Donor Selection for HSCT Prof. Ilona Hromadníková, Ph.D. Department of Molecular Biology and Cell...
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Transcript of Donor Selection for HSCT Prof. Ilona Hromadníková, Ph.D. Department of Molecular Biology and Cell...
![Page 1: Donor Selection for HSCT Prof. Ilona Hromadníková, Ph.D. Department of Molecular Biology and Cell Pathology Third Medical Faculty, Charles University in.](https://reader036.fdocuments.us/reader036/viewer/2022062421/56649e215503460f94b0ce96/html5/thumbnails/1.jpg)
Donor Selection for HSCT
Prof. Ilona Hromadníková, Ph.D.
Department of Molecular Biology and Cell PathologyThird Medical Faculty, Charles University in Prague
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Selection criteriaSelection criteria• HLA compatibility
If selection is possible among more donors - consideration of:
• ageyounger is more suitable, usually not donors > 60 yearsCzech Donor Register: Donor age has to be between 18 – 35 years when firstly included into the registr.
• sexfemale 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
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If selection is possible among more donors - consideration of :
• AB0- and Rh- blood groupsnot significant risk factor, however, necessary to knowif incompatible – remove erythrocytes and plasma
• immunity against CMVrisk 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
Selection criteriaSelection criteria
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Contra-indication for donationContra-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 diseasesfresh heart attackrespiratory insufficiencymalignanciespregnancy
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HLA system of histocompatibilityHLA system of histocompatibility
• huge gene polymorphism• Tx - important antigens HLA class I A, B, C
HLA class II DR, DP, DQ
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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 and chains: HLA DR, DP, DQ• HLA DR: chain is monomorphic, additional gene for chain
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Polymorphism Polymorphism HLA locus Antigenic variants DNA variantsHLA-A 25 83HLA-B 53 186HLA-C 11 42HLA-DR (only chain) 20 221HLA-DQ ( and chains) 9 49HLA-DP ( and chains) 6 88
antigenic variants (specificities) – due to differences in amino acid composition in or 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
Clinical genetics, 2001
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Nomenclature of HLA systemHLA 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*0401class subtype chain / number marking particular geneserologic specificity specific allele
labelling w = working labelling
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• high polymorphism number (not all!) causes variability in structure of surface HLA proteins
• HLA alleles transmitted together like haplotypes
• each parent has 2 expressed haplotypeschild 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 variantsexample: 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
Polymorphism and inheritance of HLA Polymorphism and inheritance of HLA haplotypeshaplotypes
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Incompatibility in Tx can lead to:Incompatibility in Tx can lead to:
• graft failurerole of HLA-A, -B, -C, -DR mismatches, total number of disparities
influences the risk of graft failure
• GvHD developmentmismatches in HLA class I and/or II increase risk of aGvHD
• decreased survivalmismatches in HLA-A, -B, -C, -DR but not -DQ, -DP decrease
survival according to U.S. study
outcome differs according to individual studies
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HLA typingHLA typingSerotyping
• 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
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HLA typingHLA typing
Serotyping
• Routine typing of HLA class I:
120 typing sera in microtitration platetested T lymphocytes are added, after incubation complement is added positivity = serum antibodies bind to T lymphocytes, lysed by complementevaluation using fluorescent microscope (dye binding to DNA in lysed cell)
• Routine typing of HLA class II:
60 typing sera in paneltested B lymphocytes are added, microcytotoxic testHLA DR, DQ testing
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HLA genotypingHLA genotypingDNA (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 („4-digit
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
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HLA typing - exampleHLA typing - exampleSerotyping 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)
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HLA typing of blood relativesHLA 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
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HLA typing of blood relativesHLA typing of blood relativesIHBT, 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 - „2-digits“ typing
• unclarity in haplotype identification: genotyping of individual alleles (high resolution, „4-digits“ typing)
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Primary sample submission form for HLA genotyping genotypizaci
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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
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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" typingpair - 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:
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HLA typing of unrelated donors
Example patient: A*0201, B*3501,1501, Cw* 0401,0303… CMVneg, BG Anegdonor #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)
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Tx from HLA-identical sibling
HLA
A *0201 *6801 B *3501 *3906 Cw *0401/04 * 1203 DRB1 *0101 *0801 DQB1 *0501 *0402
DPB1
A dtto B dtto Cw dtto DRB1 dtto DQB1 dtto
DPB1 dtto
donor: sister weight: 36 kg age: 9 Recipient DonorBlood group 0 Rh + 0 Rh +
Match 10/10
Patient: M/4, MDSgraft: BM
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Tx from unrelated donor
Donor code: DERKS 900111472 Sex: F
weight: 87 age: 40
Recipient DonorBlood group A Rh - A Rh +
HLA A *0201 *2402 A *0201 *0301B *2705 *4101 B *2705 *4101Cw *0202 *1602 Cw *02 *17DRB1 *0101 *0404 DRB1 *0101/17-19 *0404
DQB1 *0501 *0302 DQB1 *0501 *04
DPB1 * * DPB1 * *
Match 7/10
Patient: M/16, pre-B ALL CR2graft: PBSC
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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
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Donor centres in CRhttp://www.kostnidren.cz/registr/
http://www.czechbmd.cz/• Brno• České Budějovice • Hradec Králové • Most • Olomouc • Ostrava • Plzeň • Praha • Ústí nad Labemcooperation with other donor centres in regions
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Global Donor RegistryGlobal 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 000USA – 14 registries, cca 5,5 million donors
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Anthony Nolan TrustAnthony 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/