HLA & HLA antigen typing

methods

DR. LAKSHMAN PRAKASH S

1ST YEAR PG
MD IHBT
 OBJECTIVE
 Introduction
 Genomic organization of the HLA system
 Structure of HLA molecules
 Inheritance
 HLA nomenclature
 Expression of HLA
 Function of HLA
 Clinical laboratory testing for HLA
 Clinical relevance of HLA system
 1. INTRODUCTION

 Human leukocyte antigen (HLA) testing is a specialized branch of immunology.

 HLA is the human version of the Major Histocompatibility Complex (MHC)

 In 1958, Dausset, Snell, and Rappaport found HLA antigens in human

leucocytes for the first time.

 In the same year, van Rood et al. reported the production of antibodies against
leucocytes in the sera of multi-transfused individuals and multiparous women.
 INTRODUCTION

 Based on these discoveries, these antigenic structures were named as HLAs

because they were first identified on leucocytes. Later, it was observed that
these antigens were present not only on leucocytes but also on tissue cells.

 HLA contains loci of graft rejection, hence also termed as “transplant antigens”.

 The recognition of HLA antigens is more appropriately described as “Tissue

Typing” or “HLA Typing”
 2. GENOMIC ORGANIZATION OF THE HLA SYSTEM
 The HLA refers to a cluster of highly conserved polymorphic genes on the short arm of 6 th
chromosome that spans approximately 4000-kilo bases of deoxyribonucleic acid (DNA).
 HLA antigens are cell surface glycoproteins.
The HLA is classified into three regions based on their structure and function:

a) The class I region:

It is mostly telomeric.
CLASSICAL NON-CLASSICAL
HLA-A HLA-E
HLA-B HLA-F
HLA-C HLA-G
SEVERAL PSEUDO-GENES
b) The class II region:

In the centromeric to telomeric direction, the first is the HLA class II region
comprising α-and β-chains.

CLASSICAL NON-CLASSICAL
HLA-DR HLA-DM
HLA-DQ HLA-DO
HLA-DP
c) The class III region:
• It is sandwiched between the class II and class I region,
• Does not encode HLA molecules
• Contains genes for functionally unrelated genes such as
 Components of complement pathway (C2 and C4)
 Heat shock proteins (HSF)
 Tumor necrosis factor (TNF)
Common definitions in Human Leucocyte Antigen genetics :

TERM DEFINITION

Allele A unique sequence of an HLA gene defined by

molecular methods
Antigen Antibody-defined protein
Haplotype HLA genes inherited as a chromosomal unit
Genotype Molecularly defined HLA allele or sequence
Phenotype Serologically defined HLA protein or antigen
 3. STRUCTURE OF HLA MOLECULES

HLA CLASS I HLA CLASS II

Bond Non-covalent bond Non-covalent bond
Polypeptide • Polymorphic α-chain • Polymorphic α-chain (α1,α2)
chains (α1,α2,α3) • Polymorphic β-chain (β1,β2)
• Non-polymorphic β-chain
(β2 micro globin)
Encoded on chromosome 15

α-chain 44 kDa 24–32 kDa

β-chain 12-kDa 29–32 kDa
Binds to 8-10 amino acid peptides 13-25 amino acid peptides
 HLA CLASS I HLA CLASS II
Peptide presenting Between α1 and α2 Between α1 and β1
groove
Present to CD8+ cytotoxic T cells CD4+ T helper cells
 4. INHERITANCE
HLA HAPLOTYPE:
The entire set of A, B, C, DR, DQ, and DP genes located on one chromosome is
known as a haplotype.
LINKAGE DISEQUILIBRIUM:
 An important characteristic of HLA antigens is the existence of linkage
disequilibrium.
 Possible random combinations of antigens from different HLA loci on an HLA
haplotype are enormous.
 E.g., HLA-A1, B8, DR17 is the most common HLA haplotype among Caucasians,
with a frequency of 5%
 In North Indian population, the three most common haplotypes are
HLA-A33-B44-DR7
HLA-A33-B58-DR3
HLA-A2-B50-DR3
CROSS-REACTIVITY:
 Cross-reactivity is the phenomenon whereby one antibody reacts with several
different antigens, usually at one locus.
 Antibodies bind to specific sites on these molecules, and it would be expected that
many different antigens would share a site (epitope).
 Cross-Reactivity is the sharing of epitopes between antigens.
 The term CREG is often used to describe the “Cross Reacting Groups” of antigens.
 5. HLA NOMENCLATURE

At present, two systems are used:

 Immunologically defined nomenclature
 Sequence-defined allelic nomenclature
Immunologically defined nomenclature:
 Based on the identification of HLA antigens on the surface of leucocytes.
 The rule that follows in this are:

IDENTIFIES
HLA is separated by a hyphen from a capital letter The locus encoding distinct HLA class I (-A, -B, -C)
or class II (-DR, -DQ, -DP) antigens.

The letter is followed by a number Serologic family of alleles sharing epitopes

recognized by alloantibodies or alloreactive cytotoxic
T-cells

For e.g., HLA-DR3; where HLA stands for “Human Leucocyte Antigen”
DR: The name of the specific locus
3: The number refers to the actual antigen at the locus.
Sequence-defined allelic nomenclature:

 Based on the molecular identification of nucleotide sequences in genomic DNA.

HLA: prefix for an HLA gene

HLA-A: a particular HLA locus i.e. A
HLA-A*01: a group of alleles that encode the A01 antigen (Allele family)
HLA-A*01:01: a specific HLA allele
HLA-A*01:01:111: an allele that differs by a synonymous mutation from HLA-
A*01:01:110
HLA-A*01:01:111:01 an allele that contains a mutation outside the coding region
from HLA-A*01:01:111:02
HLA-A*01:01:111:01A an allele encoding a protein with an aberrant or doubtful
expression, where the mutation is found outside the coding
region
After the numerical designation, further letters are used as follows

(N) non-coding sequences (S) secreted, soluble but not

or null alleles a surface expression
(L) low expression (Q) questionable expression
(C) cytoplasmic expression (A) aberrant or doubtful
only expression
 6. EXPRESSION OF HLA

HLA CLASS I HLA CLASS II

Expressed on the surface of almost all Expressed only on B lymphocytes,
nucleated cells and platelets. antigen-presenting cells (monocytes,
macrophages, and dendritic cells), and
activated T lymphocytes.
 7. FUNCTION OF HLA

HLA CLASS I HLA CLASS II

Binds to 8–10 amino acid peptides 13–25 amino acid
peptides
Resulting from Proteasomal degradation Endosomal degradation of
of cytoplasmic proteins exogenous and
endogenous proteins
Presents to CD8+ cytotoxic T-cells CD4+ T helper cells
Function Alerting T-cells to virally Elicit immune responses
infected cells to organisms such as
pyogenic bacteria
 8. CLINICAL LABORATORY TESTING FOR HLA

HLA antigen typing techniques:

I. Serologic HLA typing technique
II. Molecular HLA typing techniques
 Serologic HLA typing technique
In the serological (lymphocytotoxicity) test, lymphocytes are used because of
their excellent expression of HLA antigens and ease of isolation.

METHOD:
Lymphocytes are added to sera

If the serum contains an antibody, that will bind to HLA antigen

When complement is added, it binds to positive cells only

Causes membrane damage

The damaged cells suffer sufficient membrane damage to uptake vital stains such
as eosin or fluorescent stains such as ethidium bromide

Microscopic identification of the stained cells indicates the presence of a specific

HLA antibody

Sera + eosin/ Read

Target complement fluorescent cell
Lymphocyte stain lysis
 Molecular HLA typing techniques
 The polymerase chain reaction (PCR) has been developed for investigating the
DNA nucleotide sequence.
Method:
Obtain double stranded DNA from the nuclei of an individual

Denature (by heat)

into
single-stranded DNA

Add oligonucleotide primer sequences

(Act as a starting point for reconstructing double-stranded DNA at that site)
 DNA polymerase and deoxy-ribonucleotide triphosphates are then added

To become double-stranded DNA

From one copy of DNA, it is thus possible to make two. Those two copies can then,
in turn, be denatured, re-associate with primers and produce four copies.
This cycle can then be repeated until there are sufficient copies of the selected DNA
to isolate on a gel and then sequence or type.

There are several PCR based methods in use. They are:

A. Sequence-specific priming (SSP) typing
B. Sequence-specific oligonucleotide (SSO) typing
C. Sequence-based typing (SBT)
 A. Sequence-specific priming (SSP) typing
 Genomic DNA (gDNA) is isolated from the sample.
 PCR amplified DNA using Sequence-specific primers (SSP).
 Primers are designed with specificity dependent nucleotides on the 3’ end.
 PCR product visualized directly on gel electrophoresis.
INTERPRETATION:
INTERPRETATION:
ADVANTAGES:
 Cheaper than other HLA techniques
 Easily performed
 Does not require expensive equipment
 Simple and easy to interpret

DISADVANTAGES:
 Subjective in interpretation
 Labour-intensive
 Time-consuming
 B. Sequence-specific oligonucleotide (SSO) typing
Hybridisation of PCR amplified genomic DNA using labelled sequence-specific
oligonucleotide probes.
Method:
DNA extraction PCR amplification Hybridization

Amplified DNA is immobilized on a solid support (nylon membrane)

Hybridized with a battery of sequence specific oligonucleotide probes (SSOP)

(direct hybridization).

Fluorochromes are linked with the probes to allow their detection by the
chemiluminescence technique.
Alternatively, SSO probes can be immobilized on a solid support, for example, colour-
coded microspheres, and hybridized with labelled PCR product (reverse hybridization).
The higher the number of probes better is the resolution level.
ADVANTAGE:
 Suitable for a higher number of samples.

DISADVANTAGES:
 May lead to false-positive results.
 Hybridization temperature is critical, and could lead to false-negative
hybridization.
 Lacks accuracy in precise allelic typing.
 C. Sequence-based typing (SBT)
 Based on “chain termination method” by sanger sequencing that is performed by
the random incorporation of four types of di-deoxy-nucleotide triphosphate
(ddNTP) labeled with four different fluorescent dyes.
(adenine, ddATP; cytosine, ddCTP; guanine, ddGTP; and thymidine, ddTTP)
 The chain-terminated DNA fragments are separated by size via gel
electrophoresis.
 DNA samples are loaded into one end of a gel matrix, and an electric current is
applied.
 The labeled di-deoxy-nucleotides attached to the DNA fragments will be arranged
from smallest to largest i.e., from bottom to top
 DNA polymerase only synthesizes DNA in the 5’–3’ direction
 Reading the gel bands from the smallest to largest DNA fragment, the 5’–3’
sequence of the original DNA strand can be determined.
 A computer reads each band of the gel, in order, using fluorescence to identify
each terminal ddNTP.
ADVANTAGES:
 More reliable and specific than other methods
 Used for perfect matching
 New alleles can be detected quite easily

DISADVANTAGE:
 High-end equipment is needed
 D. Next Generation Sequencing (NGS)
Massively parallel sequencing technology
 Ultra-high throughput
 Scalability
 Speed

The NGS method combines the techniques

developed in Sanger sequencing to process
millions of reactions in parallel,
resulting in very high speed and throughput
at a reduced cost.
Steps in NGS:

NGS involves
 DNA fragmentation,
 Library preparation,
 Massive parallel sequencing,
 Bioinformatics analysis, and
 Variant/mutation annotation
and interpretation
Method:
The sequencing library is prepared by fragmenting the amplified genomic DNA and
ligating specialized adapters with indices to both fragment ends

The sequencing library is loaded into a flow cell and the fragments are hybridized to
the flow cell surface.

Each bound fragment is amplified into a clonal cluster.

Sequencing reagents, including fluorescent-dye labeled nucleotides, are added and

the first nucleotide is incorporated.

The flow cell is imaged and the emitted light from each fragment cluster is recorded.
 The emission wavelength and intensity are used to identify the incorporated
nucleotide.

This cycle is repeated several times.

Reads are aligned to a reference sequence from the IPD-IMGT/ HLA Database.

After alignment, differences between the reference genome and the newly
sequenced reads can be identified.
ADVANTAGES:
 Higher sequencing depth enables higher sensitivity
 Higher mutation resolution
 More data produced with the same amount of input DNA
 Higher sample throughput

DISADVANTAGE:
 Less cost-effective for sequencing low numbers of targets (1–20 targets)
 Time-consuming for sequencing low numbers of targets (1–20 targets)
 HLA antibody screening assays
 Anti-HLA antibodies are generally defined as Panel Reactive Antibodies
(PRA). They are one of the immunological factors affecting graft survival.

 These antibodies play a critical role in solid organ transplantation and also
in haematopoietic stem cell transplantation (HSCT).

 The humoral response directed against foreign HLA molecules can be

encountered during pregnancies, blood transfusions, and/or previous
transplantations.
Lymphocytotoxicity assay:
• Preformed HLA antibodies can be detected by testing the patient’s serum
against a panel of lymphocytes with known HLA types.

Panel reactive antibody screening testing:

• Based on enzyme-linked immunosorbent assay (ELISA) and fluorescence
based flow cytometry or single antigen bead (SAB) technologies.

Compatibility testing assays (between donor and recipient):

• Tissue typing of the recipient and donor determines their HLA match.
• In addition to the determination of the HLA compatibility, screening for anti-HLA
antibodies and cross-matching are performed to assess the risk for rejection.
i. Lymphocytotoxicity crossmatch or complement-dependent cytotoxicity
crossmatch (CDCXM):
• CDC is a robust method for detecting and defining complement-fixing IgG
(IgG1 and IgG3) and IgM antibodies directed against HLA and/or non-HLA
targets (including autologous reactive antibodies).

ii. Flow cytometry crossmatch (FCXM):

• FC-XM test is an antibody-binding assay
• FCXM is a more sensitive method compared to the CDCXM test.
• Thus, donor-specific antibodies (DSAs) that CDCXM cannot detect can be
detected by the FCXM technique.
iii. Donor specific antibody crossmatch (DSA-XM):
• DSA-XM tests consist of beads coated with antihuman Class I and Class II
antibodies To isolate HLA molecules from a specific donor and to be
used in donor specific crossmatches.

iv. Virtual crossmatch:

• The strategy of virtual crossmatching, or selecting donor-recipient pairs based
upon knowledge of donor HLA type and recipient allo-antibody profile.
 9. Clinical relevance of HLA system
HLA and Transfusion:
• The HLA system can cause adverse immunologic effects in transfusion
therapy.
• These effects are mediated by HLA antibodies developed against the donor
leucocytes contained in the cellular blood components.
• These HLA antibodies induced from previous allo-sensitization (pregnancy,
transfusion, or transplant) can cause :
i. Platelet transfusion immune refractoriness
ii. Febrile non-haemolytic transfusion reaction
iii. Transfusion associated graft-versus host disease (TA-GvHD)
iv. Transfusion-related acute lung injury (TRALI)
v. Neonatal alloimmune thrombocytopenia (NAIT)
HLA and Disease Association:

Presence of the HLA Possible Diseases

antigen in the patient
B27 Ankylosing spondylitis / Reiter’s syndrome / Anterior uveitis
B5 Behcet’s disease
B8 Celiac disease / Myasthenia gravis / Sjogren’s syndrome /
Addison’s disease
DR2 Good pasture’s syndrome / Multiple sclerosis
DR3 Type 1 diabetes mellitus / Sjogren’s syndrome /
Addison’s disease
DR4 Rheumatoid arthritis / Pemphigus
DR5 Scleroderma / Hashimoto’s thyroiditis
HLA and Transplant:

 Renal transplantation
 Liver transplantation
 Bone marrow transplantation (BMT)/ Haematopoietic stem cell transplantation
(HSCT)
 Heart, lung and corneal transplantation

HLA and Paternity:

HLA typing of parents and the disputed child may be helpful to confirm the real
parents.
Thank You
References:
 DGHS
 Harmening
 AABB
 The Human Leukocyte Antigen System: Nomenclature and DNA-Based
Typing for Transplantation | By Andrés Jaramillo and Katrin Hacke
 Human Leucocyte Antigen (HLA), Major Histocompatibility complex (MHC) |
Elementary Immunology by Prof. Dr. Riaz A. Bhutta
 Marrow Notes