PURPOSE OF HLA TYPING, CONDITIONS THAT REQUIRING TRANSPLANTATION, THE PROCESS OF HLA TYPING, HLA TYPING IMPORTANT ROLE, SEROLOGICAL TEST, Microlymphocytotoxic test, MIXED LYMPHOCYTE REACTION, Molecular HLA typing, PCR BASED METHODS/ THREE CATEGORIES, STEPS OF MOLECULAR CLONING, Sequence specific priming, Hybridization with sequence specific oligonucleotide probes (SSOP), SEQUENCE BASED HLA TYPING, CLINICAL SIGNIFICANCE OF HLA TYPING,

1. TRANSPLANTATION IMMUNOLOGY- MLR, HLA
TYPING
PRESENTED BY
S.R. BHARATHKUMAAR,
I M.SC BIOTECHNOLOGY 2019-
2021 ,
BHARATHIAR UNIVERSITY.

2. What Is the HLA System?
The HLA system refers to a group of related genes that play an
important role in the immune system.
Together, the proteins made from these genes form something
called the major histocompatibility complex (MHC). These
proteins are attached to almost all of the cells of our body
(excluding red blood cells).

3. HLA stands for human leukocyte antigen
HLA typing is a kind of genetic test used to identify certain
individual variations in a person’s immune system.
The process is critical for identifying which people can safely
donate bone marrow, cord blood or an organ to a person who
needs a transplant.
HLA typing is also sometimes called HLA matching.

4. PURPOSE OF HLA TYPING
• The most common reason for HLA typing is to help determine
which people can provide the safest tissue transplants (solid
organ or hematopoietic stem cell transplantation).
• Potential tissue recipients must have the typing, as must anyone
who might potentially want to donate tissue.
• HLA type included in a bone marrow registry, for stem cell
transplantation.
• HLA typing may also be performed on terminally ill or recently
deceased people who will be serving as organ donors.
• The best possible donors have HLAs that closely match the
HLA patterns of the recipient.

5. CONDITIONS THAT REQUIRING TRANSPLANTATION
 There are many different health conditions that may need to be
treated through a transplant.
 For example, various types of blood cancers and genetic blood
disorders are treated through stem cell transplantation (taken
either from the bone marrow or from the peripheral blood).
 A solid organ transplant might be necessary for any essential
organ that has become severely damaged.
 This might happen through trauma, infection, autoimmune
disease, genetic illness, toxins, or many other disease processes.
 For example, one might need a kidney, liver or lung transplant if
one’s own organs are very functioning very poorly.

6. THE PROCESS OF HLA TYPING
HLA typing assesses the particular HLA genes that have inherited
Because there are a number of different HLA genes, as well as different
variations of these genes, there are very many different possible color
combinations that together make up your specific HLA type.
HLA typing also usually includes testing for antibodies targeted to specific
HLA proteins. Antibodies are made by part of the immune system.
If a person already has an antibody against an HLA protein it may attack that
protein if it is transplanted. This may cause the transplant to fail.
So generally, shouldn’t receive a transplant from someone if already have an
antibody against one of their HLA proteins.

7. CONT…….
HLA typing also often includes something called lymphocyte
cross matching. Lymphocytes are a type of immune cell.
Lymphocyte cross matching checks to see if the recipient has an
antibody against a protein on the donor’s lymphocytes.
If so, that person generally shouldn’t receive a transplant from
that particular person. These people are at high risk of a
transplant that won’t be successful.

8. Is HLA Typing the Same as Blood Typing?
No. HLA is much more complicated than blood typing because
there are many more HLA markers that make a person’s cells
unique.
There are only eight basic blood types, and many people can
safely receive more than one type of blood (depending on their
type).
To receive only blood from a person, do not need to be an HLA
match, because HLA is not present on red blood cells.
To receive a solid organ transplant, the recipient must have a
compatible blood type with the donor, as well as the best HLA
match possible.
For stem cell donations, one needs a very strong HLA match, but
blood type is not as important as it is for solid organ transplants.

9. How Are HLA Genes Inherited?
Because the HLA genes are located close together on DNA, they are usually
inherited as a group.
HLA type is composed of the set of HLA genes you inherited from mother
and the HLA genes you inherited from father.
Biological parents always share half of their HLA proteins with their children.
This is also called a “half match.” Conversely, a child always is a half match
with their parents.
The set of HLA alleles found on one chromosome is called haplotype.
Determination of haplotype is important for identification of HLA identical
siblings because sharing of antigen from different haplotypes is common.

10. HLA TYPING IMPORTANT ROLE
The primary target of immune responses to allogeneic transplants
Critical for response to antigenic stimuli
Implicated in genetic susceptibility to auto immune disease.

11. METHODS OF
HLA TYPING
SEROLOGICAL
(Micro
cytotoxicity test)
MIXED
LYMPHOCYTE
TEST
MOLECULAR
METHODS

12. SEROLOGICAL TEST
Potential donor′s and recipient's WBC′s added to the different
wells of microtitre plates
Antibodies specific for HLA class I and II added
After incubation complement is added
Cytotoxicity is assessed by uptake or exclusion of dye

13. SEROLOGY
HLA Typing is done serologically by MICROCYTOTOXICITY
(microlymphocytotoxicity) which tests for complement
mediated lysis of peripheral blood lymphocytes with a
standard set of typing sera.
Viable peripheral blood lymphocyte are obtained by
discontinuous density gradient centrifugation using Ficol/
Tryosil or Ficol / Sodium Metrizoate at a density of 1.077 at
19⁰C-22⁰C

14. Microlymphocytotoxic test
3 stages
• Viable lymphocyte are incubated with HLA specific antibodies. If
the specific antigen is present on the cell the antibody is bound.
• Rabbit serum as a source of complement is added, incubate. If
antibody is bound HLA antigen on the cell surface it activates the
complement which damages the cell membrane making it
permeable to vital stains.
• Results are visualized by adding dye usually a flurochrome eg.
Ethidium Bromide although both trypan blue and Eosin Y have
been used .

15. • If the reaction has takes place the EB enters the cell and binds to
the DNA
• For ease double staining is normally used. A cocktail of Ethidium
Bromide and Acridine Orange, quenched using Bovine
Hemoglobin to allow simultaneous visualization of both living and
dead cells.
• White blood cells from potential donors and the recipient are
added to separate wells of a microtiter plate. The example depicts
the reaction of donor and recipient cells with a single antibody
directed against an HLA-A antigen.
• The reaction sequence shows that if the antigen is present on the
lymphocytes, addition of complement will cause them to become
porous and unable to exclude the added dye.

16. Presence or absence of
various MHC alleles
determined by antibody
mediated cytotoxicity

18. Cont…….
• Test is left for 10 minutes and then read using an inverted
fluorescent microscope.
• A mixture of T and B lymphocytes can be used for HLA class I
typing.
• B lymphocytes are required for HLA class II typing by
serology. (Normal population 85-90% to T and 10-15% B cells)
• This can be achieve using a number of methods

19. In the past neuraminidase treated sheep red blood cell resetting
and nylon wool have been used.
Immunomagnetic bead separation is the current method of choice.
It utilizes polystyrene microspheres with a magnet able core
coated in monoclonal antibody for a HLA class II b chain
monomorphic epitope. Positive selection

20. N

22. MIXED LYMPHOCYTE REACTION
o It has been observed the lymphocytes from one donor, when
cultured with lymphocytes from an unrelated donor, are
stimulated to proliferate.
o It has been established that this proliferation is primarily due to
a disparity in the Class II MHC (DR) antigens and T cells of
one individual interact with allogeneic class-II MHC antigens
bearding cells (B cells, dendritic cells, Langerhans cells, etc…)
this reactivity was termed with mixed leukocyte reaction (MLR)
and has been used for studying the degree of histocompatibility.

23. In this test, the lymphocyte (responder cells) are mixed with
irradiated or mitomycin C treated leukocytes from the recipient,
containing B-lymphocytes and monocytes (stimulator cells).
The responder T cells will recognize the foreign class II antigens
found on the donor and undergo transformation (DNA synthesis and
enlargement: blastogenesis) and proliferation (mitogenesis) occurs
The T cells that respond to foreign Class II antigens are typically
CD4+ TH-1 type cells
These changes are recorded by the addition of radioactive (tritiated,
3H)

25. NN

26. Molecular HLA typing
DNA based procedures has increased the accuracy of HLA typing
and lead to the identification of serologically undetected alleles and
many subtypes of serological specificities.
DNA based methods of type for HLA alleles have therefore focused
in the analysis of nucleotide variation occurring in both exon 2 and
3 of class I genes and exon-2 of class II genes.

27. PCR BASED METHODS/ THREE CATEGORIES
 Polymorphism is identified directly as part of the PCR process,
although there are post amplification steps e. g (SSP)
 Product containing internally located polymorphism that can be
identified by a second technique e. g PCR sequence specific
oligonucleotide probing (SSOP), PCR- RFLP,PCR followed by
sequencing.
 Conformational analysis

28. STEPS OF
MOLECULAR
CLONING
• Extraction of genomic DNA
• Amplification of the genes of interest
and
• Detection of sequence polymorphism
that define the alleles.

29. DNA EXTRACTION
Genomic DNA extracted from nucleated cells; the purity of the
DNA extracted may be an important factor for successful results
GENE AMPLIFICATION/PCR
The specificity of the amplification can be locus specific e. g (HLA-
A, HLA-B, HLA-DRB1), group specific… e.g. (DRB1-01, DRB1-
02) Or allele specific DRB1-0401, DRB1-0402
The primers are selected to amplify a single allele or a group of
alleles
For PCR, the specificity is determined by the sequence of the
primers and amplification condition. Most typing scheme require
conditions the avoid the co-amplification of pseudogene.

30. Amplification of exon 2 (approximately 270bp ) of HLA class II
is sufficient to achieve the highest resolution level.
For typing HLA class I, both exon 2 and 3 and the intervening
intron (fragment longer than 900bp ) are amplified by single pair
of primers.
Detection of sequence polymorphism that define the alleles
• Sequence specific primers (SSP) group and alleles specific
primers
• Hybridization with sequence specific oligonucleotide probes
(SSOP)
• Sequence based typing (SSP)

31. Sequence specific priming
• The procedure relies on the
specificity of primer extension that
is matched or mismatched with the
template at its 3’end.
• A combination of 2 primers
designed for each of 2 polymorphic
sequence motifsin cis allows the
identification of an allele or a group
of alleles that are characterized by
these 2 motifs.
• The method is rapid and ideally suited
for small numbers of samples. Because very few sequences are
absolutely allele specific, SSP combine several primer to
discriminate a particular allele unambiguously.

32. Hybridization with sequence specific oligonucleotide probes (SSOP)
The procedure relies on the locus-specific
amplification of the genomic DNA segment
comprising the polymorphic sites of HLA alleles.
Amplified DNA is then immobilized on a
solid support, usually a nylon membrane,
and then hybridized with a battery of
(SSOP) (direct hybridization).
Fluorochromes are linked with the probes to
allow their detection by chemiluminescence.
Alternatively, SSO probes can be immobilized
on a solid support, for example color-coded microspheres,
and hybridized with labeled PCR product (reverse hybridization).
The higher the number of probes the better the resolution level. Usually 50-
100 probes per locus are used for intermediate/high resolution typing,
however, only the probe completely matched with the target sequence
amplified will hybridize and give a positive signal.

33. SEQUENCE BASED HLA TYPING
• Sequence based HLA typing involves determining the nucleotide
sequence of an amplified segment of HLA gene
• SBT presents the advantages over the other procedure because of
the relatively fast (24-48 hours), high level resolution
• More reliable and specific method

34. CONFORMATIONALANALYSIS
Different mutation generates specific conformational changes in
PCR products, these are identified by electrophoresis analysis.
E.g.….. heteroduplex analysis, single strand conformation
polymorphism (SSCP), denaturing gradient gel electrophoresis
(DGGE), temperature gradient gel electrophoresis (TGGE)

36. CLINICAL SIGNIFICANCE OF HLA TYPING
In organ transplantation
In transfusion therapy
Disease association
Disputed paternity
In cancer prevention
Anthropological studies

37. Thank you