1. PRINCIPLES OF HLA TYPING; HLA MATCHING IN HSCT David Smillie H & I, NHSBT, Sheffield

2. successful HSCT depends on many factors
(disease, stage, age, treatment regime etc)
not least is HLA compatibility between patient and donor!

3. HLA TYPING REPORT – a collection of letters and numbers, how do we arrive at this and what use is it?

4. 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!

5. AMINO ACID POLYMORPHISM (this is what the immune system recognises)
HLA molecule
e.g HLA-A1
e.g HLA-A2

6. DNA POLYMORPHISM (resolved by DNA typing)
SNP = Single Nucleotide Polymorphism
alleles differ by 1 or more SNP

7. HLA ANTIGENS ON NUCLEATED CELLSDR C DQ B DP
paternal haplotype A A
maternal haplotype DP B DQ C DR

8. INHERITANCE OF HLA HAPLOTYPES
Father + Mother = 4 haplotypes (25% chance of identical sib)
PARENTS
A* B* C* DRB1* DQB1*
(a)
a b c d
CHILDREN
(b)
(c)
(d)
(r)
a/c
a/d
b/c
b/d
b/r

9. ORGANISATION OF HLA GENES CHROMOSOME 6

10. HLA TYPING METHODS 1950’s discovery of HLA system
1960’s serological typing
1980’s first HLA genes cloned, sequenced
1990’s DNA/PCR based HLA typing
sequence entire MHC (HGP)
2000 database of all HLA alleles
2000’s SBT, Luminex SSO

11. HLA TYPING BY SEROLOGY (Complement Dependent Cytotoxicity - using HLA-A as an example)
anti HLA-A1
anti HLA-A2
anti HLA-A3
anti HLA-A24
alloantisera
patient/donor lymphocytes
(e.g. A2)
add complement

12. 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

13. 3 LEVELS OF RESOLUTION
low resolution (2 digit) - identifies broad families of alleles belonging to the same serotypic group (e.g. A*02)
intermediate resolution (allele string) - identifies alleles that have common sequence determinants and thus share hybridisation pattern (e.g. A*02:05/08/22)
high resolution (minimum 4 digit) - identifies single allele

14. 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

15. 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)

16. PRINCIPLE OF DNA TYPING (using HLA-A gene as an example)
allele-specific sequences (primer/probe)
A*03
A*24
conserved sequence

17. 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

18. DONOR SELECTION

19. GUIDELINES FOR HLA MATCHING IN HSCT

20. MATCHED DONOR OF CHOICE
HLA identical sibling
confirmed by family studies
identical for other genes in MHC region
HLA identical family member
differences at other gene loci possible
HLA identical unrelated donor
differences at other gene loci probable
HLA mismatched unrelated donor
cord blood unit(s)

21. HSCT – TYPICAL HLA TYPING PROTOCOL
PATIENT & FAMILY
LOW RESR HLA-A, B, C, DRB1, DQB1
NO MATCH
SBT RECIPIENT HLA-A, B, C, DRB1, DQB1
MATCH
HAPLOTYPE ASSIGNMENT
MUD SEARCH
BBMR/AN/WBMR/BMDW
CONFIRMATORY TESTING
DONOR & RECIPIENT
SBT DRB1 (& TO ESTABLISH HAPLOTYPES)
SELECT LOW RES MATCHED DONORS
MUD’s: CONFIRMATORY LOW RES & SBT HLA-A,B,C,DRB1,DQB1, CMV, BLOOD GROUP etc
TRANSPLANT

22. HLA MATCHING IN RELATED HSCT

23. FAMILY WITH 4 HAPLOTYPES (1 HLA identical sibling)
DRB1*
DQB1*
Patient * 02 29 44 51 15 16 07 -
Sib 1 *
Sib 2 24 06
Sib 3 13
Sib 4
Sib 5
Sib 6

24. FAMILY WITH 5 HAPLOTYPES
(0 HLA matches!)
HLA: A* B* C*
DRB1*
DQB1*
Patient 02 11 35 52 04 12 01 15 05 06
Sib 1 03 07 08 - 13
Sib 2
Sib 3
Sib 4
Sib 5
Sib 6

25. 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

26. 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

27. 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

28. 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)

29. 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

30. TOTAL STEM CELL PROVISION WORLDWIDE
WMDA ANNUAL REPORT

31. PROBLEMS ASSOCIATED WITH UNRELATED DONOR SEARCHING
problems are:
incomplete registry data (e.g. no HLA-C or DQB1)
HLA polymorphism (only 40-50% Caucasians have 10/10 HLA match, other groups less)
rare alleles/allelic variants
ethnicity
linkage disequilibrium
donor drop out

32. 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

33. PROBLEMS ASSOCIATED WITH UNRELATED DONOR SEARCHING (2)
HLA polymorphism
rare alleles/allelic variants
(5,880 Class I, 1647 Class II alleles)
linkage disequilibrium

34. BECAUSE OF RARE ALLELES
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
13:01
14:01/54
05:03
06:03
DEDKM
02:01 06 GB
02#1 - 07 02 14 05
DEDKM 03
40:01
#1 Not HLA A*02:05

35. BECAUSE OF RARE ALLELES
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 TO 01
15:10
12:03/06
04:02 11
03:02
AKB 25
04:01
CBU’s (matching for HLA-A, B & DRB1 only)
HLA:
Panel A* B* C*
DRB1*
DQB1*
Vol
(ml)
TNC
(107) SH
N/A
03:01
24:02
15:18
18:01
05:01
07:04
04:07
13:01 06
ACCB 03
38:01
35:23
07:02
12:03
03:02
06:03
121
318
UICB 24 35 - 04
N/T 30
208
AUCB 27 60
91.9

36. 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
12:03

37. 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

38. LINKAGE DISEQUILIBRIUM HLA-B & C

39. LINKAGE DISEQUILIBRIUM HLA-DRB1 & DQB1

40. COMPOUNDING EFFECT OF LINKAGE DISEQUILIBRIUM
HLA: A* B* C*
DRB1*
DQB1*
KaM
02:01
68:01
27:05
44:02 02 07
08:03
13:02 03
06:04
GB (AN)
11:01
1/ (BBMR)
68:02 27 44 05 08
13:01 04
06:03
DE-BBB 12355 -
DE-DKM 68 01
DE-DKM
DE-DKM

41. 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 41

42. 16th IHW PROJECT INTO HLA MISMATCHING

43. 16th IHW PROJECT - RESULTS

44. HLA MISMATCHING – NO CONSENSUS IN UK!

45. HLA NOMENCLATURE