1. Pre-HSCT Donor & Patient Investigations: HLA Typing BBMT Society of Nigeria. 15-27 July 2013 Pre-HSCT Donor & Patient Investigation: HLA Typing Anthony Oyekunle Senior. Lecturer & Consultant Haematologist Obafemi Awolowo University, Ile-Ife

2. Pre-HSCT Donor & Patient Investigations: HLA Typing 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

3. Pre-HSCT Donor & Patient Investigations: HLA Typing BBMT Society of Nigeria: HSCT Workshop Stem Cell Transplant Centre, Benin 15-27 July 2013 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).

4. Pre-HSCT Donor & Patient Investigations: HLA Typing BBMT Society of Nigeria: HSCT Workshop Stem Cell Transplant Centre, Benin 15-27 July 2013 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.

5. Pre-HSCT Donor & Patient Investigations: HLA Typing BBMT Society of Nigeria: HSCT Workshop Stem Cell Transplant Centre, Benin 15-27 July 2013 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

6. Pre-HSCT Donor & Patient Investigations: HLA Typing BBMT Society of Nigeria: HSCT Workshop Stem Cell Transplant Centre, Benin 15-27 July 2013 Dr A A Oyekunle 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

7. Pre-HSCT Donor & Patient Investigations: HLA Typing BBMT Society of Nigeria: HSCT Workshop Stem Cell Transplant Centre, Benin 15-27 July 2013 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

8. Pre-HSCT Donor & Patient Investigations: HLA Typing BBMT Society of Nigeria: HSCT Workshop Stem Cell Transplant Centre, Benin 15-27 July 2013 Dr A A Oyekunle SSP: Agarose Gel Electrophoresis

9. Pre-HSCT Donor & Patient Investigations: HLA Typing BBMT Society of Nigeria: HSCT Workshop Stem Cell Transplant Centre, Benin 15-27 July 2013 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

10. Pre-HSCT Donor & Patient Investigations: HLA Typing BBMT Society of Nigeria: HSCT Workshop Stem Cell Transplant Centre, Benin 15-27 July 2013 Dr A A Oyekunle

11. Pre-HSCT Donor & Patient Investigations: HLA Typing BBMT Society of Nigeria: HSCT Workshop Stem Cell Transplant Centre, Benin 15-27 July 2013 Dr A A Oyekunle Hurley CK et al. Biol Blood Marrow Transplant 2003;9(10):610-5.11 HLA Typing of Potential Donors

12. Pre-HSCT Donor & Patient Investigations: HLA Typing BBMT Society of Nigeria: HSCT Workshop Stem Cell Transplant Centre, Benin 15-27 July 2013 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

13. Pre-HSCT Donor & Patient Investigations: HLA Typing BBMT Society of Nigeria: HSCT Workshop Stem Cell Transplant Centre, Benin 15-27 July 2013 Dr A A Oyekunle HLA Typing: Resolution Low (A2 & A*02) Intermediate (DRB1*11:02/03) High (A*02:01 & A*02:01:01)

14. Pre-HSCT Donor & Patient Investigations: HLA Typing BBMT Society of Nigeria: HSCT Workshop Stem Cell Transplant Centre, Benin 15-27 July 2013 Dr A A Oyekunle 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

15. Pre-HSCT Donor & Patient Investigations: HLA Typing BBMT Society of Nigeria: HSCT Workshop Stem Cell Transplant Centre, Benin 15-27 July 2013 Dr A A Oyekunle Allo-HSCT Immunology Alloreactivity; basic fxns of Immune sys. Alloreactivity; basic fxns of Immune sys. Special manifestations of Alloreactivity Special manifestations of Alloreactivity Graft rejection GvHD & GvL Immune reconstitution: gradual replacement of host by donor immune sys. Immune reconstitution: gradual replacement of host by donor immune sys.

16. Pre-HSCT Donor & Patient Investigations: HLA Typing BBMT Society of Nigeria: HSCT Workshop Stem Cell Transplant Centre, Benin 15-27 July 2013 Dr A A Oyekunle THANKYOU The OAUTHC HLA Lab Team