Pre-HSCT Donor & Patient Investigations: HLA Typing BBMT Society of Nigeria. 15-27 July 2013...

Pre-HSCT Donor & Patient Investigations: HLA Typing

BBMT Society of Nigeria. 15-27 July 2013

Pre-HSCT Donor & Patient Investigation: HLA Typing

Anthony OyekunleSenior. Lecturer & Consultant Haematologist

Obafemi Awolowo University, Ile-Ife


BBMT Society of Nigeria: HSCT Workshop

Stem Cell Transplant Centre, Benin 15-27 July 2013

Dr A A Oyekunle

Transplant Immunology Histocompatibility depends on MHC genes (HLA)

12 MHC genes on 6p; 3.6Mb Most polymorphic loci of human genome; 2584 alleles Phenotypes fewer; fewer still detectable by serology Usually inherited “en bloc“ → HLA haplotypes ABDR match (familial) equivalent to genotypic identity (12/12) Recombination events are rare; 1-2% HLA-A/B & -B/DRB1

HLA I: α-chain (with 3 domains) + β²-microglobulin (Chromosome 15)

α1 & α2 domains form peptide-binding site HLA + 8-10aa from antigen bind TCR on CD8+ CTL

HLA II: α + β-chain; 2 domains each APCs: dendritic, B-cells, activated T-cells & macrophages HLA + 10-30aa from antigen bind TCR on CD4+ TH-cells




Dr A A Oyekunle

Schematic representation of the 12 HLA loci in the MHC region that comprise more than 200 genes on 6p. The corresponding serologic antigens & alleles defined by DNA sequences are also shown

Culled from Hematopoietic Stem Cell Transplantation; ESH-EBMT (Tiercy 2008).




Dr A A Oyekunle

Methods for HLA Typing

Serology: simple, low-cost, commercial trays, for HLA-A & B Low-resolution, viable lymphocytes, cross-reactivity

Cellular typing CTLpf & HTLp assays; in-vitro, mismatch are “no vetoes“ Mixed lymphocyte culture (MLC); >25% pos → class II disparity Role as final cellular crossmatch; if several matches found

DNA typing: PCR, HLA gene segments, nucleotide sequence High-tech, expensive Routine for both classes I & II; in most HLA labs Presently available in Nigeria at

Tissue Typing Lab, OAUTHC, Ife. Safety Molecular Pathology Lab, Enugu.




Dr A A Oyekunle

Serological Typing

Antigens are defined using complement-dependent lymphocytotoxicity assay & a panel of HLA-alloantisera

HLA allo-Ag typically express multiple epitopes Public specificities are common to several antigens E.g. A19 shared by A29, A30, A31, A32, A33 & A74 This defines crossreactive groups (CREGs) of antigens Private specificities are unique to a single antigen




Dr A A Oyekunle

Percent dead cells Score Interpretation0 -10% 1 negative

11 - 20% 2 possibly negative21 - 50% 4 weakly positive51 - 80% 6 positive81 - 100% 8 strongly positive

0 not interpretable

Cells are examined under a phase-contrast fluorescent microscope: pink cells are positive. If ≥60% cells are stained, they are considered positive for the HLA antigen.

Serological Typing




Dr A A Oyekunle

DNA Typing Methods: SSP & SSOP PCR-based HLA typing Partial sequence information allowing inference of HLA allele

SSP (sequence-specific primers) typing SSOP (sequence-specific oligonucleotide probe) hybridization

DNA amplification; PCR techniques, primers, thermocycler Locus-, group-, or allele-specific

SSP Sequence-specific primer panel to detect polymorphisms at a locus PCR product used in gel electrophoresis HLA type assigned from pattern of presence & size of PCR products

SSOP HLA gene amplified, blotted on membrane (forward blot) Complimentary probes applied to membrane; hybridization HLA type determined by pattern of hybridization Reverse blot; PCR product applied to immobilized probe




Dr A A Oyekunle

SSP: Agarose Gel Electrophoresis




Dr A A Oyekunle

DNA Typing Methods: Sequence-based typing (SBT)

SBT: involves the determination of the sequence of an HLA gene locus

Provides the highest resolution HLA typing possible. Polymorphic regions are amplified by PCR and then

sequenced SBT involves PCR amplification of the locus eg HLA

DRB1; followed by determination of the base sequence The sequence is then compared with the published

database of DRB1 genes sequences to assign alleles Also allows for detection of new alleles Can be fully automated, but not widely available




Dr A A Oyekunle




Dr A A Oyekunle Hurley CK et al. Biol Blood Marrow Transplant 2003;9(10):610-5.11

HLA Typing of Potential Donors




Dr A A Oyekunle

HLA Nomenclature Complex and potentially confusing! Designated to the WHO’s HLA Nomenclature Committee Originally assigned based on phenotype analysis by serology




Dr A A Oyekunle

HLA Typing: Resolution

Low (A2 & A*02) Intermediate (DRB1*11:02/03) High (A*02:01 & A*02:01:01)




Dr A A Oyekunle

Immuno-incompatibility in TransplantationVector of incompatibility

Host-vs-Graft MM: Donor alleles absent in recipient, correlates with graft rejectionGraft-vs-Host MM: Recipient alleles absent in donor, correlates with GvHD/GvL

Relation Age Sex HLA* A B C DRB1 DQB1 RemarksPatient 47 M 3001 15(71),58(17) 0302 0302,07 0201,03Daughter 15 F 02,30(19) 15(71),58(17) 03,07 03,15 02,06

Mismatch 0 0 0 1 1 2 GvH Ag mmDaughter 12 F 30(19),33(19) 14(64),15(71) 03,08 03,15 02,06

Mismatch 0 1 0 1 1 3 GvH Ag mmSibling Ad M 3001,3601 5701 0302 0301 0604

Mismatch 0 1 0 1 2 4 GvH Ag mmSibling Ad F 3601 5101 0302 0302 0201

Mismatch 1 2 0 1 1 5 GvH Ag mmSibling Ad F 3601 1802 0701 0301 0604,0302

Mismatch 1 2 1 1 1 6 GvH Ag mmSibling Ad M 8001 5701 0101 0302 0604

Mismatch 1 1 1 1 2 6 GvH Ag mmSibling Ad M 3601 5101 0101 0301 0604,0504

Mismatch 1 2 1 2 2 8 GvH Ag mm




Dr A A Oyekunle

Allo-HSCT ImmunologyAllo-HSCT Immunology Alloreactivity; basic fxns of Immune sys.Alloreactivity; basic fxns of Immune sys. Special manifestations of AlloreactivitySpecial manifestations of Alloreactivity

Graft rejectionGraft rejectionGvHD & GvLGvHD & GvL

Immune reconstitution: gradual replacement of host Immune reconstitution: gradual replacement of host by donor immune sys.by donor immune sys.




Dr A A Oyekunle

THANKTHANK

YOUYOU

The OAUTHC HLA Lab Team

Pre-HSCT Donor & Patient Investigations: HLA Typing BBMT Society of Nigeria. 15-27 July 2013...

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