HUMAN LEUCOCYTIC ANTIGEN TYPING

(HLA-TYPING)

MUHAMMAD TARIQ
Medical Technologist, Dept of Blood Bank (SBT)
HMC Peshawar
M Sc. (Medical Technology). PhD. Fellow
(Genetics)
 HUMAN LEUCOCYTIC ANTIGEN (HLA)

• HLA stands for human leukocyte antigen, a group

of proteins present on the surface of all cells on the
human body.

• The human leukocyte antigen (HLA) system is the

name of the major histocompatibility
complex(MHC) in humans.

• The super locus contains a large number of genes

 HUMAN LEUCOCYTIC ANTIGEN (HLA)

• This group of genes resides on short arm of

chromosome 6, and encodes cell-surface antigen-
presenting proteins and has many other functions.

• These protein are ALLOANTIGENS ( they differ

among members of the same species)
 Hla

• Three of the
genes which
includes HLA –
A,HLA-B and
HLA-C code for
class I MHC
proteins.

• Several HLA-D
loci determine
the class II MHC
proteins i.e
 HUMAN LEUCOCYTIC ANTIGEN (HLA)

• MHC I molecules are found on almost all

nucleated cells of the body,
• MHC II molecules are found only on antigen-
presenting cells. e.g macrophages, dendritic
cells and B cells,
 HLA
• HLAs corresponding to MHC class I (A, B, and C)
present peptides from inside the cell (including
viral peptides if present).
• These peptides are produced from digested
proteins that are broken down in the proteasomes.
• In general, these particular peptides are small
polymers, about 9 amino acids in length.
• Foreign antigens attract killer T-cells (also called
CD8 positive- or cytotoxic T-cells) that destroy
 HLA

• HLAs corresponding to MHC class II (DP, DM,

DOA, DOB, DQ, and DR) present antigens from
outside of the cell to T-lymphocytes.
• These particular antigens stimulate the
multiplication of T-helper cells, which in turn
stimulate antibody-producing B-cells to
produce antibodies to that specific antigen. Self-
antigens are suppressed by suppressor T-cells.
 Biological importance of Hla

• They are important in disease defense.

• They are the major cause of organ transplant
rejections.
• They may protect against or fail to protect (if down
regulated by an infection) against cancers.
• Mutations in HLA may be linked to autoimmune
disease (examples: type I diabetes, coeliac disease)
 HUMAN LEUCOCYTIC ANTIGEN TYPING (HLA Typing)
• Prior to transplantation surgery ,laboratory tests
called HLA Typing or Tissue Typing are
performed to determine the match between Donor
and recipient.
• Two methods are most commonly performed in
lab.
• 1) DNA Based HLA Typing .
• 2) Serologic Based HLA Typing.
• Other than these two methods Mixed Lymphocyte
culture (MLC) can be done.
 DNA BASED HLA TYPING
• DNA Extraction
• DNA quantification
• PCR with Sequence Specific Primers (SSP)
• Detection of amplify product with Agarose
Gel Electrophoresis
• Analysis with Software (SSPal Typing Analysis
Software)
Performing PCR

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 Loci reactions per the tray
Contamination No. of Reactions
well
A (24) A Loci

(48) B Loci

(24) DRB1 Loci

DRB1

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 Tray Loading Process

TBG Tray
1. Master Buffer

2. Taq DNA
polymerase

3. Sample DNA

Membrane

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Step 2: Add 10 µl of Master buffer with Taq to well 1.
Step 3: Load 2 µl of H2O into well 1.

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Step 7: Make sure the solution is mixed with primer pellets by tapping or
centrifugation.

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Step 8: Place the tray onto the
thermo cycler and place the
TBG pressure pad on top of
the tray.

Note: Tray holder or retainer must be

in place. This will prevent
uneven heating and
evaporation around the
edges.

PCR program will run for approx. 90

minutes.

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 Maxi Gel System Gel Tank

Add Comb image Add System image

Bevel Edge

Gel Tray Casting Tray

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Gel Loading Scheme (Well 1~32)
Well Gel Band
Numbering Numbering

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Gel Loading Scheme (Sample 2)
Well Gel Band
Numbering Numbering

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Load Sample 1 to the Left Side of Gel

Load 12 µl from each well into each slot in the following order

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 DNA Marker Ladder

The middle
column is for the
DNA Marker

DNA Marker
should cover
the range
50bp~500bp

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 Gel Photo Capture
CCD Camera
UV Box
C1 mode:
Automatic focusing

Electrophoresis
Agarose Gel Capture

- Turn on LCD monitor power switch

- Turn on camera power switch
- Turn on UV Box power switch
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Actual Gel Image

Add Gel Image

Gel on a UV transilluminator

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 Data Analysis
The DNA fragments amplified by allele or group specific primer
pairs are designed to have a size in the range between 70-
275 bp. Positive Positive Negative
Top Well
- The internal control fragment is 600 bp.

- Refer to the worksheet for the exact size of the

specifically amplified DNA fragment in each reaction.

- Positive Rxn = Presence of the specific band. Primer

Dimer
- Negative Rxn = Absence of the specific band. Next Well

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 Gel Interpretation
Positive Positive Negative Non-
Reaction Reaction Reaction Amplifications

Well

None
Control band None

None None
Specific band

Primer dimer

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SSPal Typing Analysis Software

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