HLA Typing for Blood Bankers

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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

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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

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Structure of HLA Class I and Class II Molecules
HLA typing targets
N Engl J Med 2000343702.
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HLA Class I Ribbon Structure
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Nomenclature Serology vs Molecular Typing

• Molecular
• DNA amplification
• DRB10401
• DRB10402
• DRB10403
• DRB10404
• DRB10405
• DRB10424

• Serology
• Ab reagents
• DR4

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Nomenclature

• HLA HLA complex
• HLA-A Locus
• HLA-A02 Ag equivalent
• HLA-A0201 Allele specificity
• HLA-A0201N Null allele
• HLA-A020101 Silent mutation
• (coding region)
• HLA-A02010101 Noncoding region
• mutation
• HLA-A020101L Low expression gene

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New Nomenclature April 2010

• HLA-Cw becomes HLA-C
• Cw0202 now C0202, represents Cw2 Ag
• Colons () to delimit separate fields
• A0301 becomes A0301
• A2601 becomes A2601
• A02 and A92 to change
• A9201 becomes A02101
• A9202 becomes A02102
• B15 and B95 to change
• B9501 becomes B15101
• B9502 becomes B15102

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Nomenclature ConfusionOne Example

• HLA-DRB10301
• HLA-DRB103
• HLA-DR17
• HLA-DR3
• This one allele could be written these four
  different ways.
• All four do not refer to the same thing.

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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
DRB10401 DRB10402 DRB10403 DRB10404 DRB10405
DRB10406
B0702 B0801 B1301 B1401 B1501
A0101 A0201 A0301
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http//www.ebi.ac.uk/imgt/hla/intro.html (1968
lt200 2007 gt7000)
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Identification of New HLA Alleles
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Family Genotype
Mother Father
Possible combinations in children (4)
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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

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Crossover
Mother Father
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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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All dead Red Score 8
All alive Green Score 1
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HLA Typing by Molecular Methods

• Pros
• More accurate and precise than serology
• e.g. DRB1 gt300 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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Sequence-Specific Primer PCR (SSP) Gel
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ML H G F E D C B A
1 2 3 4





Class II DQB1 High Resolution Gel
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DQB10202
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Sequence-Specific Oligonucleotide Probe
Hybridization (SSO, SSOPH)
Multiple Microparticles (Luminex)
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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

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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 ?

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DNA Typing Resolution

• Low
• serologic equivalent
• DRB104, DRB113
• Intermediate
• DRB10401 or 0403 or 0404
• DRB10401/03/04
• High
• allele level
• DRB10401
• DRB10401/36/45/52/56

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DNA Typing Resolution

• Low
• serologic equivalent
• DRB104, DRB113
• Intermediate
• DRB10401 or 0403 or 0404
• DRB10401/03/04
• High
• allele level
• DRB10401
• DRB10401/36/45/52/56

ambiguities
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High Resolution Typing

• Type to a single common allele
• Rare allele definition
• For Class I lt 150,000 alleles
• For DRB1 lt 1100,000 alleles
• B1501, 3501
• B1501/29/33/34, 3501/40N/42

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Sequence-Based Typing (SBT)
Sanger sequencing (chain-termination method)
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Sequence-Based Typing (SBT)
Homozygous sequence
Heterozygous sequence
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A0101, 0201
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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

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Clinical Applications of HLA Typing

• Population studies
• Disease associations
• Pharmacogenomics
• Platelet transfusion
• TRALI risk reduction
• Transplantation
• hematopoietic stem cell
• solid organ

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Population StudiesHLA Antigen Frequencies ()
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HLA and Disease Associations

• Disease HLA RR
• Ankylosing spondolytis B27 gt100
• Narcolepsy DRB11501 94
• Celiac disease DQB10201 11
• Rheumatoid arthritis DRB104 11
• Multiple sclerosis DRB11501 5
• HIV slow progress DQB10605 9
• HIV fast progress B07 3

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HLA-B27 and Ankylosing Spondylitis

• A.S.
• gt90
• B27
• HLA-B27
• 8 of population
• 2 of B27 dev AS

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Ingelman-Sundberg M. Pharmacogenomic Biomarkers
for Prediction of Severe Adverse Drug Reactions
NEJM 2008358637-639
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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

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Histocompatible Platelets

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

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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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ASCP, 2012
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Transfusion-Related Fatalities Reported to FDA
FY2008-FY2012
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Transfusion-Related Fatalities Reported to FDA
FY2002-FY2012 TRALI Cases
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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?

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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)

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HLA Antibodies

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

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HLA Typing and Transplantation

• Solid Organs
• Kidney
• Liver
• Heart
• Lung
• Pancreas
• Hematopoietic stem cells
• Bone marrow
• Peripheral blood
• Cord blood

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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

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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 _at_ HLA-C, DQB1, DPB1

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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

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Cadaver Kidney TransplantsHLA-ABDR Mismatches
(MM)
HLA DNA Typing Review and Transplantation.
Immunity, Vol. 14, 347356, April, 2001
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Allogeneic Hematopoietic Stem Cell
Transplantation (Allo HSCT)
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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!

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Choosing a Matched Unrelated Donor (MUD)
HLA-A HLA-B HLA-DR
Pt 0201, 0301 0702, 4402 0301, 1301
D1 0201, 0301 0702, 4402 0301, 1301
D2 02, 03 07, 44 03, 13
D3 2, 3 7, 44 3, 13
D4 01, 03 07, 44 0301, 1302
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Case 1How 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

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Case 1How 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

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Case 2Extended 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

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Case 2Extended 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

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Case 2Extended Family Typing
Pt Sib 1 Sib 2
Sib 3
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Case 2Extended Family Typing
Parent 1 Parent 2
Pt Sib 1 Sib 2
Sib 3
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Case 2Extended Family Typing
perfect match
Parent 1 Parent 2
Pt Sib 1 Sib 2
Sib 3
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Unrelated Donor Searches

• HLA matched unrelated donors MRD
• Available donors
• NMDP gt 9.5 million volunteer donors
• Worldwide 20 million total
• Cord blood gt 550,000
• Chance of finding A, B, DR match
• 60-80 with one million donors
• Chance best
• Caucgt Am. Indiangt Hisp/Asiangt Afr-Amer
• February 2012

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Matching for Stem Cell TransplantationHLA 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

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Which HLA Loci Impact Survival?
A B C DR DQ
JMDP Yes Yes No No No
FHCRC Yes Yes Yes Yes Yes
NMDP Yes Yes Yes Yes No
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Antigen vs Allele Mismatch

• Pt A0101, 0201 B0702, 0801
• Donor 1 A 1, 2 B 7, 8
• Low resolution (LR) match
• Donor 2 A0101, 0301 B0702, 0801
  
  Antigenic (LR)
  mismatch
• Donor 3 A0101, 0202 B0702, 0801
  Allelic (HR) mismatch

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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?

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Acceptable Mismatches?

• Locus important?
• A vs B vs C vs DRB1 gt DQB1
• Marrow ADR MM worse than BC
• PBSC C MM worse than others
• Cord C Ag MM increases TRM
• Specific mismatches important?
• A0201 vs 0202 vs 0203
• No preformed anti-donor-specific HLA Ab (DSA)

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HLA Typing Guidelinesfor 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)

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HLA Typing Guidelinesfor HSC Transplantation
Using URD

• Search all donor registries worldwide
• Matched URD
• molecular HLA typing gt 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

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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?

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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-A02 and A02
  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
  20101152704-2708.

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Non-Inherited Maternal Antigens (NIMA)
HLA-A HLA-B HLA-DRB1
Patient 02, 24 18, 35 0101, 1104
UCB donor unit 02, 32 18, 35 0101, 1104
UCB donors mother 02, 24 07, 35 0101, 1301
HLA-A24 is not carried by UCB donor but is
carried by UCB donors mother and the pt this is
a NIMA-matched UCBT.
Van Rood JJ, et al. Proc Natl Acad Sci USA
200910619952. Rocha V, et al. Biol Blood Marrow
Transplant 2012 July 17 Epub.
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NIMA Mismatch
HLA-A HLA-B HLA-DRB1
Patient 02, 11 18, 35 0101, 1104
UCB donor unit 02, 32 18, 35 0101, 1104
UCB donors mother 02, 24 07, 35 0101, 1301
HLA-A11 is not carried by UCB donor or the UCB
donors mother this is a NIMA-mismatched UCBT.
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NMDP
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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