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HLA Typing for Blood Bankers

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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
HLA typing targets N Engl J Med 2000;343:702.

4 HLA Class I Ribbon Structure


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

7 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


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.

Chromosome 6 Class II Class III Class I DP DQ DR B C A B1 A B1 A 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

12 http://www. ebi. ac. uk/imgt/hla/intro
(1968: <200; 2007: >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 sibs Chance (%)

16 Crossover Mother Father


18 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)


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



24 Sequence-Specific Primer PCR (SSP) Gel

25 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

26 * *

27 * DQB1*02:02

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


30 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

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 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

33 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

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 Narcolepsy DRB1*15: Celiac disease DQB1*02: Rheumatoid arthritis DRB1* Multiple sclerosis DRB1*15: HIV slow progress DQB1*06: HIV fast progress B*

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:

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


48 ASCP, 2012

49 Transfusion-Related Fatalities Reported to FDA FY2008-FY2012

50 Transfusion-Related Fatalities Reported to FDA FY2002-FY2012

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 Clinically insignificant
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 HLA-C, DQB1, DPB1

57 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

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-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

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 Sib Sib 3

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

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

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


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?

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: 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:

80 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.

81 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.



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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