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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.

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Presentation on theme: "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."— Presentation transcript:

1 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

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

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

4 HLA Class I Ribbon Structure

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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

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

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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

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.

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

12 http://www.ebi.ac.uk/imgt/hla/intro.htmlhttp://www.ebi.ac.uk/imgt/hla/intro.html (1968: 7000)

13 Identification of New HLA Alleles

14 Family Genotype Mother Father Possible combinations in children (4)

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

16 Crossover Mother Father

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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)

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

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

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24 Sequence-Specific Primer PCR (SSP) Gel

25 Class II DQB1 High Resolution Gel 12341234 ML H G F E D C B A * ** * *

26 * *

27 * DQB1*02:02

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

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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

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 

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

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

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

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

36 Sequence-Based Typing (SBT) Homozygous sequence Heterozygous sequence

37 A*01:01, 02:01

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

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

40 Population Studies: HLA Antigen Frequencies (%)

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

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

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

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

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

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

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48 ASCP, 2012

49 Transfusion-Related Fatalities Reported to FDA FY2008-FY2012

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

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?

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)

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

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

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

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

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

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

59 Allogeneic Hematopoietic Stem Cell Transplantation (Allo HSCT)

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!

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

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

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

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

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

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

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

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

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

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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

72 Which HLA Loci Impact Survival? ABCDRDQ JMDPYesNo FHCRCYes NMDPYes No

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

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?

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)

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)

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

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?

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.

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 donor’s 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 donor’s mother and the pt; this is a NIMA-matched UCBT.

81 NIMA Mismatch HLA-A*HLA-B*HLA-DRB1* Patient02, 1118, 3501:01, 11:04 UCB donor unit02, 3218, 3501:01, 11:04 UCB donor’s mother02, 2407, 3501:01, 13:01 HLA-A*11 is not carried by UCB donor or the UCB donor’s mother; this is a NIMA-mismatched UCBT.

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83 NMDP

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


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