Download HLA-A, -B

Donor Selection for HSCT
Prof. Ilona Hromadníková, Ph.D.
Department of Molecular Biology and Cell Pathology
Third Medical Faculty, Charles University in Prague
Selection criteria
• HLA compatibility
If selection is possible among more donors consideration of:
• age
younger is more suitable, usually not donors > 60 years
Czech Donor Register: Donor age has to be between 18 – 35 years
when firstly included into the registr.
• sex
female to male BMT – risk of GvHD especially when female is
alloimmunized against H-Y and other antigens of minor histocomp.
system, male to female with SAA BMT – higher rejection risk
Selection criteria
If selection is possible among more donors consideration of :
• AB0- and Rh- blood groups
not significant risk factor, however, necessary to know
if incompatible – remove erythrocytes and plasma
• immunity against CMV
risk of infection transfer, when donor pos. and recipient neg.
CMV positivity → unfavourable influence on development and
course of GvHD even if no clinical symptoms of infection
Contra-indication for donation
• infectious diseases transmitted by blood
antibody screening: syphilis, hepatitis B, C, HIV-1/HIV-2, CMV
(IgM, IgG)
• because of general anaesthesia mainly:
cerebrovascular diseases
fresh heart attack
respiratory insufficiency
malignancies
pregnancy
HLA system of histocompatibility
• huge gene polymorphism
• Tx - important antigens HLA class I A, B, C
HLA class II DR, DP, DQ
HLA class I
• expressed on cell surface of nucleated cells, thrombocytes
• antigen presentation to CD8+ T lymphocytes
• cca 20 genes, most important HLA A, B, C
HLA class II
•
•
•
•
expressed on antigen presenting cells (DC, B lympho, macrophages)
antigen presentation to CD4+ T lymphocytes
3 gene pairs coding a and b chains: HLA DR, DP, DQ
HLA DR: a chain is monomorphic, additional gene for b chain
Polymorphism
HLA locus
HLA-A
HLA-B
HLA-C
HLA-DR (only b chain)
HLA-DQ (a and b chains)
HLA-DP (a and b chains)
Antigenic variants
25
53
11
20
9
6
DNA variants
83
186
42
221
49
88
Clinical genetics, 2001
antigenic variants (specificities) – due to differences in amino acid composition in
a or b chain, determined by serology
DNA variants – in HLA alleles defined by serology were found further variations in
DNA sequence
Example: HLA B27 allele is serologically unique, however 12 different variations in
nucleotide sequence were found
Nomenclature of HLA system
HLA A*0101
locus serologic specificity
specific allele defined by nucleotide sequencing
Example:
HLA A*0101, HLA A*0102 – same serologic specificity A1 differing in nucleotide
sequence
HLA DRB1*0401
class subtype
serologic specificity
chain a/b
number marking particular gene
specific allele
labelling w = working labelling
Polymorphism and inheritance of HLA
haplotypes
• high polymorphism number (not all!) causes variability in
structure of surface HLA proteins
• HLA alleles transmitted together like haplotypes
• each parent has 2 expressed haplotypes
child will have one or another → 25% chance exists, that two siblings will
have identical HLA haplotype as one of the parents
AB + CD → AC / AD / BC / BD
• high variability in profile and frequence of HLA variants
example: HLA A2 most frequent in all populations
HLA A24 in Caucasians, not in afro-Americans and Asians
• ethnically different distribution of particular HLA alleles and
haplotypes
Incompatibility in Tx can lead to:
• graft failure
role of HLA-A, -B, -C, -DR mismatches, total number of disparities
influences the risk of graft failure
• GvHD development
mismatches in HLA class I and/or II increase risk of aGvHD
• decreased survival
mismatches in HLA-A, -B, -C, -DR but not -DQ, -DP decrease
survival according to U.S. study
outcome differs according to individual studies
HLA typing
Serotyping
• identification of HLA class I and II
• discrimination of protein molecules on the basis of diversity in
antigenic characteristics
• using typing antisera panel (commercial trays), blood of
multiparous women (1 father) – serum contains sufficient
amount of antibodies against HLA molecules of foetus inherited
from the father
• very laborious procedure
• less time-consuming than genotyping
• certain degree of inaccuracy
HLA typing
Serotyping
• Routine typing of HLA class I:
120 typing sera in microtitration plate
tested T lymphocytes are added, after incubation complement is added
positivity = serum antibodies bind to T lymphocytes, lysed by complement
evaluation using fluorescent microscope (dye binding to DNA in lysed cell)
• Routine typing of HLA class II:
60 typing sera in panel
tested B lymphocytes are added, microcytotoxic test
HLA DR, DQ testing
HLA genotyping
DNA (genotyping)
• using PCR and specific primers for individual allele, DNA isolation from blood
• 2 approaches:
low resolution – identification of broad families of alleles that cluster into
serotypes („2-digit typing“, e.g. A*02 = A2 in serology)
high resolution – identification of the individual alleles within each serotype („4digit typing“, e.g. A*0201)
• more time-consuming
• simple method, easy automatization
• more sensitive method
Example: serotyping defines A*02, another 64 known alleles (A*0201-0264)
genotyping defines A*0201 allele
Clinical scheduling usually:
HLA class I serotyping, HLA class II genotyping
HLA typing - example
Serotyping defines individual serotype:
A1,A3,B7,B8,DR3,DR15(2),DQ2,DQ6(1)
Genotyping specifies HLA phenotype in individual:
A*0101, *0301, Cw*0701,*0702, B*0702,*0801,
DRB1*0301,*1501, DQA1*0501,*0102, DQB1*0201,*0602
composed from 2 haplotypes from the parents:
A*0101 : Cw*0701 : B*0801 : DRB1*0301 : DQA1*0501 : DQB1*0201
(by serotyping A1-Cw7-B8-DR3-DQ2)
A*0301 : Cw*0702 : B*0702 : DRB1*1501 : DQA1*0102 : DQB1*0602
(by serotyping A3-Cw7-B7-DR15-DQ6)
HLA typing of blood relatives
Typing of patient and related donor
• HLA-A, -B, -DR typing of two digits behind * mostly sufficient
for identification of maternal and paternal haplotypes
• confirmation of genotypic identity for the whole set of HLA
genes (A,B,C,DR,DP,DQ) on both chromosomes = match 12/12
• HLA-DP usually not tested – match 10/10
HLA typing of blood relatives
IHBT, National reference laboratory for DNA diagnostics
Department of HLA analysis
• genotyping always with indication for HSCT, concurrently blood
taking from primary blood relatives (siblings, parents, eventually
children of the patient)
• standard typing of HLA-A, -B, -DRB1 on the level of allele groups
(low resolution) and identification of both haplotypes
= serotyping of HLA class I and genotyping of HLA class II - „2digits“ typing
• unclarity in haplotype identification: genotyping of individual
alleles (high resolution, „4-digits“ typing)
Primary sample submission form for HLA genotyping
genotypizaci
After determination of
match/mismatch with relatives:
a) HLA-identical related donor in close family found
(i.e. sibling, parent)
blood taking for confirmatory examination, low resolution genotyping in loci:
HLA-A,-B,-C,-DRB1,-DQB1
(prior to starting conditioning regimen before Tx)
a) HLA-identical related donor in close family not found
searching in extended family - uncle, cousin... (HLA-A,-B,-DRB1 low
resolution) or indication for unrelated HSCT (registry searching)
after finding the donor:
confirmatory examination by high resolution genotyping of loci:
HLA-A,-B,-Cw,-DRB1,-DQB1
HLA typing of unrelated donors
• has to be typed: HLA- A*,B,Cw* , DRB1* a DQB1*
• matching degree expressed as a 10/10, 9/10, 8/10 match
• optional examination of genes DPB1*, DRB3*-5* or DQA1*
when several matched donors identified (also match in: AB0, age, sex, CMV status)
• match evaluated according to results of „4-digits" typing
pair - DRB1*1101 vs. DRB1*1103 mismatched
• HLA mismatch preferences for possible donor choice:
Cw*>A*B*,DQB1*>DRB1
Cw* the most accepted mismatch, DRB1 least accepted mismatch
• non-HLA mismatches (blood group, CMV) can influence the
choice of the donor more than HLA mismatch hierarchy, see the
example:
HLA typing of unrelated donors
Example
patient:
A*0201, B*3501,1501, Cw* 0401,0303… CMVneg, BG Aneg
donor #1:
A*0201, B*3503, 1501, Cw*0401, 0303, CMVneg , BG Aneg
donor #2:
A*0201, B*3501, 1501, Cw*0401, 0304, CMVpos, BG Bpos
-> donor #1 is preferred despite of HLA-B gene mismatch (CMV
+ABO compatibility)
Tx from HLA-identical sibling
Patient: M/4, MDS
graft: BM
donor: sister
Blood group
HLA
Match 10/10
weight:
36
kg
age: 9
Recipient
0
Rh +
Donor
0 Rh +
A *0201 *6801
A
dtto
B *3501 *3906
B
dtto
Cw *0401/04 * 1203
Cw
dtto
DRB1 *0101 *0801
DRB1 dtto
DQB1 *0501 *0402
DQB1 dtto
DPB1
DPB1 dtto
Tx from unrelated donor
Patient: M/16, pre-B ALL CR2
graft: PBSC
Donor code: DERKS 900111472
Sex: F
weight: 87
Blood group
HLA
Match 7/10
A
Recipient
Rh
age: 40
-
A
Donor
Rh
+
A
B
Cw
DRB1
*0201
*2705
*0202
*0101
*2402
*4101
*1602
*0404
A
B
Cw
DRB1
*0201
*2705
*02
*0101/17-19
*0301
*4101
*17
*0404
DQB1
*0501
*0302
DQB1
*0501
*04
DPB1
*
*
DPB1
*
*
Searching in donor registries
Czech republic has 3 registries
• Czech Stem Cell Registry (IKEM, Prague)
• Cord Blood Bank (IHBT, Prague)
• Czech National Marrow Donor Registry (CNMDR, Pilsen)
-
established in 1992 in Pilsen by Bone Marrow Transplant Foundation, 7 donor
centers created in CR
1993 cooperation via Bone Marrow Donor Worldwide located in Leiden,
Netherlands
1997 cooperation contract with the American National Marrow Donor Program
until 1998 funded entirely by Bone Marrow Transplant Foundation
2000 concluded mutual contracts with health insurance companies – cover
some of the expenses in conjunction with active search for the most suitable
donors from the Registry as well as the expenses connected with their more
detailed HLA examination
Donor centres in CR
http://www.kostnidren.cz/registr/
http://www.czechbmd.cz/
• Brno
• České Budějovice
• Hradec Králové
• Most
• Olomouc
• Ostrava
• Plzeň
• Praha
• Ústí nad Labem
cooperation with other donor centres in regions
Global Donor Registry
• Bone Marrow Donor Worldwide
14,093,962 (13,684,277 donors and 409,685 CBU's)
(last updated: 22-Feb-2010)
60 bone marrow registries from 44 states
+ 42 cord blood registries from 26 states
Germany cca 3,7 million donors, CR cca 55 000
USA – 14 registries, cca 5,5 million donors
Anthony Nolan Trust
• first bone marrow donors registry
• established in 1974 in Great Britain (Westminster Children‘s
Hospital)
• by Anthony Nolan‘s mother (WAS, 1971 – 1979)
• presently one of the biggest registries in the world
• http://www.anthonynolan.org.uk/