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

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

Structure of HLA Class I and Class II Molecules HLA typing targets N Engl J Med 2000;343:702.

HLA Class I Ribbon Structure

Nomenclature: Serology vs Molecular Typing DNA amplification DRB1*0401 DRB1*0402 DRB1*0403 DRB1*0404 DRB1*0405… DRB1*0424 Serology Ab reagents DR4

Nomenclature HLA HLA complex HLA-A Locus HLA-A*02 Ag equivalent HLA-A*02:01 Allele specificity HLA-A*02:01N Null allele HLA-A*02:01:01 Silent mutation (coding region) HLA-A*02:01:01:01 Noncoding region mutation HLA-A*02:01:01L Low expression gene

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

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.

GENES OF THE HUMAN MHC SYSTEM Chromosome 6 Class II Class III Class I DP DQ DR B C A B1 A1 B1 A1 B1 B3/4/5 A C4 TNF 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

http://www. ebi. ac. uk/imgt/hla/intro http://www.ebi.ac.uk/imgt/hla/intro.html (1968: <200; 2007: >7000)

Identification of New HLA Alleles

Family Genotype Mother Father Possible combinations in children (4)

Chance of Finding an HLA-Matched Sibling 1 – (0.75)n n = no. of siblings No. of sibs Chance (%) 1 25 2 44 3 58 4 68 5 76 6 82 7 87 8 90 9 92 10 94

Crossover Mother Father

HLA Typing Methods Serologic assays Cellular assays Molecular assays Microlymphocytotoxicity test Cellular assays Mixed lymphocyte culture Molecular assays Sequence-specific primer (SSP) Sequence-specific oligonucleotide probe (SSOP) Sequence-based testing (SBT)

All dead = Red Score =8 All alive = Green Score = 1

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

Sequence-Specific Primer PCR (SSP) Gel

Class II DQB1 High Resolution Gel ML H G F E D C B A 1 2 3 4 * * * * * Class II DQB1 High Resolution Gel

* *

* DQB1*02:02

Sequence-Specific Oligonucleotide Probe Hybridization (SSO, SSOPH) Multiple Microparticles (Luminex)

SSO Pros and Cons Pros Cons Fairly rapid High volume High(er) resolution Reasonable cost/test Cons Single typing is more expensive Requires more DNA Expensive equipment

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 

DNA Typing Resolution Low Intermediate High 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

DNA Typing Resolution Low Intermediate High 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

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

Sequence-Based Typing (SBT) Sanger sequencing (chain-termination method)

Sequence-Based Typing (SBT) Homozygous sequence Heterozygous sequence

A*01:01, 02:01

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

Clinical Applications of HLA Typing Population studies Disease associations Pharmacogenomics Platelet transfusion TRALI risk reduction Transplantation hematopoietic stem cell solid organ

Population Studies: HLA Antigen Frequencies (%)

HLA and Disease Associations Disease HLA RR Ankylosing spondolytis B*27 >100 Narcolepsy DRB1*15:01 94 Celiac disease DQB1*02:01 11 Rheumatoid arthritis DRB1*04 11 Multiple sclerosis DRB1*15:01 5 HIV slow progress DQB1*06:05 9 HIV fast progress B*07 3

HLA-B27 and Ankylosing Spondylitis A.S. >90% B27+ HLA-B*27 ~8% of population ~2% of B*27+ dev AS

Ingelman-Sundberg M. Pharmacogenomic Biomarkers for Prediction of Severe Adverse Drug Reactions NEJM 2008;358:637-639

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

Histocompatible Platelets HLA-matched Crossmatch-compatible HLA antigen-negative HSCT donor Blood relative donor Not for potential HSCT recipient

“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

ASCP, 2012

Transfusion-Related Fatalities Reported to FDA FY2008-FY2012

Transfusion-Related Fatalities Reported to FDA FY2002-FY2012 TRALI Cases

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?

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)

HLA Antibodies Clinically significant Clinically insignificant IgG type Clinically insignificant Autoantibodies Non-HLA antibodies

HLA Typing and Transplantation Solid Organs Kidney Liver Heart Lung Pancreas Hematopoietic stem cells Bone marrow Peripheral blood Cord blood

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

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

Transplant HLA and ABO Matching HLA ABO Kidney No* Yes Liver No Yes Heart No Yes Lung No Yes Pancreas No* Yes Cornea No No Stem cell Yes No *HLA matching preferred but not required

Cadaver Kidney Transplants HLA-A+B+DR Mismatches (MM) HLA DNA Typing Review and Transplantation. Immunity, Vol. 14, 347–356, April, 2001

Allogeneic Hematopoietic Stem Cell Transplantation (Allo HSCT)

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!

Choosing a Matched Unrelated Donor (MUD) HLA-A HLA-B HLA-DR Pt 02:01, 03:01 07:02, 44:02 03:01, 13:01 D1 D2 02, 03 07, 44 03, 13 D3 2, 3 7, 44 3, 13 D4 01, 03 03:01, 13:02

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

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

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

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

Case 2: Extended Family Typing Pt Sib 1 Sib 2 Sib 3

Case 2: Extended Family Typing Parent 1 Parent 2 Pt Sib 1 Sib 2 Sib 3

Case 2: Extended Family Typing perfect match Parent 1 Parent 2 Pt Sib 1 Sib 2 Sib 3

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

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

Which HLA Loci Impact Survival? B C DR DQ JMDP Yes No FHCRC NMDP

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

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?

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)

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)

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

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?

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.

Non-Inherited Maternal Antigens (NIMA) HLA-A* HLA-B* HLA-DRB1* Patient 02, 24 18, 35 01:01, 11:04 UCB donor unit 02, 32 UCB donor’s mother 07, 35 01:01, 13:01 HLA-A*24 is not carried by UCB donor but is carried by UCB donor’s mother and the pt; this is a NIMA-matched UCBT. 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.

NIMA Mismatch HLA-A* HLA-B* HLA-DRB1* Patient 02, 11 18, 35 01:01, 11:04 UCB donor unit 02, 32 UCB donor’s mother 02, 24 07, 35 01:01, 13:01 HLA-A*11 is not carried by UCB donor or the UCB donor’s mother; this is a NIMA-mismatched UCBT.

NMDP

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