1.
Dr Nisha Singh
Guide:Dr S .S RautHLA TYPING AND ITS IMPLICATIONS
Dr. Nisha Singh
Guide: Dr S S Raut

2.
 History
 MHC
 Immunity and MHC
 MHC & HLA
 Structure & Functions
 Nomenclature
 Typing
 Implications
History
MHC
Immunity and MHC
MHC & HLA
Structure & Functions
Nomenclature
Typing
Implications

3.
History
Early work of Gorer (1930) on antigens responsible for allograft
rejection in mice led to the discovery of the ‘MAJOR
HISTOCOMPATIBILITY COMPLEX (MHC)’.
Development of congenic & recombinant strains of mice by Snell
enabled the detailed analysis of various loci of this complex.

4.
Dausset pioneered studies on HUMAN LEUKOCYTE
ANTIGENS MAJOR
HISTOCOMPATIBILITY ANTIGENS in human being.
Benacerraf & colleagues established genetic basis of
immune response.
For their work on MHC & genetic control of immune
response, Snell, Dausset & Benacerraf were awarded
Nobel Prize for medicine in 1980.

5.
•Human leukocyte antigen (HLA) complex is the
locus of genes that encode for proteins on the
surface of cells responsible for regulation of the
immune system in humans.
•Located on short arm of chromosome 6
•Inherited as haplotypes (i.e. set of alleles) one from
each parent
•They are co-dominantly expressed

6.
HLA proteins found in the membranes (outer coating) of nearly
every cell in the body (all cells that have a nucleus). These
antigens are in especially high concentration on the surface of
white blood cells(leukocytes).
The HLA loci are part of the genetic region known as the major
histocompatibility complex

7.
 The MHC system in humans was subsequently discovered in
early 1950s.
 The MHC has genes (including the HLA) that form part of the
normal function of the immune system.
 The MHC is an extreme gene-dense region of the genome, and
it can be divided into three sub-regions; the class I, the class
and the class III regions. All encoded by a gene complex
located on the short arm of 6.
The MHC system in humans was subsequently discovered in
early 1950s.
The MHC has genes (including the HLA) that form part of the
normal function of the immune system.
The MHC is an extreme gene-dense region of the genome, and
it can be divided into three sub-regions; the class I, the class
and the class III regions. All encoded by a gene complex
located on the short arm of 6.

8.
 The class I region of approximately 2000 kilobases include;the
polymorphic HLA-A, B, C loci; non classical class HLAE,F,
G,..
 The class II region of approximately 1000 kilobases
include;HLA-DR,DQ an DP loci, ……….and non classical
class HLA class II HLA-DM, DO,.
 The class III region of approximately 1000 kilobases encode
genes with diverse functions and does not contain any HLA
genes. Contain loci responsible for the
complement,hormones,…

9.
Class I
Encoded by A,B,C etc loci
Expressed on all nucleated cells and platlets
 Present antigenic peptides
from within the cells ( endogenous)
to CD8 + T cell
 MHC restriction of cell mediated cytotoxicity , the
acceptance and rejection of grafts
 These antigens also function as complement of harmone.

10.
Class ІІ: - or D region –DR, DQ,
& DP loci
Protein that regulate the immune response
These are found only on the cell of immune system such as macrophage,
dendritic cell activated T cell and B cell.
These are the heterodimer consists of an alpha and beta chains.
Each chain has two domain – a proximal constant region and a distal
variable region .The distal region constitute the antigen-binding site for
reorganization by CD4 lymphocyte.
The immune response genes, which control immunological response to
specific antigen, are situated in this class 2 antigen.
These are responsible for the graft versus host response and mixed
leucocytes reaction (MLR).

11.
Class ІІІ: - or complement region---
This group contains genes for C2 and C4 of classical
pathway, properdin factor of alternative, heat shock protein,
and tumor necrosis factor
These are important component of complement system and
are responsible for various cellular activities.
HLA loci are multiallelic, that is the genes
occupying the locus can be any one of the of several
alternative forms (alleles). As each allele determines a
distinct product (antigen), the HLA system is very
pleomorphic .For examples, at least 24 distinct allele have
been identified at HLA locus A and 50 at B.

12.
Glycoproteins, heterodimers (two chains)
Structure of HLA molecules of both classes enables antigen
binding and contact with T cell receptors. Extracellulary
located peptide binding cleft polymorphic (predominantly in
the cleft).
Nonpolymorphic part of the molecule contains binding sites
for the T cell molecules CD4 and CD8

13.
HLA class 1 and class 2 molecules

14.
HLA is extremely polymorphic. It is one of the most
polymorphic genetic system in man. Associated with each of
the loci is a set of alleles, each of which give rise to the
production of a unique antigenic specificity that is expressed
on cell surface and that can be detected by specific
antibodies or immunologically activated T cells.

15.
Limited diversity in HLA gene polymorphism due to breeding bottle-neck in
recent past leaves cheetahs exceptionally susceptible to viral infections. (6th
Ed. P. 206)

16.
Most individuals inherit a set of non recombined HLA alleles from each
parent.
These genes are codominantly expressed. Thus if the HLA types of family
member are determined, segregation of HLA types within the family can
be used to construct the HLA types from each chromosome
The set of HLA alleles found on one chromosome is called a haplotype.
Determination of haplotype is important for identification of HLA
identical siblings because sharing of antigen from different haplotypes is
common.

17.
 The primary target of immune responses to allogenic
transplants.
 Critical for response to antigenic stimuli.
 Implicated in genetic susceptibility to autoimmune disease.

18.
A. Serological ( microcytotoxicity test)
B. Mixed lymphocyte reaction
C. Molecular methods

19.
Potential donor’s and recepient’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.

20.
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 lymphocytes are obtained by
discontinuous density gradient centrifugation using Ficoll /
Tryosil or Ficoll / Sodium Metrizoate at a density of 1.077 at
19º - 22ºC.

21.
Microlymphocytotoxic test: 3 stages
1.Viable lymphocytes are incubated with HLA specific
antibodies. If the specific antigen is present on the cell the
antibody is bound.
2.Rabbit serum as a source of complement is added, incubate.
If antibody is bound to the HLA antigen on the cell surface it
activates the complement which damages the cell membrane
making it permeable to vital stains.
3.Results are visualised by adding dye usually a
fluorochrome eg Ethidium Bromide although both Trypan
Blue and Eosin Y have been used in the past

22.
If the reaction has taken 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 Haemoglobin to allow simultaneous visualisation of
both living and dead cells.

23.

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.

24.
Presence or absense of various MHC
allels determined by antibody
mediated cytotoxicity

25.
Because cells express
numerous HLA
antigens, they are tested
separately with a
battery of antibodies
specific for various
HLA-A antigens.
Here, donor 1 shares
HLA-A antigens
recognized by antisera
in wells 1 and 7 with the
recipient, whereas
donor 2 has none of
HLA-A antigens in
common with the
recipient.

26.
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% T and 10-15% B cells)
This can be achieved using a number of methods.

27.
 .
 In the past neuraminidase treated sheep red blood cell
rosetting and nylon wool have been used.
 Immunomagnetic bead separation is the current method of
choice.
 It utilises polystyrene microspheres with a magnetisable
core coated in monoclonal antibody for a HLA Class II b
chain monomorphic epitope. Positive selection

28.
It has been observed that lymphocytes from one donor, when
cultured with lymphocytes from an unrelated donor, are
stimulated to proliferate.
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 antigen
bearing cells (B cells, dendritic cells, langerhans cells, etc.).
This reactivity was termed mixed leukocyte reaction (MLR)
and has been used for studying the degree of
histocompatibility

29.
In this test, the test lymphocytes (responder cells)are mixed
with irradiated or mitomycin C treated leukocytes from the
recipient, containing B-lymphocytes and monocytes
(stimulator cells). The cells are cultured for 4 6 days.
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).
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) thymidine into the culture and monitoring its
incorporation into DNA.

30.
 Pros:
 Easily performed does
not require expensive
equipment.
 Takes around three
hours to perform.
 Low level resolution,
with good antisera
reliable results.
 Cons:
 Requires large volumes
of blood
 Requires viable
lymphocytes
 Difficult to find good
antisera for rarer
antigens in different
populations

31.
Allele A
Recipient cell sharing
Class II MHC of donors
No reaction
Irradiation
Allele A
Donor cells
Allele B
Recipient cells lacking
Class II MHC of donor
Activation & proliferation
of recipient cells
[ 3H] thymidine
Incorporation of radioactivity into
Cell Nuclear DNA

32.
DNA based procedures has increased the accuracy of HLA
typing and lead to the identification of serologically
undetected alleles and of many subtypes of serological
specificities.
DNA based methods to 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.

33.
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

34.
Extraction of genomic DNA
Amplification of the genes of interest and
 Detection of sequence polymorphism that
define the alleles.

35.
.
Genomic DNA extracted from nucleated cells.
The purity of the DNA extracted may be an important
factor for successful results

36.
 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 that avoid the coamplification of
pseudogene.

37.

Amplification of exon 2 (Approximately 270 bp) 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 900 bp) are
amplified by single pair of primers.

38.
Sequence specific primers (SSP) (group and alleles specific
primers).
Hybridization with Sequence specific oligonucleotide probes
(SSOP).
Sequence based typing (SSP)

39.
SSP is a rapid method of typing that uses sets of primer pairs
to amplify specific region of genomic DNA.
The efficiency of the amplification reaction is controlled by
the primers that amplify conserved sequences of a selected
gene.

40.
SSOP: this method involves selective amplification of target
followed by hybridization to a panel of oligonucleotide probes.
Specificity for a particular HLA locus was achieved by
selecting PCR primers specific for a sequence in the conserved
region of the second exon.

41.
Sequence based HLA typing involves determining the
nucleotide sequence of an amplified segment of an HLA gene.
SBT presents the advantages over the other procedure because
of the relatively fast (24-48)hrs, high level resolution.
More reliable and specific method

42.
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) and temperature
gradient gel electrophoresis (TGGE).

43.
DNA is isolated and amplified in multiple wells, each
containing specific primer complementary to particular HLA
allele
Contents of the well run by electrophoresis through agarose
gel
If DNA is amplified with the specific primer, appear as band
on gel

44.
Pros:
 Does not require viable
cells.
 Samples do not have to
arrive in the lab the day
they are taken.
 PCR SSOP good for batch
testing.
 Can be semi automated.
Cons:
 Requires good quality
DNA.
 Require a degree of
redundancy within the
primers used.
 Sequence of alleles must
be known.

45.
 In organ transplantation
 In transfusion therapy
 Disease association
 Disputed paternity
 In cancer prevention
 Anthropological studies

46.
•HLA typing is performed for kidney, bone marrow, pancreas
and heart transplants etc.
•HLA typing provides evidence of tissue compatibility.
• HLA antigens expressed on the surface of the lymphocytes
of the recipient are matched against those from various
donors.
• HLAA, HLA B and HLA DR: major transplantation
antigens.

47.
.

48.

Transplant success rate increases with the number of
identical HLA antigens.
Reduces the risk of complications after transplant,
especially graft-versus-host disease (GVHD). GVHD is a
potentially serious complication. GVHD occurs when the
immune cells, which are part of the donated marrow or
cord blood, attack your body.

49.
Repeated transfusions may lead to alloimmunization( i.e.
development of antibodies after exposure to non self
antigens)
Alloimmunization may lead to platelet refractoriness

50.
Platelet refractoriness can be avoided by prior:
ABO compatibility testing
HLA typing( screening for anti HLA antibodies)
Transfusion-Related Acute Lung Injury
donor HLA antibodies react against recipient antigens
Can also be avoided by HLA typing

51.
•An assosciation has been observed between HLA types and
certain autoimmune diseases like ankylosing spondilytis.
•Evidences show that these autoimmune disease have multiple
genetic and environmental factors.
•Thus HLA plays an important role not exclusive role in
disease causation.
•Thus HLA typing supports the diagnosis of a disease.

52.
Disease HLA Relative risk*
Ankylosing spondyloarthritis
B27 87.4
Uveitis B27 10
Goodpasture syndrome DR2 15.9
Multiple sclerosis DR2 4.8
Graves-Basedow disease DR3 3.7
Systemic lupus erythematodes DR3 5.8
Myasthenia gravis DR3 2.5
Pemphigus DR4 14.4
Rheumatoid arthritis DR4 4.2
Hashimoto thyreoiditis DR5 3.2

53.

To determine the parentage (paternity testing).
The HLA antigens of the mother, child, and alleged
father are compared.
When an HLA antigen of the child cannot be attributed
to the mother or the alleged father, then the latter is
excluded as the father of the child.

54.

HLA-mediated diseases are directly involved in the
promotion of cancer. Gluten-sensitive enteropathy is
associated with increased prevalence of enteropathy-
associated T-cell lymphoma
HLA molecules play a protective role, recognizing increases
in antigens that are not tolerated because of low levels in the
normal state. Abnormal cells might be targeted for apoptosis,
which is thought to mediate many cancers before diagnosis.

55.
 Immunology –by – Janis Kuby
 Essential immunology ---Ivan M. Roitt
 Clinical immunology ---STITES WELLS
 Text book of Microbiology – Paniker
 Pathology –Robins
 Lawlor DA, Zemmour J, Ennis PD, ET AL. Evolution of
Class I MHC genes and proteins; from natural selection to
thymic selection. Annu Rev Immunol 1990;8:23-63.
 So A. Genetic polymorphism and regulation of expression
of HLA region genes. In: Lecher A. HLA and Disease. San
Diego; Academic Press. Inc., 1994: 1-34.
 Daniel P. Stites,Abba T. Terr. Basic Human Immunology:
ISBN. 0838505430.
 Dyer P, Middleton D Histocompatibility testing .A practical
Approach. IRL Press,1993.
 Annia Ferrer et al., Genomic structure of the human MHC.
Biotechnologia Applicada. 2005,vol.22,No.2