Donor Selection for HSCT

Prof. Ilona Hromadníková, Ph.D.

Department of Molecular Biology and Cell PathologyThird Medical Faculty, Charles University in Prague

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

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

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

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

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

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

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

• 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

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

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

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

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

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)

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

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)

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" 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:

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)

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

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

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 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

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

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/