Organ Transplantation and the HLA system • Understand what the HLA system is and what it means to be histocompatible. • Have a basic understanding of the organisation and inheritance of the MHC. •Give an overview of pre-transplantation tests and understand their significance. •Demonstrate an understanding of the molecular principle behind SSP-PCR and its use in HLA typing.
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Organ Transplantation and the HLA system
• Understand what the HLA system is and what it means to be histocompatible.
• Have a basic understanding of the organisation and inheritance of the MHC.
•Give an overview of pre-transplantation tests and understand their significance.
•Demonstrate an understanding of the molecular principle behind SSP-PCR and its use in HLA typing.
• Allograft transplantation: transfer of an organ from one individual to another
• Most common transplanted organ is the kidney.
• Immune response dictates whether body will accept or reject a graft
What is Transplantation?
The HLA system• Human leukocyte antigens otherwise known as MHC molecules• Highly polymorphic Glycoprotein complexes• ~ 200 genes on chromosome 6• 3 clusters:
MHC class I – HLA-A, HLA-B, HLA-CMHC class II – HLA-DR, HLA-DP, HLA-DQMHC class III – Soluble components
HLA inheritance and familial matching
• We express two alleles at each loci: one inherited from either parent.•Co-dominant expression• <0.5% recombination frequency
Homozygous parents
Heterozygous children
HLA inheritance and familial matching
•‘Haplotype’: a combination of alleles at adjacent loci on a chromosome that are inherited together.
•Breeding between two heterozygous individuals results in children with 4 possible genotypes
•There is a 1 in 4 chance of a complete match between two siblings
DonorHLA type: A1, -, B8, 39, DR1, 3.Homozygous at the A locus for A1Heterozygous at the B locus for B8 and B39Heterozygous at the DR locus for DR1 and DR3
RecipientHLA type: A1, 24, B39, 44, DR1, 11.Heterozygous at each loci1 mismatches for the B locus1 mismatch for the DR locus
HLA typing and nomenclatureheterozygous
Pre-transplant tests
• %PRA: Recipient serum screened for antibodies towards HLA antigens
• ABO blood typing
• HLA tissue typing
• Serum cross-matching
%PRA
Polymerase Chain Reaction
One cycle consists of...
Step 1 – denature dsDNA at 98o
Step 2 – annealing of primers by reducing the temperature
Step 3 – extension of primers by heat stable DNA polymerase
Gel electrophoresis
•DNA can be stained using ethidium bromide
•DNA fragments separated by size.
•Negatively charged DNA moves through the gel towards the anode