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.

11. GENES OF THE HUMAN MHC SYSTEM
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
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 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?B C DR DQ
JMDP
Yes No
FHCRC
NMDP

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.

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