PRINCIPLES OF HLA TYPING;
HLA MATCHING IN HSCT
David Smillie
H & I, NHSBT, Sheffield
Histocompatibility & Immunogenetics
Blood and Transplant
• successful HSCT depends on many factors
(disease, stage, age, treatment regime etc)
• not least is HLA compatibility between patient
and donor!
Histocompatibility & Immunogenetics
Blood and Transplant
HLA TYPING REPORT – a collection of letters and
numbers, how do we arrive at this and what use is it?
Histocompatibility & Immunogenetics
Blood and Transplant
DEFINITIONS
• HLA = Human Leucocyte Antigen
• membrane glycoproteins on all nucleated cells
• 6 ‘classical’ HLA loci, Class I (A,B,C) & Class II (DR,DQ,DP)
each encoded by separate genes
• recognised by the immune system as ‘self’ or ‘non self’
• this determines histocompatibility = acceptance/rejection of
foreign tissue (e.g. transplant) - host vs graft, graft vs host, graft
vs leukaemia)
• cellular immunity
• antibody response
• most polymorphic system in human genome - challenges for
HLA typing and donor selection!
Histocompatibility & Immunogenetics
Blood and Transplant
AMINO ACID POLYMORPHISM
(this is what the immune system recognises)
HLA molecule
e.g HLA-A1
Histocompatibility & Immunogenetics
e.g HLA-A2
Blood and Transplant
DNA POLYMORPHISM
(resolved by DNA typing)
• SNP = Single Nucleotide Polymorphism
• alleles differ by 1 or more SNP
Histocompatibility & Immunogenetics
Blood and Transplant
HLA ANTIGENS ON NUCLEATED CELLS
DR
DQ
C
DP
B
A
DP
A
B
DQ
paternal
haplotype
maternal
haplotype
C
DR
Histocompatibility & Immunogenetics
Blood and Transplant
INHERITANCE OF HLA HAPLOTYPES
Father + Mother = 4 haplotypes (25% chance of identical sib)
PARENTS
A*
a b
c d
B*
C* DRB1* DQB1*
(a) 01
08
07
03
02
(b) 03
07
07
15
06
(c) 02
44
05
04
03
(d) 30
13
06
10
05
(r) 02
44
05
10
05
CHILDREN
a/c
a/d
b/c
b/d
Histocompatibility & Immunogenetics
b/r
Blood and Transplant
ORGANISATION OF HLA GENES
CHROMOSOME 6
Histocompatibility & Immunogenetics
Blood and Transplant
HLA TYPING METHODS
1950’s
1960’s
1980’s
1990’s
1999
2000
2000’s
discovery of HLA system
serological typing
first HLA genes cloned, sequenced
DNA/PCR based HLA typing
sequence entire MHC (HGP)
database of all HLA alleles
SBT, Luminex SSO
Histocompatibility & Immunogenetics
Blood and Transplant
HLA TYPING BY SEROLOGY
(Complement Dependent Cytotoxicity - using HLA-A as an example)
anti HLA-A1
• alloantisera
anti HLA-A2
anti HLA-A3
anti HLA-A24
• patient/donor
lymphocytes
(e.g. A2)
• add complement
Histocompatibility & Immunogenetics
Blood and Transplant
ADVANTAGES OF DNA BASED
TECHNIQUES
• not dependent on cell viability or cell surface
expression of antigens
• standardisation of reagents (synthetic c.f. alloantisera,
complement)
• more accurate and more precise
Histocompatibility & Immunogenetics
Blood and Transplant
3 LEVELS OF RESOLUTION
1. low resolution (2 digit) - identifies broad
families of alleles belonging to the same
serotypic group (e.g. A*02)
2. intermediate resolution (allele string) identifies alleles that have common sequence
determinants and thus share hybridisation
pattern (e.g. A*02:05/08/22)
3. high resolution (minimum 4 digit) identifies single allele
Histocompatibility & Immunogenetics
Blood and Transplant
LEVELS OF RESOLUTION FOR HSCT
• European Federation for Immunogenetics (EFI)
Standards v5.6 (stipulated by JACIE)
• related donor - ‘adequate testing to definitively
establish HLA identity by descent’
• unrelated donor - ‘low resolution HLA-A/B/C
(2 digit) and high resolution DRB1 typing (4 digit)’
• confirmatory typing
Histocompatibility & Immunogenetics
Blood and Transplant
HLA TYPING BY DNA
TECHNOLOGY – ACRONYMS!
gene polymorphism detected by:
• primer specificity (PCR-SSP)
• probe specificity (PCR-SSOP) e.g. Luminex
(primers/probes are short lengths of synthetic
DNA which hybridise only to their exact
complementary sequence and this hybridisation
can be detected)
• sequencing based typing (SBT)
Histocompatibility & Immunogenetics
Blood and Transplant
PRINCIPLE OF DNA TYPING
(using HLA-A gene as an example)
A*01
A*02
A*03
allele-specific sequences (primer/probe)
A*24
conserved sequence
Histocompatibility & Immunogenetics
Blood and Transplant
HIGH RESOLUTION HLA TYPING WHY
SEQUENCING BASED TYPING ?
• complete view of HLA gene sequence (cf PCR-SSP,
SSOP etc); detects new alleles
• ‘gold standard’ for HSCT
Histocompatibility & Immunogenetics
Blood and Transplant
DONOR SELECTION
Histocompatibility & Immunogenetics
Blood and Transplant
GUIDELINES FOR HLA MATCHING IN HSCT
Histocompatibility & Immunogenetics
Blood and Transplant
MATCHED DONOR OF CHOICE
1. HLA identical sibling
– confirmed by family studies
– identical for other genes in MHC region
2. HLA identical family member
– differences at other gene loci possible
3. HLA identical unrelated donor
– differences at other gene loci probable
4. HLA mismatched unrelated donor
5. cord blood unit(s)
Histocompatibility & Immunogenetics
Blood and Transplant
HSCT – TYPICAL HLA TYPING PROTOCOL
PATIENT & FAMILY
LOW RESR HLA-A, B, C, DRB1, DQB1
MATCH
HAPLOTYPE ASSIGNMENT
CONFIRMATORY TESTING
DONOR & RECIPIENT
SBT DRB1 (& TO ESTABLISH
HAPLOTYPES)
TRANSPLANT
Histocompatibility & Immunogenetics
NO MATCH
SBT RECIPIENT HLA-A, B, C,
DRB1, DQB1
MUD SEARCH
BBMR/AN/WBMR/BMDW
SELECT LOW RES MATCHED
DONORS
MUD’s: CONFIRMATORY LOW
RES & SBT HLAA,B,C,DRB1,DQB1, CMV, BLOOD
GROUP etc
Blood and Transplant
HLA MATCHING IN RELATED HSCT
Histocompatibility & Immunogenetics
Blood and Transplant
FAMILY WITH 4 HAPLOTYPES
(1 HLA identical sibling)
HLA:
A*
B*
DRB1*
C*
DQB1*
Patient *
02
29
44
51
15
16
07
-
02
-
Sib 1 *
02
29
44
51
15
16
07
-
02
-
Sib 2
24
29
07
44
07
16
07
15
02
06
Sib 3
02
-
13
51
06
15
07
-
02
-
Sib 4
24
29
07
44
07
16
07
15
02
06
Sib 5
02
-
13
51
06
15
07
-
02
-
Sib 6
02
-
13
51
06
15
07
-
02
-
Histocompatibility & Immunogenetics
Blood and Transplant
FAMILY WITH 5 HAPLOTYPES
(0 HLA matches!)
A*
HLA:
B*
DRB1*
C*
DQB1*
Patient
02
11
35
52
04
12
01
15
05
06
Sib 1
01
03
07
08
07
-
03
13
02
06
Sib 2
01
11
08
52
07
12
03
15
02
06
Sib 3
01
-
08
-
07
-
03
-
02
-
Sib 4
02
03
07
35
04
07
01
13
05
06
Sib 5
01
03
07
08
07
-
03
13
02
06
Sib 6
01
11
08
52
07
12
03
15
02
06
Histocompatibility & Immunogenetics
Blood and Transplant
HLA MATCHING IN UNRELATED HSCT
• donor identification via national/international registries
• best results - allele match at 5 loci (A,B,C,DRB1,DQB1 =10/10)
• Caucasian patients have a 40-50% chance of having a high
resolution matched donor at HLA-A, -B, -C, -DRB1 and -DQB1
(10/10 match)
• the chance of a 10/10 match in other ethnic groupings is lower
• comparable disease free survival in good risk patients
• increased frequency of post-transplant complications
Histocompatibility & Immunogenetics
Blood and Transplant
UNRELATED DONOR MATCHING TYPICAL STRATEGY
• HLA-A, B, C, DRB1 & DQB1 (5 loci = 10 alleles) at low resolution
• if matched at low resolution, proceed to SBT (minimum A, B,
DRB1)
• if matched at high resolution, select on:
• gender
• CMV
• blood group
• if not matched at high resolution
• widen search (BMDW ~20 million)
• single allele mismatch
• single or double CBU, 6/6 > 5/6 > 4/6 and cell dose
Histocompatibility & Immunogenetics
Blood and Transplant
UK REGISTRIES
UK Stem Cell Strategic Forum 2010
Recommendations – Transplantation:
• streamline registry activities in the UK
• data collection and outcome monitoring at every stage
• alternative donor clinical trials network
• cord blood transplantation concentrated into designated Centres
of Excellence
Histocompatibility & Immunogenetics
Blood and Transplant
UK REGISTRIES
UK Stem Cell Strategic Forum 2010
Recommendations – Cord Blood:
• increase from ~8000 to 50,000 high dose units in 5 years
• 30 to 50% of donations from black and ethnic minority women
• newly banked units to have > 90 x 107 TNC (ethnic minority
donors) or 120 x 107 TNC (Caucasian donors)
Histocompatibility & Immunogenetics
Blood and Transplant
UK STEM CELL STRATEGIC
FORUM 2011 (£4 million)
• align provision of stem cell donations - AN to become the single
contact point for all searches (access >700,000 adult donors)
• select 20,000 young adult donors with common phenotypes for high
resolution HLA typing
• increase collection at 8 cord blood collection sites, additional 2,000
CBU’s per year
• genotype prediction algorithm to speed up searches (? 2012) probability estimates for finding a 10/10 donor based on HLA
haplotype and allele frequencies in relevant population is highly
predictable
Histocompatibility & Immunogenetics
Blood and Transplant
TOTAL STEM CELL PROVISION
WORLDWIDE
WMDA ANNUAL REPORT
Histocompatibility & Immunogenetics
Blood and Transplant
PROBLEMS ASSOCIATED WITH
UNRELATED DONOR SEARCHING
problems are:
1. incomplete registry data (e.g. no HLA-C or DQB1)
2. HLA polymorphism (only 40-50% Caucasians have
10/10 HLA match, other groups less)
• rare alleles/allelic variants
• ethnicity
• linkage disequilibrium
3. donor drop out
Histocompatibility & Immunogenetics
Blood and Transplant
PROBLEMS ASSOCIATED WITH
UNRELATED DONOR SEARCHING (1)
incomplete registry data
• HLA
• not all donors typed by DNA techniques
• not all donors typed for DRB1
• not all donors typed for C &/or DQB1
• very few donors high resolution typing
• gender, blood group, ethnicity, CMV not always
available
• costs
Histocompatibility & Immunogenetics
Blood and Transplant
PROBLEMS ASSOCIATED WITH
UNRELATED DONOR SEARCHING (2)
HLA polymorphism
• rare alleles/allelic variants
(5,880 Class I, 1647 Class II alleles)
• linkage disequilibrium
Histocompatibility & Immunogenetics
Blood and Transplant
NO 10/10 DONOR
BECAUSE OF RARE ALLELES
HLA:
A*
B*
C*
DRB1*
DQB1*
JM
02:05
03:01
07:02
40:02
02:02
07:02
13:01
14:01/54
05:03
06:03
DEDKM 2127057
02:01
03:01
07:02
40:02
02:02
07:02
13:01
14:01/54
05:03
06
GB 1300733
02#1
-
07
40:02
02
07
13:01
14
05
06
DEDKM 620547
02#1
03
07
40:01
03
07
13:01
14
05
06
#1
Not HLA A*02:05
Histocompatibility & Immunogenetics
Blood and Transplant
NO 10/10 DONOR
BECAUSE OF RARE ALLELES
MUD’s
HLA:
A*
B*
C*
DRB1*
DQB1*
SH
03
24
15:18
18
05
07
04:07
13:01
03:01
06
TO03 3992
01
24
15:10
18
03
12:03/06
04:02
11
03:02
05
AKB-142342
24
25
15:18
18
07
12:03/06
04:01
13:01
03:02
06
CBU’s (matching for HLA-A, B & DRB1 only)
HLA:
Panel
SH
N/A
6504125
A*
B*
C*
DRB1*
DQB1*
Vol
(ml)
TNC
(107)
03:01
24:02
15:18
18:01
05:01
07:04
04:07
13:01
03:01
06
N/A
N/A
ACCB
03
24:02
38:01
35:23
07:02
12:03
04:07
13:01
03:02
06:03
121
318
999293104
UICB
03
24
35
-
04
04:07
13:01
N/T
30
208
290055467
AUCB
03
24
27
35
04:07
13:01
N/T
60
91.9
Histocompatibility & Immunogenetics
N/T
Blood and Transplant
SUITABLE DONOR
DESPITE RARE ALLELES
HLA:
A*
B*
C*
DRB1*
DQB1*
DM
02:11
11:01
35:03
40:06
04:01
15:02
10:01
15:01
05:01
06:01
DEDKM 3571314
02:11
11:01
35:03
40:06
04:01
15:02
10:01
15:01
05:01
06:01
0564-3760-1
02:11
11:01
35:03
40:06
12:03
15:02
10:01
15:01
05:01
06:01
Histocompatibility & Immunogenetics
Blood and Transplant
LINKAGE DISEQUILIBRIUM
• some alleles occur more frequently together than expected by random
association
• extended (ancestral) haplotypes e.g.A*01, B*08, C*07, DRB1*03,
DQB1*02
• commonly found HLA-B & C, HLA-DRB1 & DQB1
• patients with common HLA-B and -C or HLA-DRB1 and -DQB1
associations have a positive impact on the likelihood of finding a donor
• patients with uncommon HLA-B and -C or HLA-DRB1 and -DQB1
associations have a negative impact on the likelihood of finding a donor
Histocompatibility & Immunogenetics
Blood and Transplant
LINKAGE DISEQUILIBRIUM HLA-B & C
B*27
C*01
B*27
C*02
B*55
C*03:03(Cw9)
B*60
C*03:04(Cw10)
B*35
C*04
B*44:02
C*05
B*57
C*06:02
B*07
C*07
B*08
C*07
B*14
C*08
B*15:02
C*08
B*52
C*12:02
B*38
C*12:03
B*51
C*15
B*44:03
C*16:01
B*41
C*17
Histocompatibility & Immunogenetics
Blood and Transplant
LINKAGE DISEQUILIBRIUM HLA-DRB1 & DQB1
DRB1*01
DQB1*05
DRB1*15
DQB1*06:02
DRB1*16
DQB1*05
DRB1*03:01 (DR17)
DQB1*02:01
DRB1*03:02 (DR18)
DQB1*04
DRB1*04
DQB1*03:01/03:02
DRB1*11
DQB1*03:01
DRB1*12
DQB1*03:01
DRB1*13
DQB1*06:03/06:04
DRB1*14
DQB1*05
DRB1*07
DQB1*02:02/03:03
DRB1*08
DQB1*04/03:01
DRB1*09
DQB1*03:03
DRB1*10
DQB1*05
Histocompatibility & Immunogenetics
Blood and Transplant
COMPOUNDING EFFECT OF LINKAGE
DISEQUILIBRIUM
HLA:
A*
B*
DRB1*
C*
DQB1*
KaM
02:01
68:01
27:05
44:02
02
07
08:03
13:02
03
06:04
GB 1545503 (AN)
02:01
68:01
27:05
44:02
02
07
11:01
13:02
03
06:04
1/026701 (BBMR)
02
68:02
27
44
02
05
08
13:01
04
06:03
DE-BBB 12355
02
68:01
27
44
05
-
08
13:01
03
06:03
DE-DKM 336800
02
68
27
44
01
07
08
13:02
04
06:04
DE-DKM 513225
02
68:01
27
44
01
05
08
13:01
04
06:03
DE-DKM 2473710
02
68:01
27
44
05
07
08
13:01
04
06:03
0213-6872-5
02
68:01
27
44
03
07
08
13:02
04
06:04
0491-2420-9
02
68
27
44
01
07
08
13:02
04
06:04
Histocompatibility & Immunogenetics
Blood and Transplant
WHAT IS THE RISK OF HLA
MISMATCHING ?
• graft failure (rejection)
• GVHD (but GVL; ?HLA-DPB1)
• selecting a mismatch at 1 locus may affect
other loci due to linkage disequilibrium
Histocompatibility & Immunogenetics
Blood and Transplant
16th IHW PROJECT INTO HLA
MISMATCHING
Histocompatibility & Immunogenetics
Blood and Transplant
16th IHW PROJECT - RESULTS
Grades III-IV
aGVHD
Relapse
Survival
HLA mismatch
OR, P value
HLA 10/10 (10,930)
1
1
1
Single HLA-A (1,430)
1.64, <0.001
1.10, 0.11
1.36, <0.001
Single HLA-B (651)
1.88, <0.001
0.93, 0.47
1.38, <0.001
Single HLA-C (2,600)
1.55, <0.001
1.00, 1
1.29, <0.001
Single HLA-DRB1 (383)
1.47, <0.003
1.04, 0.74
1.14, 0.07
Single HLA-DQB1(1,139) 1.00, 0.99
1.07, 0.29
1.05, 0.27
Single DQB1 mismatch is tolerated the best
OR, P value
HLA 10/10 (10,930)
1
1
1
Single Class I (4,681)
1.62, <0.001
1.02, 0.58
1.32, <0.001
Single Class II (1,522)
1.11, 0.15
1.06, 0.29
1.07, 0.07
Class I mismatches are more detrimental than Class II
Histocompatibility & Immunogenetics
Blood and Transplant
HLA MISMATCHING – NO CONSENSUS IN UK!
Histocompatibility & Immunogenetics
Blood and Transplant
HLA NOMENCLATURE
Histocompatibility & Immunogenetics
Blood and Transplant