible HLA antigen-negative HSCT donor Blood relative donor Not for potential HSCT recipient Slide 46 HLA-Matched Platelets Patient: A1,2; B7,8 Matching grade A: perfect match; A1,2; B7,8 donor B: crossreactive (X) or unidentified (U) BIX, BIU; A1,3; B7,8 or A1,-; B7,8 donor B2X, B2U: A1,3; B7,27 or A1,-; B7,- donor C: one MM Ag; A1,2; B7,44 donor D: two MM Ag; A1,24; B7,44 donor Slide 47 Slide 48 ASCP, 2012 Slide 49 Transfusion-Related Fatalities Reported to FDA FY2008-FY2012 Slide 50 Transfusion-Related Fatalities Reported to FDA FY2002- FY2012 TRALI Cases Slide 51 TRALI and HLA Antibodies Implicated components RBCs, PLTs, FFP TRALI risk: key factors Patient susceptibility first hit Antibody presence and titer Antibody specificity cognate pt Ag? Slide 52 HLA Antibody Detection Antigen nonspecific Cytotoxicity (NIH, variations: wash, DTT, extended incubations, antiglobulin) Flow cytometry (T cell / B cell) Antigen specific ELISA (yes / no, PRA%, specificity) Flow cytometry (beads: PRA%, specificity) Multiplex (Luminex, protein chips) Slide 53 HLA Antibodies Clinically significant IgG type Clinically insignificant Autoantibodies Non-HLA antibodies Slide 54 HLA Typing and Transplantation Solid Organs Kidney Liver Heart Lung Pancreas Hematopoietic stem cells Bone marrow Peripheral blood Cord blood Slide 55 Solid Organ Transplantation ABO compatibility essential Organ size requirements Cold ischemia time: organ to recipient Medical urgency / time on waiting list HLA matching for pt Ab / donor Ag essential HLA flow cytometric XM = standard of care Recipient serum and donor lymphs HLA compatibility beneficial Required for renal transplants matching: graft survival, meds Slide 56 Solid Organ Transplantation United Network for Organ Sharing (UNOS): federal contract HLA typing must be by molecular +/- serologic methods HLA-A, B, C, DRB1, DRB3/4/5, DQB1 typing HLA-A, B, Bw4/6, Cw, DR51/52/53, DQ Ags reported +/-HLA-DPB1 typing for heart and/or lungs Zero Ag MM (6/6) for HLA-A, B, DR Ags only Zero Ag MM for MM @ HLA-C, DQB1, DPB1 Slide 57 Transplant HLA and ABO Matching HLAABO Kidney No* Yes Liver No Yes Heart No Yes Lung No Yes Pancreas No* Yes Cornea No No Stem cellYes No *HLA matching preferred but not required Slide 58 Cadaver Kidney Transplants HLA-A+B+DR Mismatches (MM) HLA DNA Typing Review and Transplantation. Immunity, Vol. 14, 347356, April, 2001 Slide 59 Allogeneic Hematopoietic Stem Cell Transplantation (Allo HSCT) Slide 60 HSCT: Donor Selection HLA compatibility essential ABO compatibility not required Siblings: best chance for identity 25% chance of matching any one sib ~30% of patients have a matched sib Monozygotic twin not preferred? Other family members may match pt No MRD? Search for MUD ASAP! Slide 61 Choosing a Matched Unrelated Donor (MUD) HLA-AHLA-BHLA-DR Pt02:01, 03:0107:02, 44:0203:01, 13:01 D102:01, 03:0107:02, 44:0203:01, 13:01 D202, 0307, 4403, 13 D32, 37, 443, 13 D401, 0307, 4403:01, 13:02 Slide 62 Case 1: How Many Haplotypes? Pt: A1, 24; B7, 8; DR3, 4 Sib 1: A2, 11; B40, 55; DR2, 7 Sib 2: A24, 28; B8, 44; DR4, 13 Sib 3: A2, 28; B40, 44; DR2, 13 Slide 63 Case 1: How Many Haplotypes? Pt: A1, 24; B7, 8; DR3, 4 Sib 1: A2, 11; B40, 55; DR2, 7 Sib 2: A24, 28; B8, 44; DR4, 13 Sib 3: A2, 28; B40, 44; DR2, 13 Slide 64 Case 2: Extended Family Typing Pt: A 1, 24; B 8, 48; DR 3, 7 Sib 1: A 1, 2; B 8, 35; DR 2, 3 Sib 2: A 2, 24; B35,48; DR 2, 7 Sib 3: A 2, 24; B35,48; DR 2, 7 Slide 65 Case 2: Extended Family Typing Pt: A 1, 24; B 8, 48; DR 3, 7 Sib 1: A 1, 2; B 8, 35; DR 2, 3 Sib 2: A 2, 24; B35,48; DR 2, 7 Sib 3: A 2, 24; B35,48; DR 2, 7 Slide 66 Case 2: Extended Family Typing Pt Sib 1 Sib 2 Sib 3 Slide 67 Case 2: Extended Family Typing Pt Sib 1 Sib 2 Sib 3 Parent 1 Parent 2 Slide 68 Case 2: Extended Family Typing Pt Sib 1 Sib 2 Sib 3 Parent 1 Parent 2 perfect match Slide 69 Unrelated Donor Searches HLA matched unrelated donors = MRD Available donors: NMDP: > 9.5 million volunteer donors Worldwide: ~20 million total Cord blood: > 550,000 Chance of finding A, B, DR match: ~60-80% with one million donors Chance best: Cauc> Am. Indian> Hisp/Asian> Afr-Amer February 2012 Slide 70 Slide 71 Matching for Stem Cell Transplantation HLA Factors DNA-based testing methods Goal: match 8/8 HLA-A, B, C, DRB1 All loci equally important Match 10/10 A, B, C, DRB1, DQB1? Allele level matching needed Allele mismatch = antigen mismatch Minimize the number of mismatches Slide 72 Which HLA Loci Impact Survival? ABCDRDQ JMDPYesNo FHCRCYes NMDPYes No Slide 73 Antigen vs Allele Mismatch Pt: A*01:01, 02:01; B*07:02, 08:01 Donor 1: A 1, 2; B 7, 8 Low resolution (LR) match Donor 2: A*01:01, 03:01; B*07:02, 08:01 Antigenic (LR) mismatch Donor 3: A*01:01, 02:02; B*07:02, 08:01 Allelic (HR) mismatch Slide 74 Molecular vs Serologic HLA Typing: Benefits of Better Matching Improved rate of engraftment Decreased incidence/severity aGVHD Decreased incidence/severity cGVHD Improved rate of overall survival Serologic typing sufficient for MRD? Slide 75 Acceptable Mismatches? Locus important? A vs B vs C vs DRB1 > DQB1 Marrow: A+DR MM worse than B+C PBSC: C MM worse than others Cord: C Ag MM increases TRM Specific mismatches important? A*02:01 vs 02:02 vs 02:03 No preformed anti-donor-specific HLA Ab (DSA) Slide 76 HLA Typing Guidelines for HSC Transplantation Type patient for A, B, C, DRB1 +/- DQB1 Type siblings for A, B, C, DRB1 +/- DQB1 (may screen with HLA-LR class I or II) No sibling donor (MRD)? Consider MUD No MUD? Consider cord or MMUD Molecular methods preferred High resolution (allele matching) Slide 77 HLA Typing Guidelines for HSC Transplantation Using URD Search all donor registries worldwide Matched URD molecular HLA typing > serology choose young, male / nonparous female consider CMV status, donor/pt size, ABO/Rh Mismatched URD mismatch rare alleles ethnic group matching preferred Consider cord blood donor Slide 78 HLA-DPB1: Need to Match? Studies have suggested that DPB1 matching does not impact overall survival DPB1 match increases relapse risk DPB1 mismatch increases aGVHD and TRM Lack of tight DPB1 linkage with other loci decreases the ease of finding a DPB1 match Only ~20% of 10 of 10 matched transplants will be matched for DPB1 Permissive mismatches? Slide 79 HLA Alloantibodies in HSCT Presence of recipient HLA alloantibodies are not predictive of graft failure Donor-specific HLA Abs (DSA) are predictive of graft failure (e.g., recipient anti-A*02 and A*02 donor pair) HLA antibody evaluations should be a part of the routine workup for unrelated stem cell transplantation The detection of donor-directed, HLA-specific alloantibodies in recipients of unrelated HCT is predictive of graft failure. Blood 2010;115:2704-2708. Slide 80 Non-Inherited Maternal Antigens (NIMA) HLA-A*HLA-B*HLA-DRB1* Patient02, 2418, 3501:01, 11:04 UCB donor unit02, 3218, 3501:01, 11:04 UCB donors mother02, 2407, 3501:01, 13:01 Van Rood JJ, et al. Proc Natl Acad Sci USA 2009;106:19952. Rocha V, et al. Biol Blood Marrow Transplant 2012; July 17 Epub. HLA-A*24 is not carried by UCB donor but is carried by UCB donors mother and the pt; this is a NIMA-matched UCBT. Slide 81 NIMA Mismatch HLA-A*HLA-B*HLA-DRB1* Patient02, 1118, 3501:01, 11:04 UCB donor unit02, 3218, 3501:01, 11:04 UCB donors mother02, 2407, 3501:01, 13:01 HLA-A*11 is not carried by UCB donor or the UCB donors mother; this is a NIMA-mismatched UCBT. Slide 82 Slide 83 NMDP Slide 84 HLA Typing Summary HLA typing nomenclature Low, intermediate, high resolution typing Clinical application of HLA typing Role in TRALI risk mitigation Important role in donor selection for solid organ transplant and HSCT Optimal matching between pt and donor