Pre-HSCT Donor & Patient Investigations: HLA Typing
BBMT Society of Nigeria. 15-27 July 2013
Pre-HSCT Donor & Patient
Investigation: HLA Typing
Anthony Oyekunle
Senior. Lecturer & Consultant Haematologist
Obafemi Awolowo University, Ile-Ife
Pre-HSCT Donor & Patient Investigations: HLA
Typing
Transplant Immunology
BBMT Society of Nigeria: HSCT Workshop
Stem Cell Transplant Centre, Benin 15-27 July 2013
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Histocompatibility depends on MHC genes (HLA)
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12 MHC genes on 6p; 3.6Mb
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HLA I: α-chain (with 3 domains) + β²-microglobulin
(Chromosome 15)
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Most polymorphic loci of human genome; 2584 alleles
Phenotypes fewer; fewer still detectable by serology
Usually inherited “en bloc“ → HLA haplotypes
ABDR match (familial) equivalent to genotypic identity (12/12)
Recombination events are rare; 1-2% HLA-A/B & -B/DRB1
α1 & α2 domains form peptide-binding site
HLA + 8-10aa from antigen bind TCR on CD8+ CTL
HLA II: α + β-chain; 2 domains each
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APCs: dendritic, B-cells, activated T-cells & macrophages
HLA + 10-30aa from antigen bind TCR on CD4+ TH-cells
Dr A A Oyekunle
Pre-HSCT Donor & Patient Investigations: HLA
Typing
BBMT Society of Nigeria: HSCT Workshop
Stem Cell Transplant Centre, Benin 15-27 July 2013
Schematic representation of the 12 HLA loci in the MHC region that comprise more than
200 genes on 6p. The corresponding serologic antigens & alleles defined by DNA
sequences are also shown
Dr A A Oyekunle
Culled from Hematopoietic Stem Cell Transplantation; ESH-EBMT (Tiercy 2008).
Pre-HSCT Donor & Patient Investigations: HLA
Typing
Methods for HLA Typing
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Low-resolution, viable lymphocytes, cross-reactivity
Cellular typing
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Stem Cell Transplant Centre, Benin 15-27 July 2013
Serology: simple, low-cost, commercial trays, for HLA-A & B
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BBMT Society of Nigeria: HSCT Workshop
CTLpf & HTLp assays; in-vitro, mismatch are “no vetoes“
Mixed lymphocyte culture (MLC); >25% pos → class II disparity
Role as final cellular crossmatch; if several matches found
DNA typing: PCR, HLA gene segments, nucleotide sequence
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Dr A A Oyekunle
High-tech, expensive
Routine for both classes I & II; in most HLA labs
Presently available in Nigeria at
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Tissue Typing Lab, OAUTHC, Ife.
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Safety Molecular Pathology Lab, Enugu.
Pre-HSCT Donor & Patient Investigations: HLA
Typing
Serological Typing
BBMT Society of Nigeria: HSCT Workshop
Stem Cell Transplant Centre, Benin 15-27 July 2013
Complement
antibody
Positive reaction to antibody
kills cells. Dead cells pick up dye.
Buffy coat
from patient
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Negative reaction to antibody:
cells survive and exclude dye.
Antigens are defined using complement-dependent
lymphocytotoxicity assay & a panel of HLA-alloantisera
HLA allo-Ag typically express multiple epitopes
Public specificities are common to several antigens
E.g. A19 shared by A29, A30, A31, A32, A33 & A74
This defines crossreactive groups (CREGs) of antigens
Private specificities are unique to a single antigen
Dr A A Oyekunle
Pre-HSCT Donor & Patient Investigations: HLA
Typing
Serological Typing
Percent dead cells Score Interpretation
0 -10%
11 - 20%
21 - 50%
51 - 80%
81 - 100%
Dr A A Oyekunle
1
2
4
6
8
0
negative
possibly negative
weakly positive
positive
strongly positive
not interpretable
BBMT Society of Nigeria: HSCT Workshop
Stem Cell Transplant Centre, Benin 15-27 July 2013
Cells are examined under a phasecontrast fluorescent microscope:
pink cells are positive.
If ≥60% cells are stained, they are
considered positive for the HLA
antigen.
Pre-HSCT Donor & Patient Investigations: HLA
Typing
BBMT Society of Nigeria: HSCT Workshop
Stem Cell Transplant Centre, Benin 15-27 July 2013
DNA Typing Methods: SSP & SSOP
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PCR-based HLA typing
Partial sequence information allowing inference of HLA allele
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DNA amplification; PCR techniques, primers, thermocycler
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Locus-, group-, or allele-specific
SSP
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SSP (sequence-specific primers) typing
SSOP (sequence-specific oligonucleotide probe) hybridization
Sequence-specific primer panel to detect polymorphisms at a locus
PCR product used in gel electrophoresis
HLA type assigned from pattern of presence & size of PCR products
SSOP
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HLA gene amplified, blotted on membrane (forward blot)
Complimentary probes applied to membrane; hybridization
HLA type determined by pattern of hybridization
Reverse blot; PCR product applied to immobilized probe
Dr A A Oyekunle
Pre-HSCT Donor & Patient Investigations: HLA
Typing
BBMT Society of Nigeria: HSCT Workshop
Stem Cell Transplant Centre, Benin 15-27 July 2013
SSP: Agarose Gel Electrophoresis
Dr A A Oyekunle
Pre-HSCT Donor & Patient Investigations: HLA
Typing
BBMT Society of Nigeria: HSCT Workshop
Stem Cell Transplant Centre, Benin 15-27 July 2013
DNA Typing Methods:
Sequence-based typing (SBT)
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SBT: involves the determination of the sequence of an
HLA gene locus
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Provides the highest resolution HLA typing possible.
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Polymorphic regions are amplified by PCR and then
sequenced
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SBT involves PCR amplification of the locus eg HLA
DRB1; followed by determination of the base sequence
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The sequence is then compared with the published
database of DRB1 genes sequences to assign alleles
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Also allows for detection of new alleles
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Can be fully automated, but not widely available
Dr A A Oyekunle
Pre-HSCT Donor & Patient Investigations: HLA
Typing
Dr A A Oyekunle
BBMT Society of Nigeria: HSCT Workshop
Stem Cell Transplant Centre, Benin 15-27 July 2013
Pre-HSCT Donor & Patient Investigations: HLA
Typing
BBMT Society of Nigeria: HSCT Workshop
Stem Cell Transplant Centre, Benin 15-27 July 2013
HLA Typing of Potential Donors
HLA Locus
Matching
Resolution of Testing
A
B
C
DRA
DRB1
DRB3, DRB4, DRB5
DQA1
DQB1
DPA1
DPB1
Recommended
Recommended
Recommended
No
Recommended
Unknown*
No
Uncertain*
No
Uncertain
Allele level
Allele level
Allele level
Allele level
*Unkno wn indicates that the effect o f matching has no t been evaluated; uncertain indicates that
studies disagree as to the impo rtance o f these lo ci in matching.
Dr A A Oyekunle
Hurley CK et al. Biol Blood Marrow Transplant 2003;9(10):610-5.11
Pre-HSCT Donor & Patient Investigations: HLA
Typing
HLA Nomenclature
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BBMT Society of Nigeria: HSCT Workshop
Stem Cell Transplant Centre, Benin 15-27 July 2013
Complex and potentially confusing!
Designated to the WHO’s HLA Nomenclature Committee
Originally assigned based on phenotype analysis by serology
Dr A A Oyekunle
Pre-HSCT Donor & Patient Investigations: HLA
Typing
HLA Typing: Resolution
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Low (A2 & A*02)
Intermediate (DRB1*11:02/03)
High (A*02:01 & A*02:01:01)
Dr A A Oyekunle
BBMT Society of Nigeria: HSCT Workshop
Stem Cell Transplant Centre, Benin 15-27 July 2013
Pre-HSCT Donor & Patient Investigations: HLA
Typing
BBMT Society of Nigeria: HSCT Workshop
Stem Cell Transplant Centre, Benin 15-27 July 2013
Immuno-incompatibility in Transplantation
Vector
of incompatibility
Host-vs-Graft MM: Donor alleles absent in recipient,
correlates with graft rejection
Graft-vs-Host MM: Recipient alleles absent in donor,
correlates with GvHD/GvL
Relation
Patient
Daughter
Age Sex
47 M
15 F
HLA*
Mismatch
Daughter
12
F
Mismatch
Sibling
Ad
M
Mismatch
Sibling
Ad
F
Mismatch
Sibling
Ad
F
Mismatch
Sibling
Ad
M
Mismatch
Sibling
Ad
M
Mismatch
Dr A A Oyekunle
A
3001
02,30(19)
0
30(19),33(19)
0
3001,3601
0
3601
1
3601
1
8001
1
3601
1
B
15(71),58(17)
15(71),58(17)
0
14(64),15(71)
1
5701
1
5101
2
1802
2
5701
1
5101
2
C
0302
03,07
0
03,08
0
0302
0
0302
0
0701
1
0101
1
0101
1
DRB1
0302,07
03,15
1
03,15
1
0301
1
0302
1
0301
1
0302
1
0301
2
DQB1
0201,03
02,06
1
02,06
1
0604
2
0201
1
0604,0302
1
0604
2
0604,0504
2
Remarks
2 GvH Ag mm
3 GvH Ag mm
4 GvH Ag mm
5 GvH Ag mm
6 GvH Ag mm
6 GvH Ag mm
8 GvH Ag mm
Pre-HSCT Donor & Patient Investigations: HLA
Typing
BBMT Society of Nigeria: HSCT Workshop
Allo-HSCT Immunology
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Stem Cell Transplant Centre, Benin 15-27 July 2013
Alloreactivity; basic fxns of Immune sys.
Special manifestations of Alloreactivity
Graft rejection
GvHD & GvL
Immune reconstitution: gradual replacement of host by donor
immune sys.
Dr A A Oyekunle
Pre-HSCT Donor & Patient Investigations: HLA
Typing
BBMT Society of Nigeria: HSCT Workshop
Stem Cell Transplant Centre, Benin 15-27 July 2013
The OAUTHC HLA Lab Team
THANK
YOU
Dr A A Oyekunle