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Pre-HSCT Donor & Patient Investigation: HLA Typing

Pre-HSCT Donor & Patient Investigation: HLA Typing. Anthony Oyekunle Senior. Lecturer & Consultant Haematologist Obafemi Awolowo University, Ile-Ife. Transplant Immunology. Histocompatibility depends on MHC genes (HLA) 12 MHC genes on 6p; 3.6Mb

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Pre-HSCT Donor & Patient Investigation: HLA Typing

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  1. Pre-HSCT Donor & Patient Investigation: HLA Typing Anthony Oyekunle Senior. Lecturer & Consultant Haematologist Obafemi Awolowo University, Ile-Ife

  2. 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

  3. 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).

  4. 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.

  5. Complement antibody Positive reaction to antibody kills cells. Dead cells pick up dye. Buffy coat from patient Negative reaction to antibody: cells survive and exclude dye. 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

  6. Serological Typing 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.

  7. 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

  8. SSP: Agarose Gel Electrophoresis

  9. 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

  10. HLA Typing of Potential Donors

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

  12. HLA Typing: Resolution • Low (A2 & A*02) • Intermediate (DRB1*11:02/03) • High (A*02:01 & A*02:01:01)

  13. Immuno-incompatibility in Transplantation Vector of incompatibility Host-vs-Graft MM: Donor alleles absent in recipient, correlates with graft rejection Graft-vs-Host MM: Recipient alleles absent in donor, correlates with GvHD/GvL

  14. Allo-HSCT Immunology • Alloreactivity; basic fxns of Immune sys. • Special manifestations of Alloreactivity Graft rejection GvHD & GvL • Immune reconstitution: gradual replacement of host by donor immune sys.

  15. The OAUTHC HLA Lab Team THANK YOU

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