2. first human leukocyte antigens (HLAs) were discovered in 1958 by Jean Dausset,
Rose Payne, and Jon van Rood. characterized using antibodies
in sera obtained from multiparous women andfrom patients after multiple blood
transfusions
3. THE HLA SYSTEM
recognized as the human equivalent of the major histocompatibility complex
(MHC), previously identified in inbred rodents, the products of which control the
recognition of self and foreign antigen
encoded on the short arm of chromosome 6
most intensively studied region of the human genome.
The region spans over 4 mega bases and contains in excess of 250 expressed
genes, making it the most gene dense region characterized to date.
about 28% ofthese genes encode proteins with immune-related functions.
4.
5. complex multigene family consisting of more than 10 loci.
HLA types are codominantly inherited on a maternal and paternal haplotype and
transmitted as a single Mendelian trait
6. HLA CLASS I
span 2 megabases at the telomeric end of the 6p21.3 region of chromosome 6.
This region encodes the classical transplantation antigens (HLA-A, -B, and -C) that are expressed on virtually all
nucleated cells.
Genes of the HLA class I loci encode the 44 kD heavy chains which associate with intracellular peptides present
within the cytoplasm
α1 and α2 chains are highly polymorphicand fold to form a peptide-binding cleft that accommodates peptides of 8–
10 amino acids in length that are mostly derived from endogenous proteins present within the cell cytoplasm.
major areas of amino acid polymorphism line the sides and base of the cleft and thereby govern the peptide-
binding repertoire of the HLA molecule
α3 domain proximal to the cell membrane is highly conserved and acts as a ligand for CD8 expressed on T
lymphocytes.
The tertiary structure is stabilized on the cell surface by non-covalent association with β2-microglobulin, a non-
polymorphic 12 kD protein encoded on chromosome 15
7. CLASS I LOCI
HLA-H, -J, -K, and -L pseudogenes
HLA-N, -P, -S, -T, U, -V, -W, -X, -Y, and -Z gene fragments that are not
transcribed or translated.
HLA-G expressed on placental trophoblast cells, implicating a
possible involvement in fetal– maternal development
HLA-E, -F, and -G Limited polymorphism and are known to act as ligands
natural killer (NK) cell inhibitory receptors (e.g., CD94)
8. HLA Class II
consists of three main loci: HLA-DR, -DQ, and -DP.
The glycoprotein products are heterodimers with non-covalently associated alpha and beta chains of molecular weight
approximately 33 kD and 28 kD, respectively.
Both chains have two extracellular immunoglobulin- like domains, a transmembrane region and a cytoplasmic tail
The membrane distal domains α1 and α1 form a peptide binding , accommodating peptides of 10–20 amino acids that
are derived predominantly from ingested (endocytosed or phagocytosed) extracellular (exogenous) proteins
The α1 domains of HLA-DR, -DQ, and -DP are highly polymorphic and so govern the peptide binding repertoire. They
are constitutively expressed on cells with immune function such as B lymphocytes, activated T lymphocytes, and antigen
presenting cells (monocytes, macrophages, and cells of dendritic lineage).
The conserved membrane proximal domain associates with CD4 on T lymphocytes with predominantly helper/inducer
function and thereby confers HLA class II restriction and forms the basis of cellular and humoral immunity to circulating
pathogens such as bacteria.
20. HLA-SPECIFIC ALLOSENSITIZATION
Routes of Sensitization
blood transfusion
Pregnancy
previous organ transplantation-poorly HLAmatched kidneys can result in allosensitization to the
mismatched donor HLA.
idiopathic (natural) HLAspecific antibodies result from cross-reactivity with infectious agents,
reactive with specific epitopes expressed on denatured HLA proteins but absent on HLA proteins in their
native form do not react with native antigen in cell-based assays and are considered non-harmful to a
transplanted kidney
21. Antibody Detection and Specificity
Definition
In order to perform effective antibody screening, serum samples should be obtained regularly
from patients on the transplant waiting list to provide a historical and contemporary
assessment (within the last 3 months) of antibody specificity
If a potential sensitization event occurs, additional samples are required, e.g., 14 and 28 days
following a transfusion with blood products
23. The CDC crossmatching technique
• Pioneered by Terasaki and colleagues in the 1960s
• scored on the percentage of dead cells
0-no dead cells
2- 20% lysis (generally taken as the cut-off for a
positive result)
8 -all cells having lysed ,strongest possible
reaction
TITRED CROSSMATCH
dilution usually performed to 1 in 2, 4, 8, 16, 32,
64 and so on.
high titre of high avidity DSAb, it may be that
many dilutions are required for the test to
become negative (e.g. 1 in 128)
Low titres- negative crossmatch could be
achieved with a desensitization protocol
24.
25. Interpretation of CDC-XM results
T cell XM B cell XM Explanation
Negative Negative No significant DSA
Very low titre DSA
Non-complement fixing DSA
Positive Positive HLA antibodies
Non-HLA antibodies IgG
Autoantibodies
IgM antibody
Recent treatment with
thymoglobulin/
alemtuzumab
Negative Positive DSA to HLA class II only
Low titre HLA class-I DSA
Treatment with rituximab
26.
27. The Flow-Cytometry Crossmatch (FCXM)
1990’s
depends on donor lymphocytes
being incubated with recipient
serum and flourocein dye
semi-quantitative and more
objective
28.
29.
30. Solid Phase Immunoassay (SPI)
commercial kits of purified recombinant HLA molecules coated on a microtitre
plate or synthetic beads
specific for HLA antibodies
Can be done by
ELISA-purified recombinant HLA molecules coated on a microtitre plate
more sensitive than CDCXM
LUMINEX- synthetic beads, more sensitive than CDCXM AND FCXM
31.
32. SAB
relevant beads are all coated with a single cloned antigen.
most precise and specific in detecting DSA against a specific antigen
33.
34. Epitope matching
limited area of the HLA molecule comprising a 15-25 amino acid sequence
Can be private and public
public epitopes result in numerous cross-reactions during HLA testing that result in
false positive results
can be done both in the laboratory (in-vitro) and from a computed based system (in-
silico)
highly specific SAB system is the commonest in-vitro epitope based matching system
The most widely used computer-based algorithm is the ‘HLA Matchmaker
algorithm’- allows donor and recipient matching based on epitopes and has been
used especially in relation to transplanting highly sensitized recipients.
35. The Virtual Crossmatch (VXM)
Advancement from wet XM
The antigens against which DSA are detected are referred to as Unacceptable
Antigens (UA), which can trigger HAR
compares the recipient’s UA against the HLA screening of the potential donor,
by a virtual matching process rather than an actual laboratory ‘wet’ crossmatch
36. Panel Reactive Antibodies
Testing recipient sera against a panel of donor lymphocytes from a sample of
donors representing the potential local donor population
recipient’s %PRA indicates the probability of having a positive crossmatch against
a given donor from that population and thereby the chances of receiving a
crossmatch negative kidney
