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

powpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.pptpowpoint Pringciples ò hla typing hla matching in hsct david smillie.ppt

PRINCIPLES OF HLA TYPING;

HLA MATCHING IN HSCT

David Smillie

H & I, NHSBT, Sheffield

• successful HSCT depends on many factors

(disease, stage, age, treatment regime etc)

• not least is HLA compatibility between patient

and donor!

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

• 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

AMINO ACID POLYMORPHISM (this is what the immune system recognises)

HLA molecule

DNA POLYMORPHISM (resolved by DNA typing)

DRDQ

DP

ABCDR

DQ

paternal haplotype

HLA ANTIGENS ON NUCLEATED CELLS

INHERITANCE OF HLA HAPLOTYPES

01 08 07 03 02A* B* C* DRB1* DQB1*

03 07 07 15 06

02 44 05 04 03

30 13 06 10 05

(a)(b)(c)(d)

PARENTS

CHILDREN

Father + Mother = 4 haplotypes (25% chance of identical sib)

a b c d

ORGANISATION OF HLA GENES

CHROMOSOME 6

1950’s discovery of HLA system

1960’s serological typing

1980’s first HLA genes cloned, sequenced

1990’s DNA/PCR based HLA typing

HLA TYPING BY SEROLOGY(Complement Dependent Cytotoxicity - using HLA-A as an example)

ADVANTAGES OF DNA BASED

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

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

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)

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

DONOR SELECTION

GUIDELINES FOR HLA MATCHING IN HSCT

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

HSCT – TYPICAL HLA TYPING PROTOCOL

PATIENT & FAMILY LOW RESR HLA-A, B, C, DRB1, DQB1

SELECT LOW RES MATCHED

DONORS

MUD’s: CONFIRMATORY LOW

RES & SBT

HLA-TRANSPLANT

HLA MATCHING IN RELATED HSCT

-FAMILY WITH 4 HAPLOTYPES

(1 HLA identical sibling)

FAMILY WITH 5 HAPLOTYPES

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

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

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

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)

UK STEM CELL STRATEGIC

• 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

TOTAL STEM CELL PROVISION

WORLDWIDE

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

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

PROBLEMS ASSOCIATED WITH UNRELATED DONOR SEARCHING (2)

HLA polymorphism

• rare alleles/allelic variants (5,880 Class I, 1647 Class II alleles)

• linkage disequilibrium

HLA: A* B* C* DRB1* DQB1*

JM 02:0 5 03:0 1 07:0 2 40:0 2 02:0 2 07:0 2 13:01 14:01/54 05:0 3 06:03 DEDKM 2127057 02:0 1 03:0 1 07:0 2 40:0 2 02:0 2 07:0 2 13:01 14:01/54 05:0 3 06

GB 1300733 02 #1 - 07 40:0 2 02 07 13:01 14 05 06 DEDKM 620547 02 #1 03 07 40:0 1 03 07 13:01 14 05 06

#1 Not HLA A*02:05

NO 10/10 DONOR BECAUSE OF RARE ALLELES

HLA: A* B* C* DRB1* DQB1*

DM 02:11 11:01 35:03 40:06 04:0 1 15:02 10:01 15:01 05:01 06:01 DEDKM

3571314

02:11 11:01 35:03 40:06 04:0

1 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

SUITABLE DONOR DESPITE RARE ALLELES

• 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

LINKAGE DISEQUILIBRIUM HLA-B & C

LINKAGE DISEQUILIBRIUM HLA-DRB1 & DQB1

COMPOUNDING EFFECT OF LINKAGE

DISEQUILIBRIUM

KaM 02:0 1 68:01 27:0 5 44:0 2 02 07 08:0 3 13:02 03 06:04

GB 1545503 (AN) 02:0 1 68:01 27:0 5 44:0 2 02 07 11:01 13:02 03 06:04 1/026701 (BBMR) 02 68:0 2 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

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

16th IHW PROJECT INTO HLA

MISMATCHING

16th IHW PROJECT - RESULTS

HLA MISMATCHING – NO CONSENSUS IN UK!

HLA NOMENCLATURE