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Principles of
HLA Typing
Dr. Nibras Saleam Al-Ammar
PhD in Immunology
Pathology Department
HLA
HLA is a highly
polymorphic gene
cluster located on the
Major
Histocompatibility
Complex (MHC)
This group of
genes resides
on
chromosome
6.
MHC
Has an extension of 3.5 mega bases
& contains over 200 genes divided into three
sub-regions:
Class I
Class II
Class III
(1800kb)
(800kb)
(1100kb)
MHC class I
Loci A, B, and C
present peptides from
inside the cell
Viral infected cell or
tumor cell
attract CD8 positive- or
cytotoxic T-cells that
destroy cells.
MHC class II
Loci DP, DM,
DO(A&B), DQ(A&B),
and DR
present antigens from
outside of the cell to Tlymphocytes.
stimulate multiplication
of T-helper cells, which
in turn stimulate B-cells
to produce antibodies.
Certain HLA alleles
tend to occur
together in the same
haplotype
A haplotype
(haploid
genotype)
It is a collection of
specific alleles (DNA
sequences) in a
cluster of tightlylinked genes on a
chromosome.
The haplotype can be written as follows:
HLA- A*0101 : Cw*0701 : B*0801 :
DRB1*0301 : DQA1*0501 : DQB1*0201
OR shorthand A1::DQ2 (4,731,878
nucleotides in length).
How we inherit HLA
Father
Child
Mother
We inherit one haplotype from each parent.
Therefore, there are a total of four different
haplotype combinations from 2 parents.
Basic rule in HLA inheritance
25% chance
Same 2 haplotypes
25% chance
non of the same haplotype
50% chance of sharing one
haplotype with siblings
Therefore, you have a 1 in 4 chance of being
an identical match with your siblings.
Purpose of HLA typing
HLA typing, along with ABO (blood type)
grouping, is used to provide evidence of tissue
compatibility.
Typing is also used along with blood typing
and DNA tests to determine the parentage
(that is, for paternity testing).
Many of the MHC-region genes are involved in
immunological processes.
What are the Methods of
HLA Typing?
Serological
Cellular
(MLC)
Molecular
Serological methods used for HLA Typing:
● Microcytotoxicity assay:
Principle:
Is done by using the pattern of lysis of peripheral
blood lymphocytes in the presence of complement
and typing sera to type serologically defined HLA
antigens.
HLA class I antigens test
B
lymphocytes
are chosen
HLA class II antigens test
T
lymphocytes
are chosen
MLC
Mixed Lymphocyte Culture (MLC)
Principle:
Lymphocytes of one donor, when cultured with
lymphocytes of an unrelated donor, they get
stimulated to proliferate or become cytotoxic.
Rarely being used
now
Originally used for
Class II typing
Molecular Assays
DNA-Based
Testing.
Intermediate
& high
resolution
typing
Identified
nucleotide
polymorphisms
High resolution typing is performed using
automated nucleotide sequencing
supplemented with
SSP
SSO
SBT
Principle
Luminex
The principles of Luminex technology
The Luminex technology is based on the use of 5.6
micron polystyrene microspheres (beads) each
internally dyed with a unique combination of red
and infrared dye. The combination of different
intensities of the two dyes allows for the
identification of each bead by its unique signature
when excited by a laser beam. This permits
multiplexing of up to 100 reactions in a single tube.
The application of Luminex to
HLA typing
HLA typing using Luminex is a reverse polymerase chain
reaction sequence specific oligonucleotide (PCR-SSO)
system which involves PCR amplification of targeted regions
within the MHC class I or II regions with group specific
primers, followed by a process of probing the amplicon with
Luminex beads, each coated with sequence specific
oligonucleotide probes to identify the presence or absence of
specific alleles. The assignment of HLA type is then based on
the reaction pattern observed, compared to patterns
associated with published sequences.
The application of Luminex to the detection
and definition of HLA specific antibodies
Types of Luminex systems
Pooled antigen system
Phenotype panel system
Single antigen system
Pooled Ag &
Phenotype
panel systems
Single Ag
system
• Use affinity
purified HLA Ags
to coat Luminex
beads
• Uses
recombinant
HLA proteins
Advantages of Luminex for HLA typing
●The combination of speed & reproducibility form the
main advantages of Luminex as used in HLA typing.
● The technique is fairly robust.
● Requires very little DNA.
● Fewer staff required.
● Reduction or even elimination of the use of
agarose gel electrophoresis and its associated
use of ethidium bromide.
Disadvantages of Luminex in HLA typing
● even it is rapid, it is still not as rapid as PCRSSP (unsuitable for use in on call situation).
● Results are low to medium resolution &
therefore require further testing by SSP or SBT
to obtain high resolution results.
● produces a small number of heterozygous
ambiguities.
● Kits are relatively expensive compared to
other techniques.
The advantages of Luminex for Ab detection
It is possible to look at reactivity against
single HLA antigens.
It is It is now possible to rapidly obtain a
result of a HLA antibody investigation when a
suspected rejection episode or reduced
graft function is reported.
Disadvantages of Luminex for Ab detection
High cost kits.
another disadvantage of Luminex
platform is that its use limits the laboratory to
only being able to identify Abs only if its cogent
Ags has been included on the beads.
Standard Luminex kits give no
information of whether or not detected Abs are
complement fixing.
Next Generation Sequencing (NGS)
Next Generation Sequencing (NGS) approaches
have been developed within the last few years that
provide allele phase and unambiguous pairwise
combinations of alleles.
These technologies can reduce the cost of high
resolution typing to that of commonly used low or
medium-resolution methods while maintaining
equivalent simplicity in methods and application.
Roche Introduces Next-Generation HLA
Typing Solution for 454 Sequencing Systems
The primer sets are designed for enabling
high-resolution typing and unambiguous
allele assignment in a single run.
Thank You