Hla typing

1. HLA TYPING
B Montague

2. HLA = Human Leucocyte Antigen
system
•HLA forms part of the Major
Histocompatibiblity Complex (MHC)
•Found on the short arm of chromosome 6
•MHC antigens are integral to the normal
functioning of the immune response.
•Essential role of HLA antigens lies in the
control of self recognition and thus
defence against micro-organisms and
surveillance.

3. Interesting facts
•HLA comprises two classes: Class I
Class II
. Class I A,B,C most significant (other loci
eg E,F,G,H etc are not so important in
transplantation)
.Expressed on most nucleated cells
.Have soluble form in plasma
.Are adsorbed onto platelets (some
antigens more readily than others)

4. More interesting facts
•Erythrocytes will adsorb some Class I
antigens viz. Bg blood group system
(B7,A28, B57….)
•HLA B most polymorphic system and
studies have shown is most significant
followed by A and then C
•45Kd glycoprotein comprising three heavy
chain domains, non-covalently associated

5. •with beta-2-microglobulin (coded
chromosome 15) which plays an important
role in the structural support of the heavy
chains.
•Class I molecules are assembled within
the cell and ultimately sit on the cell
surface with a section inserted into the
lipid bilayer of the cell membrane and a
short cytoplasmic tail where they present
antigen in the form of peptide to cytotoxic
T (CD8+) cells

6. •HLA Class II five loci DR, DQ, DP, DM
and DO
•HLA DR, DQ, DP most significant
•Expressed on B lymphocytes, activated T
lymphocytes, macrophages, endothelial
cells ie immune competent cells.
•Comprise 2 chains encoded by HLA
genes, alpha and beta each with 2
domains.
•Hypervariable region is in the beta 1
domain

7. •HLA Class II present peptide in the cleft to
helper T (CD4+) cells. Thus Class II
presentation involves the helper-function
of setting up a general immune reaction
involving cytokine, cellular and humoral
defence.
•The role of Class II in initiating a general
immune response is why they only need to
be present on immunologically active
cells.

8. TYPING METHODS
•SEROLOGY used to be the ‘gold’
standard. Now being superceded by
molecular techniques as they become
more robust and time efficient
•CELLULAR rarely used now. Orginally
used for Class II typing
•MOLECULAR fast becoming the method
of choice. Many laboratories test of
choice.

9. SEROLOGY
•Complement Dependent Cytotoxicity
(CDC)
•Viable peripheral blood lymphocytes are
obtained by discontinous density gradient
centrifugation using Ficoll / Tryosil or Ficoll
/ Sodium Metrizoate at a density of 1.077
at 19º - 22ºC.
•Microlymphocytotoxic test: 3 stages

10. Microlymphocyototoxic test
•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.

11. Microlymphocyototoxic test 2
•3.Results are visualised by adding dye usually a
fluorochrome eg Ethidium Bromide although
both Trypan Blue and Eosin have been used in
the past.
•If the reaction has taken place the EB enters the
cell and binds to the DNA.
•For ease double staining is normally used. We
use a cocktail of Ethidium Bromide and Acridine
Orange, quenched using Bovine Haemoglobin to
allow simultaneous visualisation of both living
and dead cells.

12. Microlymphocytotoxicity test 3
•Test is left for 10 minutes and then read
using an inverted fluorescient 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.

13. Microlymphocytotoxicity test 4
•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.

14. Pros and cons
•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

15. Cellular typing
•Not / Rarely used by laboratories these
days.
•Requires panels of homozygous typing
cells.
•Cell culture method therefore takes a long
time. Labour intensive involves use of
radioisotopes.

16. Molecular
•All commonly used molecular methods require
good quality genomic DNA. There are numerous
methods for extraction of DNA from whole blood.
•There are ‘in house’ methods based on Miller et
al’s Salting Out which are cheap and easy but
labour intensive.
•There are also numreous commercial kits
available such as individual matrix capture
columns, beads and semi automated systems.
This however can increase the cost per
extraction from around 65p to £3.60p.

17. Molecular 2
•All methods rely on DNA extraction from
the nucleated cells following cell lysis and
protein digestion.

18. Molecular Methods
•The application of molecular techniques to HLA
typing began around 1987 when the Southern
Blot technique was used to identify restriction
fragment length polymorphisms (RFLP’s)
associated with known serological DR/DQ and
cellular Dw defined specifities.
•Around 1992 polymerase chain reaction (PCR)
methods were developed.
•Most methods currently used have a PCR
element within the technique.

19. Molecular Methods 2
•PCR
•Three steps per cycle– denaturation,
annealing and extension. Amplification is
exponential yielding 2 power n where n =
number of cycles.
•The introduction of the programmable
Thermal Cycler revolutionised the use of
PCR within the routine laboratory.

20. Molecular Methods 3
•PCT SSP (Sequence Specific Priming)
•Can be used for HLA Class I and II typing using
a panel of primer pairs either for low to medium
resolution whereby primers amplify groups of
alleles or high resolution whereby primer pairs
amplify specific alleles. Each PCR reaction takes
place in a separate tube therefore the number of
tubes depends on the level of resolution. Each
tube also contains a pair of primers for part of
the human growth hormone gene as an internal
control. These are at a much lower
concentration thus do not compete with specific
primers.

21. Molecular Methods 4
•Electrophoresis is used following amplification.
PCR product is run out on an agarose gel
containing ethidium bromide. Each product
moves according to its size and is compared to a
molecular weight marker.
•Interpretation: every tube should produce an
identical sized product as internal control and
either a specific band or not dependent on
whether the allele(s) is/are present or not.
•Results are visualised using 312nm UV
transillumination and recorded either by video
imaging or polaroid photograghy.

22. Molecular methods 5
•PCR SSOP ( Sequence Specific
Oligonucleotide Probes)
•‘Dot blot’ in house method usually
whereby one labels ones own probes with
Digoxigenin
•‘Reverse dot blot’ normally commercial
where specific oligonucleotide probes are
attached to a nylon membrane. Dynal and
Innotrans for example produce such kits.

23. Molecular Methods 6
•Amplification: DNA of interest is amplified by a single pair of
biotinylated primers which flank the whole of exon eg exon 2 of the
HLA DRB1 gene. PCR amplifies all the alleles in the exon.
•Hybridisation: PCR product is denatured and then added to a ‘well’
containing the nylon membrane with the bound probes and
incubated with hybridisation buffer . PCR product hybridises to
probes with complementary sequences.
•Excess product is washed away during a series of wash steps.
•Temperature is VERY important during these stages.
•Visualisation of results is achieved by incubating with a conjugate
and enzyme often streptavidin and horse radish peroxidase which
binds to the biotin of the PCR product and then adding a substrate.
Band with PCR product turn blue.
•Strips will have internal control bands to show the test has worked.
•Interpretation is usually achieved by entering the band pattern into a
computer programme.
•This is an excellent method for low resolution batch testing.
•Can be semi automated.

24. Pros and Cons
•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.

25. Molecular Methods 7
•Sequence Based Typing (SBT)
•DNA sequencing is the determination of the
sequence of a gene and thus is the highest
resolution possible. Sequence based typing
involves PCR amplification of the gene of
interest eg HLA DRB1 followed by determination
of the base sequence. The sequence is then
compared with a database of DRB1 gene
sequences to find comparable sequences and
assign alleles. This method also allows for
detection on new alleles.

26. Molecular Methods 8
•Other molecular methods:
•Reference Strand Conformational Analysis
(RSCA) Offers sequence level typing
without the need to sequence. Assigns
HLA type on the basis os accurate
measurement of conformation ie shape
dependent on DNA mobility in
polyacrylamide gel electrophoresis
(PAGE). Complex and difficult technique
not taken up by labs for routine use.

27. Molecular Methods 9
•Luminex technology – SSOP based. Just
beginning to be introduced into
laboratories for routine use on non urgent
samples.

28. Nomenclature
•In the 1950’s Dausset, Rose and van Rood
amongst others described leukoagglutinating
antibodies in the serum of patients who had
been pregnant or transfused and that patterns of
reaction were observed against random cell
panels.
•Numbers of investigators increased and it was
realised that collaboration was needed to
develop a standardised nomenclature and typing
methods.
•Out of this desire was born the first International
Workshop in Durham, North Carolina in 1964.
•Since then there have been 14 such workshops.

29. Nomenclature 2
•These workshops led to a logical and
consistent if somewhat complex
nomenclature.
•Within HLA A and B loci you will not find
the same number used to describe an
antigen eg HLA-A1 there is NOT an
HLA-B1 or HLA-B8 there is not an HLA-A8
and so on.
•In serology HLA C retains ‘w’ to
differentiate from the complement cascade

30. Nomenclature 3
•HLA-Aidentifies HLA A locus
•HLA-A1 serologically defined antigen
•HLA-A* asterisk denotes HLA alleles defined by
molecular methods
•HLA-A*01 2 digit resolution denotes a group of
alleles corresponds usually to serological group
– low resolution
•HLA-A*0101 4 digit resolution – sequence
variation between alleles results in amino acid
substitutions
•HLA-A010101 6 digit resolution – non coding
variation: sequence changes synonymous no
amino acid substitution

31. Nomenclature 4
•HLA-A01010101 8 digit resolution – sequence
variation occurs within the introns or 5’ / 3’
extremities of the gene
•HLA-A01010102N Null allele
•Alphabetical suffix
•NNull allele
•LLow level expression
•AAberrant expression
•CMolecule present in cytoplasm only
•SSecreted molecule present only as soluble
form