powpoint Pringciples of hla typing hla matching in hsct david smillie.ppt

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
DR

Histocompatibility & Immunogenetics

paternal
haplotype
maternal
haplotype

C

Blood and
Transplant


INHERITANCE OF HLA HAPLOTYPES
Father + Mother = 4 haplotypes (25% chance of identical sib)
PARENTS

A*

a b

c d

CHILDREN

a/c

a/d

b/c

b/d

Histocompatibility & Immunogenetics

b/r

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

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*

C*

DRB1*

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*

C*

DRB1*

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