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

Histocompatibility & Immunogenetics Blood and Transplant

PRINCIPLES OF HLA TYPING; HLA MATCHING IN HSCT

David SmillieH & 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?

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!

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

HLA molecule

e.g HLA-A1 e.g HLA-A2

DNA POLYMORPHISM(resolved by DNA typing)

• SNP = Single Nucleotide Polymorphism• alleles differ by 1 or more SNP

DPmaternal haplotype

paternal haplotype

HLA ANTIGENS ON NUCLEATED CELLS

INHERITANCE OF HLA HAPLOTYPES

01 08 07 03 02

A* B* C* DRB1* DQB1*

03 07 07 15 06

02 44 05 04 03

30 13 06 10 05

02 44 05

(r) 10 05

PARENTS

CHILDREN

a/c a/d b/c b/d b/r

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

a b c d

ORGANISATION OF HLA GENESCHROMOSOME 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

1999 sequence entire MHC (HGP)

2000 database of all HLA alleles

2000’s SBT, Luminex SSO

HLA TYPING METHODS

• alloantisera

• patient/donor lymphocytes

(e.g. A2)

• add complement

anti HLA-A1

anti HLA-A2

anti HLA-A3

anti HLA-A24

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

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

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 syntheticDNA which hybridise only to their exactcomplementary sequence and this hybridisationcan be detected)

• sequencing based typing (SBT)

allele-specific sequences (primer/probe)

conserved sequence

PRINCIPLE OF DNA TYPING(using HLA-A gene as an example)

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 CHOICE1. 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)

HSCT – TYPICAL HLA TYPING PROTOCOL

PATIENT & FAMILYLOW RESR HLA-A, B, C, DRB1, DQB1

MATCHHAPLOTYPE ASSIGNMENT

CONFIRMATORY TESTING

DONOR & RECIPIENT

SBT DRB1 (& TO ESTABLISH HAPLOTYPES)

NO MATCHSBT RECIPIENT HLA-A, B, C,

DRB1, DQB1

MUD SEARCH

BBMR/AN/WBMR/BMDW

SELECT LOW RES MATCHED DONORS

MUD’s: CONFIRMATORY LOW RES & SBT HLA-

A,B,C,DRB1,DQB1, CMV, BLOOD GROUP etc

TRANSPLANT

HLA MATCHING IN RELATED HSCT

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 -

FAMILY WITH 4 HAPLOTYPES(1 HLA identical sibling)

FAMILY WITH 5 HAPLOTYPES(0 HLA matches!)

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

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 • single or double CBU, 6/6 > 5/6 > 4/6 and cell dose

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 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

TOTAL STEM CELL PROVISION WORLDWIDE

WMDA ANNUAL REPORT

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

• costs

PROBLEMS ASSOCIATED WITH UNRELATED DONOR SEARCHING (2)

HLA polymorphism

• rare alleles/allelic variants

(5,880 Class I, 1647 Class II alleles)

• linkage disequilibrium

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

NO 10/10 DONORBECAUSE OF RARE ALLELES

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

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 03:01 06 N/A N/A

6504125 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 N/T 04:07 13:01 N/T 60 91.9

CBU’s (matching for HLA-A, B & DRB1 only)

MUD’s

NO 10/10 DONORBECAUSE OF RARE ALLELES

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

SUITABLE DONORDESPITE RARE ALLELES

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

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

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

COMPOUNDING EFFECT OF LINKAGE DISEQUILIBRIUM

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

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

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

HLA MISMATCHING – NO CONSENSUS IN UK!

HLA NOMENCLATURE

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

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