You are on page 1of 19

HLA Typing Mcthods

Submitted By:
Prajjalendra Barooah
IMT/07/8040
Section S
AIB

INTRODUCTION

The human leukocyte antigen (HLA) system is the name oI the major
histocompatibility complex (MHC) in humans. The super locus contains a large
number oI genes related to immune system Iunction in humans. This group oI
genes resides on chromosome 6, and encodes cell-surIace antigen-presenting
proteins and many other genes. The HLA genes are the human versions oI the
MHC genes that are Iound in most vertebrates.(1) The proteins encoded by certain
genes are also known as antigens, as a result oI their historic discovery as Iactors in
organ transplants. The major HLA antigens are essential elements Ior immune
Iunction.



Different classes have different functions:
HLAs corresponding to MHC class I (A, B, and C) present peptides Irom inside
the cell. (2) These peptides are produced Irom digested proteins that are broken
down in the proteasomes. In general, the peptides are small polymers, about 9
amino acids in length. Foreign antigens attract killer T-cells that destroy cells.
HLAs corresponding to MHC class II (DP,DM, DOA,DOB,DQ, and DR) present
antigens Irom outside oI the cell to T-lymphocytes. These particular antigens
stimulate the multiplication oI T-helper cells, which in turn stimulate antibody-
producing B-cells to produce antibodies to that speciIic antigen. SelI-antigens are
suppressed by suppressor T-cells.
HLAs corresponding to MHC class III encode components oI the complement
system.
HLAs have other roles. They are important in disease deIense. They may be the
cause oI organ transplant rejections. They may protect against or Iail to protect (iI
down regulated by an inIection) against cancers. They may mediate autoimmune
disease. Also, in reproduction, HLA may be related to the individual smell oI
people and may be involved in mate selection.
Aside Irom the genes encoding the 6 major antigens, there are a large number oI
other genes, many involved in immune Iunction, located on the HLA complex.
Diversity oI HLAs in the human population is one aspect oI disease deIense, and,
as a result, the chance oI two unrelated individuals with identical HLA molecules
on all loci is very low.




Role In Graft Rejection:

The HLA molecules control the immune response through recognition oI "selI"
and "non-selI¨ and the main Iunction oI the HLA molecules is presenting the
antigen to the T Lymphocytes and initiating the speciIic immune response. (5)

O HLA system constitutes an immunological barrier which must be avoided or
otherwise overcome in clinical transplantation.

O For Haemopoietic Stem Cell (HSC) transplants, the degree oI HLA
matching is critical in determining the probability oI GraIt-versus-Host
disease (GvHD).

O In an attempt to minimise these alloresponses, the HLA class I and class II
types oI the donor and recipient are matched as closely as possible.

O However, because oI extensive polymorphism, an HLA identical donor is
only rarely available.

O Most transplant recipients thereIore receive immunosuppressive drugs to
prevent or stop detrimental alloresponses, but this non-speciIic approach
also compromises beneIicial immune responses to inIection.









A Typing Methods

Traditionally, HLA antigens have been deIined using serological techniques. These
techniques rely on obtaining viable lymphocyte preparations (Ior HLA Class II
typing, B lymphocytes are needed) and the availability oI suitable antisera to
recognize the HLA antigens. The HLA loci, by virtue oI their extreme
polymorphism ensure that Iew individuals are identical and thus the population at
large is well equipped to deal with attacks. Because some HLA antigens are
recognized on all body tissues (rather than just blood cells), the identiIication oI
HLA antigens is described as 'Tissue typing¨.

During the last Iew years, DNA-based typing techniques have begun to replace the
serological techniques in clinical applications. The DNA methods were initially
applied to Class II typing, but more recently they have been used to determine
Class I alleles. While serology perIormed adequately in typing Iamily members, it
proved unsatisIactory in typing unrelated donors Ior bone marrow transplantation,
once the extent oI polymorphism was known within 'serologically identical¨
speciIicities. DNA typing also proved invaluable when serological typing was
diIIicult (poor cell viability or expression) and in conIirming or reIuting
phenotypic homozygosity
.
Although DNA techniques were introduced to many Clinical Tissue Typing
Laboratories with their involvement in the 10th International Histocompatibility
Working Group (IHWG) in 1987 |4|, studies in a limited number oI research
laboratories had preceded the Workshop by several years.









Serological Techniques

These techniques rely on obtaining viable lymphocyte preparations (Ior HLA class
II typing, B lymphocytes are needed) and the availability oI suitable antisera to
recognise the HLA antigens. The advent oI magnetic beads, coated with antibody
used Ior isolation oI B lymphocytes, made class II typing easier to accomplish.
Reliable antisera was not available commercially and laboratories needed to screen
to Iind their own reagents and exchange these with other such-minded laboratories.
This meant a diIIerence in the quality oI reagents between laboratories and led to
some laboratories producing more accurate results than others. In addition these
reagents could not be replenished. Whilst serology perIormed adequately in typing
Iamily members, it proved unsatisIactory in typing unrelated donors Ior bone
marrow transplantation, once the extent oI polymorphism was known within
'serological identical¨ speciIicities.

Serological typing was also diIIicult in those cases oI poor cell viability or poor
expression and in conIirming or reIuting phenotypic homozygosity; by
implementing a DNA technique Ior HLA-C alleles homozygosity role was reduced
in stem cell donor registry Irom 50° by serology to 21°.

The last twenty years has seen an exponential growth in the application oI DNA
technology to the Iield oI Histocompatibility and Immunogentics (H&I). Initially
this was conIined to a Iew research laboratories. However, development and
application oI several diIIerent DNA methods by many laboratories has led to the
situation whereby nearly every H&I laboratory perIorms some DNA typing Ior the
detection oI HLA alleles.


Molecular Methods of A Typing

The potential oI DNA-based HLA typing methods was realized over a decade ago
when a high degree oI correlation was observed between major histocompatibi!ity
complex (MHC) HLA class II phenotypes and genomic DNA restriction Iragment
length polymorphism (RFLP). ReIinement oI the RFLP tests to encompass HLA-
DR/Dw, DQ and DP typing, and gradual clinical evaluation culminating in the 10
th

International Histocompatibility Workshop, led to the widespread adoption oI
RFLP by 1988. A Iurther Iive years elapsed beIore the clinical relevance oI RFLP
analysis was unequivocally demonstrated on a grand scale by G. Opelz et al. in the
Collaborative Transplant Study I. This study showed the statistically signiIicant
impact oI RFLP-deIined HLA-DR matching on graIt survival in over 3000
cadaveric renal transplants Irom nearly 100 transplant centres, and re-emphasized
the Iundamental importance oI DNA-based HLA typing methods in allogeneic
transplantation.


Restriction Fragment ength Polymorphism (RFP)

Complexities oI binding patterns and inter-locus cross hybridisation oI probes
drew attention to the drawbacks oI using Iull length HLA class II cDNA probes. In
order to overcome these problems some investigators used short or exonspeciIic
probes. Even at this early stage oI development, there were indications that RFLP
was more accurate than serology. The lack oI locusspeciIic probes limited the
characterization oI class I RFLP, as cloned class I gene probes cross-hybridised
with all members oI the class I Iamily. Some HLA-A and -B probes were
constructed and RFLPs were deIined which correlated with serologically deIined
HLA-A, -B and -C alleles.

In 1987, two reports using Taq I enzyme showed how short probes Ior DRµ, DQµ
and DQu could be applied sequentially, aIter dehybridization, to a single
membrane. The recognition site oI Taq I includes the nucleotide dimer CpG and
restriction sites containing this dimer show a higher Irequency oI polymorphism in
human DNA than other restriction sites. Not only was there an excellent
correlation between RFLP and serologically determined antigens, but
heterogeneity was proven in several DR and DQ speciIicities, especially HLA-
DR6.

One oI the novel ideas was the use oI 19 base pair oligonucleotides as probes based
on sequence inIormation. As the probes also hybridised to other genomic
sequences, restriction enzyme digestion and gel electrophoresis were required to
separate the target sequence Irom the bulk oI the DNA. In addition, due to the
small number oI available copies oI the relevant DNA this approach lacked
sensitivity. A reIinement oI this method was to use the oligonucleotides to probe
total RNA, as non-speciIic binding was not Iound on Northern blots.

The RFLP methods also had their disadvantages. They did not directly identiIy the
polymorphic coding sequences within the second exon oI DR, DQ and DQa, but
relied on polymorphic restriction sites generally located outside these exons. In
addition, they required the use oI DR-DQ associations to discriminate between
certain DR alleles that had identical DR RFLP patterns. Thus, care was needed to
apply the system to non-Caucasian populations. The method was cumbersome and
could take up to 16 days to produce results Ior only 24 samples. A nonradioactive
RFLP method was describe using digoxigenin and chemiluminescence, but by this
time Iundamentally diIIerent techniques were being developed.

Eventually, RFLPs were replaced, but not beIore the results Irom their use had
stimulated the development oI better methods Ior DNA typing.


Polymerase Chain Reaction (PCR)

PCR had a revolutionary impact on molecular biology research in general and
inIluenced multiple clinical applications.

Emergence oI nucleotide sequence data Ior the alleles oI HLA genes permitted the
rapid development oI many PCR-based techniques and reagents. Conversely, the
PCR technique greatly reduced the eIIort required in subsequent sequencing oI
new alleles. PCR-based methods may be broadly classiIied into three categories:

(i) which generate a product containing internally located polymorphisms which
can be identiIied by a second technique, (e.g. PCR-sequence speciIic
oligonucleotide (SSO) probing, PCR-RFLP, PCR Iollowed by sequencing)

(ii) in which the polymorphism is identiIied directly as part oI the PCR process,
although there are post-ampliIication steps, e.g. PCR-sequence speciIic primer
(SSP)

(iii) conIor-mational analysis in which diIIerent mutations generate speciIic
conIormational changes in PCR products. The latter are identiIied by
electrophoretic analysis e.g. heteroduplex analysis.

The two main methods most Irequently adopted to clinical histocompatibility have
been SSO and SSP, although at regular intervals a novel method or a novel
variation oI an existing method are reported.


Sequence Specific Oligonucleotides (SSO)

The major diIIerences between the alternative methods Ior SSO typing are the
length and sequence oI oligonucleotide probes, and the reporter molecule and its
detection.

Initially
32
P-labelled allele-speciIic oligonucleotides were hybridised to an
ampliIied conserved region oI exon 2 oI the HLA-DQu gene, but soon aIter biotin
was used as a label. The PCR-SSO method was quickly applied to other loci; DPµ,
DQµ and DRµ with various procedures using
32
P, biotin or horseradish peroxidase-
labeled probes. Methods in the clinical laboratory have tended to use either a
substrate in a coloration development system or a substrate which generates a
chemiluminescent signal.

The SSO method can be customised Ior each application. For example,
approximation oI HLA-DR serological speciIicities requires detection oI shared
polymorphic sequences which encode the epitopes detected by antibodies. These
shared sequences identiIy Iamilies oI alleles that belong to the same serologically-
deIined speciIicity groups. This level oI typing is oIten reIerred to as 'low
resolution¨ or 'generic¨ SSO. Alternatively, 'high resolution¨ SSO typing can
distinguish all known alleles. High resolution SSO usually requires selective
ampliIication oI a group oI related alleles. For example, all HLA-DRB1*04 alleles
are speciIically ampliIied with selected PCR primers and then the DNA is
hybridized with a panel oI probes which distinguish each HLADRB1* 04 allele.

Today, most laboratories prepare one membrane Ior each probe in the assay. This
procedure is Iacilitated using a 96 well maniIold. The use oI automation not only
eliminates the reuse oI membranes aIter dehybridization, but it also minimizes
sample to sample variation in loading and provides a relatively large surIace area
to aid in evaluation oI hybridization dots oI varying intensity. Other laboratories
have used a robotic work station Ior both this and the ampliIication aspect oI the
technique.

Many laboratories reduce the number oI diIIerent wash temperatures by the
addition oI tetramethylammonium chloride (TMAC), which reduces the eIIect oI
GC content on the stability oI the hybrids and, providing the probes are oI the same
length, enables membranes to be washed at the same temperature. Some
laboratories avoid the use oI TMAC due to its toxic properties.

The SSO technique has proved very reliable, robust and accurate. Good
ampliIication always gives a clean and clear cut SSOP hybridisation while almost
all the problematic typing results encountered are due to poor ampliIication.

DNA typing techniques were initially applied to class II genes rather than class I
Ior several reasons. The requirement Ior replacing class II serology was thought to
be more urgent as serological typing Ior class II antigens was diIIicult (HLA-DR
and -DQ) or impossible (HLA-DP). Class II was thought to be more important Ior
transplantation and disease association. Furthermore DNA typing Ior class I was
destined to be more complex because sequence polymorphisms in class I genes are
located in two exons. However, once the beneIits oI developing and applying the
methods to class II loci were apparent, attention turned to class I genes. Initially
the methods were used to deIine alleles in selected speciIicity groups such as HLA-
A2, -A68, -A6962 or HLA-A2, -A3, -B44, alleles oI a single speciIicity e.g.
HLAB27, or to identiIy speciIicities diIIicult to detect by serology e.g. HLA-Cw6.
Two methods Ior the determination oI a complete locus system proved to be the
Ioundation Ior development oI SSO class I methods. These methods were later
improved by better resolution and methods oI probe labelling.


PCR-RFP

Initially this method used the availability oI sites in the nucleotide sequences to
employ restriction endonucleases which recognized allelic variations, to digest
PCR ampliIied HLA genes (HLA-DR, -DQ, - DP).However, small bands located
close to each other on the polyacrylamide gels sometimes obscure precise analysis
and some heterozygotes cannot be discriminated.

These problems have been overcome Ior HLA-DR by a modiIied PCR-RFLP
method using inIormative restriction enzymes, which have a single recognition site
present in some alleles but not in others and using group speciIic primers to avoid
cross hybridization with other genes. This method was also applied to HLA-DQB1,
-DQA1 and -DPB1 genes and simultaneous digestion oI ampliIied DNA with two
or more enzymes has been applied.One oI the Iirst indications that HLA-DP
matching may be important in bone marrow transplantation was reported using the
PCR-RFLP method.A recent innovation was the use oI consecutive rounds oI
PCRRFLP. AIter the Iirst digestion oI the PCR product, the cleaved Iragment was
extracted Irom the gel and used as template Ior a second PCR-RFLP. For the
'allele walking¨ to proceed, a previous 'cutting¨ was required.


Sequence Specific Primers (SSP)

The principal oI this method is that a completely matched primer will be more
eIIiciently used in the PCR reaction than a primer with one or several mismatches.
SpeciIicity is determined by the use oI sequence speciIic primers in which a 3`
singlebase mismatch inhibits the priming oI non-speciIic reactions. Because Taq
polymerase lacks 3` to 5` exonuclease activity, even iI primer pairs do anneal non-
speciIically, they will not ampliIy eIIiciently. Thus only the desired allele or alleles
will be ampliIied and the ampliIied product can then be detected by agarose gel
electrophoresis.

Other investigators have used multiplex PCR i.e.having several primer pairs in the
same reaction. Sizing oI the PCR product is necessary Ior interpretation,
necessitating that the gel be run longer to separate the PCR Iragments. The SSP
method is ideal Ior typing individual samples, but is costly and requires high
capacity thermal cyclers Ior larger numbers oI samples. This was reduced by
instigating a two-stage technique low resolution Iollowed by high resolution
according to the Iirst result. As the method takes less than Iive hours, it can be
applied to cadaveric transplantation.

Much oI the earlier work in class I was perIormed by Browning and colleagues
who developed a low resolution typing system Ior HLA-A, and quickly Iollowed
with a more extensive system to cover all HLA-A alleles. The same group
designed a low resolution primer panel Ior HLA-B. Others used the SSP system as
a supplement to serology by only typing Ior alleles oI certain serological
speciIicities, or only determining HLAB*27. Bunce et al. developed a SSP system
Ior HLA-C99 and a high resolution system Ior HLA-B.

The products oI many alleles oI the HLA-C locus are diIIicult to detect by
serological methods due to the low expression oI HLA-C molecules at the cell
surIace and to the corresponding lack oI suitable antisera. Thus having a DNA
typing system Ior HLAC proved very useIul. In addition, systems were developed
Ior other loci which complemented each other, so that complete HLA-A, -B. -C, -
DRB1, -DRB3, -DRB4, -DRB5 and -DQB1 typing could be perIormed
simultaneously.

This method, termed 'phototyping¨, has a resolution equivalent to high quality
serology and could be completed within three hours.



Sequence Specific Priming, Exonuclease Released
Fluorescence (SSPERF)

The SSP method suIIers Irom the disadvantage that the end-step oI gel
electrophoresis is not suitable Ior large numbers oI samples or Ior automation. A
novel method has been reported which removes the electrophoresis and combines
high throughput with speed and high resolution. The method uses Iluorogenic
probes, each oI which has a reporter and a quencher dye. When the probe is intact,
the proximity oI the two dyes results in the suppression oI the reporter
Iluorescence. During PCR-SSP, iI the target oI interest is present, the probe
speciIically anneals between the Iorward and reverse primer site. The nucleolytic
activity oI the Taq polymerase cleaves the probe, resulting in an increase in
Iluorescence. Taq polymerase does not cleave the Iree probe, the enzyme requires
sequence complementarity between the probe and template Ior cleavage to occur.
AIter cleavage, the shortened probe dissociates Irom the target and polymerization
oI the strand continues. This process occurs in every cycle and allows a direct
detection oI the PCR product.


eteroduplexes

During the primer-annealing stage oI each cycle oI the PCR, a proportion oI
coding strands oI each DRB locus allele may hybridise to the noncoding strands oI
a diIIerent DRB locus allele and vice versa. This double stranded DNA will thus be
mismatched in some regions (heteroduplexes) leading to alteration in the
conIormation oI the DNA molecule. This conIormation varies Ior each DR
haplotype and can be detected by the modiIied migration in nondenaturing
polyacrylamide gels, as the heteroduplex will move more slowly than the
homoduplexes (complementary strands). A single mismatch oI nucleotide can
cause a marked electrophoretic retardation and thus even subtypes involving a
single substitution can be detected.

Additional bands are Iormed in heterozygotes which are not present in either oI the
patterns oI the individual alleles and are caused by heteroduplex Iormation in trans
i.e. between PCR products Irom two diIIerent haplotypes. This phenomenon was
used in the DNA crossmatch test whereby DNA Irom two diIIerent individuals are
co-ampliIied in the PCR. II the individuals are identical Ior HLA-DR then the
banding pattern oI the mixture will be the same as the banding pattern oI each oI
the individuals, but iI the individuals are not identical the mixture will contain
extra bands.


Sequence Based Typing (SBT)

One oI the drawbacks oI SSO or SSP is that,although they are capable oI detecting
a single base diIIerence in DNA sequence between two alleles, they are not likely
to detect a new undeIined allele, unless the variation happens to be at the speciIic
site detected by the probe or the primer. Methods based on sequencing have come
to the Iore. The sequencing technology advanced with the introduction oI
dyelabelled primers and Iluorescent automated Sequencing. Group-speciIic
ampliIication is perIormed in order to limit the number oI allele sequences in any
sequencing template, otherwise DNA Irom both haplotypes would be present. This,
in turn, simpliIies the soItware-based allele assignments suitable computer
soItware was one oI the greatest problems with this technique. Initially in typing
Ior HLA-DR alleles PCR ampliIication used one set oI primers with common
sequence to all HLA-DRB1 alleles. However this meant the simultaneous
ampliIication oI alleles oI other HLA-DRB loci (e.g. DRB3, DRB4) and led to
diIIiculties in assignment oI the DRB1 alleles. However by using a series oI
primers, the sequence oI each being speciIic Ior some DRB1 alleles, but which do
not share sequences with other DRB genes this problem has been nulliIied.
This strategy has also been applied to SSOP typing.

The great advantage oI SBT is its accuracy. It is the only technique, which directly
detects the nucleotide sequences oI an allele, thus allowing an exact assignment. It
requires very expensive equipment. Nevertheless, it should be only a matter oI
time until generally accepted, easy to perIorm protocols will be available, thus
leading to wider use oI SBT. The advent oI capillary based sequencers has been a
tremendous boost in reducing the sophistication oI the approaches required in the
laboratory; laboratories with no previous experience in sequencing have quickly
adapted to this method.


Reference strand conformation analysis (RSCA)

ReIerence strand conIormation analysis (RSCA), initially termed double-strand
conIormation analysis (DSCA), is a modiIication oI complementary strand
analysis. DNA Irom a homozygous reIerence sample is ampliIied using primers,
one oI which is Iluorescent-labeled at its 5' end. The sample under test is ampliIied
and the PCR product is mixed with the reIerence PCR product to Iorm
heteroduplexes. These are resolved in an automated DNA sequencer with only the
Iluorescent-labeled duplexes being observed and identiIied according to the
distance they have migrated.


Use Of Denaturing Gradient Gel Electrophoresis
(DGGE)

Another method involving allele separation has recently been applied to the HLA-
B locus. AIter reverse transcription oI RNA, most exon 2 and all exon 3 are
ampliIied and the products separated using DGGE, which separates DNA
Iragments based on their sequence composition. AmpliIied products are excised
Irom the gel and the eluted DNA is reampliIied and directly sequenced.
Theoretically, 92 oI the 118 HLA-B alleles known at the time oI Eberle et al.`s
study could be typed by this method. II ambiguous pairs are still present,
heterozygous sequencing is perIormed on a short segment at the beginning oI exon
2. This increased the number oI alleles which could be typed to 111. The method
uses Taq FS dye primer chemistry which combines advantages oI Taq and
Sequenase into one enzyme, Taq FS, which has the thermo stability oI Taq and the
uniIorm nucleotide incorporation ability oI sequenase. The same group recently
applied this method to the HLA-DR locus.

Single Strand Conformation Polymorphism (SSCP)

This technique is similar to that oI heteroduplex analysis but uses single-stranded
DNA |106, 107|. The technique is useIul Ior the detection oI HLA-DQA1, -DQB1,
-DPA1 and DPB1 polymorphisms. Heteroduplex analysis can be used Ior these
loci but requires spiking. Polymorphism at HLA-DRB loci can be determined by
SSCP, but heteroduplex analysis is more inIormative. The SSCP analysis was
originally introduced to detect point mutations in oncogenes and sequence
polymorphisms in the human genome, based on the Iinding that the electrophoretic
mobility oI single stranded nucleic acid in a non-denaturing polyacrylamide gel
depends not only on size, but also on sequence. There is a limitation on the size oI
the DNA Iragment to 200-400 base pairs, making SSCP unsuitable Ior Class I
typing, and electrophoresis is a labor-intensive method which cannot be easily
automated. During the 11th IHWC, improvements in this technique were reported
that avoided the use oI radioactive materials and specialized cooling equipment. To
improve the resolution oI the system, some researchers have advocated the use oI
restriction enzyme cleavage oI the ampliIied product to resolve some patterns
which had been diIIicult to diIIerentiate. Others have used SSP ampliIication to
divide a complex series oI alleles into groups Iollowed by SSCP to distinguish the
alleles oI HLA-DRB3 and -DQB1.

Comparison of Techniques In A Typing


METOD

SUMMARY

ADVANTAGES

DISADVANTAGES
Serology
.
Serologically testing
expressed HLA antigens
on the surIace oI
lymphocytes by using
monoclonal antibodies
Quicker and cheaper
than molecular
methods.

Does not provide direct
inIormation about
sequence variation in
alleles.

Incapable oI detecting
some diIIerences in DR
molecule.
PCR-RFLP PCR ampliIied DNA
digested with restriction
enzyme to generate
speciIic restriction
pattern and alleles are
identiIied according to
pattern.
Distinguishes
polymorphisms
associated with
DR3, DR5 and DR6
haplotypes.

Higher speciIicity
than serological
methods.
Lacks accuracy in
precise allelic typing,
especially DR4
haplotypes.

Long procedure and
extensive handling oI
samples
PCR - SSO

Labelled sequence
speciIic probes are
hybridised to PCR
ampliIied DNA and
then detected.

Most speciIic
technique. High
resolution typing
withing 10 hours.
Easy handling oI
several samples in
one run
.
Does not require
controls at each
step.
Sequences oI alleles
must be known.
Hybridisation
temperature is critical,
could lead to Ialse
negative hybridisation.

Lacks accuracy in
precise allelic typing


PCR - SSP PCR ampliIied DNA
using Sequence speciIic
primers (SSP). Primers
are designed with
speciIicity-dependent
nucleotide on the 3' end.

Faster than PCR RFLP
and PCR SSO,

Faster than PCR
RFLP and PCR
SSO.

As accurate as PCR
SSO.

Cheaper than other
methods.

Unequivocal typing oI
the eight DB1806
alleles have been
observed
PCR - SBT

DNA ampliIied by PCR
using primers speciIic Ior
the site oI interest. PCR
products are puriIied and
then sequenced.

More reliable and
speciIic than other
methods.
New alleles can be
detected
quite easily.
Apparatus needed is
expensive


Choice of Method

The use oI a speciIic technique will depend on the laboratory`s requirements. The
choice will be inIluenced by clinical urgency and requirement, sample numbers,
availability oI equipment, staII skills and budget. Some laboratories, depending on
their needs, may use a combination oI methods.(3) Because oI the ease oI storage
and transport oI DNA samples, or reIerence cell lines, and the Iact that reagents
can be made and not continually searched Ior, as in serology, some laboratories
have been able to assist laboratories to set-up the techniques.

According to the clinical application, high or low resolution typing may be
required. Kidney transplant candidates, Ior instance, do not necessarily have to be
typed at a high resolution level, because only the broad` serological speciIicities
(eg.DR1-DR10) are usually taken into consideration Ior organ allocation. For
unrelated bone marrow transplantation purposes, high resolution typing is required.
Some alleles only diIIer in sequence outside these regions. A list oI these alleles
can be Iound on the IMGT/HLA database at http://www.ebi.ac.uk/imgt/hla. At
present there are 34 such pairs oI alleles (including ten in which the pair oI alleles
only diIIer in silent substitutions) in class I, but only two at HLA-DR.

Future

The chemistry oI probe synthesis has evolved to the stage where probes can be
synthesised on surIaces such as glass or silicon. The use oI large arrays oI
oligonucleotides on a solid support (DNA chips) which can then be hybridised
with a labelled target sequence should be Ieasible Ior HLA typing. The next Iew
years will see Iurther expansion. This will be the result oI the needs in other Iields
Ior technological improvements in direct sequencing technology and automation.
The tests that become available should have Ilexibility in their resolution enabling
laboratories to economically purchase what they require. For example a medium
resolution system would at present be adequate Ior renal transplantation whereas
high resolution to the allele level will probably be required Ior marrow
transplantation.

REFERENCES

1. Overview on A and DNA typing methods, Annia Ferrer, Maria E
Fernandez, Marcelo Nazabal Genomics Department, Center Ior Genetic
Engineering and Biotechnology (CIGB)

. Advances in DNA based A typing methods, JeIIerey Bidwell,
Immunology Today, Vol 15 No 7, 1994, 303-307

3. A Typing from Serology to Sequencing Era, Derek Middleton,
47the73 I7ela3/ Hist4.425atibility a3/ I22:34e3eti.s Lab47at47y, 54/
IRANIAN JOURNAL OF ALLERGY, ASTHMA AND IMMUNOLOGY
Vol. 4, No. 2, June 2005

4. Shankarkumar U, Pednekar SV, Gupte S, Ghosh K, Mohanty D. A
antigen distribution in Marathi speaking hindu population from
Mumbai, Maharashtra, India. J Hum1999; 105:367-72.

. http://www.srl.in/rd/medimail/HLA°20Typing¸Medimail¸May°202011.p
dI

. Freeman SM, Noreen HJ, Bach FH. Oligonucleotide probing. Applications
to A typing. Arch Pathol Lab Med 988; 112:22-7

.

943$  .   $0.

943 3 :2. 0020398 147 22:30 1:3.908  %0574903803.47  .70 3:2-07 41 0308 70.90 .3 .39038 .3 0308.4250   3 :2.84343.4.9.0 .81. %# &%   %0 :2.3 4907 0308  %0  0308 ..8.70 088039.94783 47.0790-7.39038 .0 8:71.07.38  %8 74:5 41 0308 708/08 43 .3/ 03.74248420   .20 41 90 2./8.8.38  %0 8:507 4.3 97.708:9419078947.439.38 .70 90 :2.3/ 2.7014:3/32489.3903 57080393 5749038 .943    .425.9-9 .079.3903   88902 8 90 3.385.4/08 ..078438 41 90 03089.90/ 94 22:30 88902 1:3.47 894.:8 .70. .4/0/-.398  %0 2.3 0:4.

0894574/:...708:9 90.90/3/.9.8.08  8. 240.90/ - . 3 9:73 892:.0/ 1742 /0890/ 5749038 9.907% .9 .4.4250  .4.9438 8 .8.3.943 41 % 0507 .4/0 .39038 1742 4:98/0 41 90 .70 574/:.9850./:.385.943  4. 542078  . 20/.90 90 2:95..04194:370..234.0 4907 7408  %0 .:9422:30 /80.3903 $01 ..850.0/32..88      ./83039 4703.9/08974. -0 90 .79.39038.4/3 90 2.. 1107039.3/2...08  8 . .3/# 5708039 .90/ 94 90 3/.88 !      " .8808.80  84  3 70574/:..0    %080 5059/08 .80/010380 .70 3:2-0741 4907 0308  2. -0 70.42502039 88902  8 .943  8/0 1742 90 030803.425430398 41 90 .70 82.943   2.39 3 /80..-4:9  .3/  .39-4/08949..0/11070391:3.39-4/ 574/:.997.89/039.47708543/3 94  .08  .9457490.:.9438430.80 /010380  %0 2.4.:08 43.88  03.47708543/394.57490.39700.90 .-03./:.074      .0/ 3 22:30 1:3.47.1.941/80.39038.47708543/3 94  .90 .3 3.0.39038 892:...9 1 /43 70:..3  .70 8:5570880/-8:55708847% .078  %0 2.3545:.3.  90 5059/08 .389 .39038 9070.84208  3 0307.0789418390:2.70 -7403 /43 3 90 57490.089..3 310.908  %080 5.397. 820 41 50450.9438 %02.90800.943  .3/   5708039 5059/08 1742 38/0 90 .:804147.389471.0 94 % 254..70.90/ 43 90  .70 25479.7 .

39 70.0 54247582  ..0 22:348:557088.50398 90701470 70..3/ 90 2.9..385.908.79.7707.1.039 47 8945 /0972039.99025994 232809080.39039490%254.480.   O 3.19 .8.07  -0.19#00.385.078:8 489 /80.3/ 343 801  .3.22:3070854380     O 88902.85488-0   O 40.5039..943  %0  240.3  /039.943           .43974 90 22:30 70854380 974: 70..398  90 /0700 41  2. . $902 0 $  97..-9 41 7..0 /7:8 94 570.. .3/70.-.4708543808 90..43943 41 801 .07.702. 3 /0907233 90 574-.-0   O 489 97.3/.22:3070854380894310.:08 .3.0.425742808-0301..1.:08 8 57080393 90 .3/39.84.97.3 1:3.4708543808  -:9 98 343 850.0/.70.80 .88 95084190/4347.2:89-0.9.3 8 .#4037.322:344.385.5574.4/0/47 4907804..88.943 41 90  240.9390850.0245409. . /4347 8 437..4203..39.943   O 47 .43899:908.:80 41 09038.

-0 254.84 574.....33 . .78  -.  - .  850..:9 5447 .390388/08.4.3 3.943  43.0/ 3..9 .94708 9 907 3.-0 03 80744.:80 8420  .36:08..9473953:370.2202-078 9 574..0 90 09039 41 54247582 .9438 147  .:.99./06:.79:0 41 907 097020 54247582038:709.-47.-9 47 05708843  .89100.36:08 70 43 4-9..88.02039 3 90  9 39073.36:08 070 3974/:.8 343 93 80744..425.. .39038 .090 80744.943.36:08 3 .431723 47 701:93 503495.. 953 .39038.7..0/ 94 2.3 :89 -44/ .-0 .0-0:394705.3/9:890545:.981.39.%53094/8  %7.550/ 94 .70 70.94708.3/ 90 .08  90 /0391.39807.8  0. 94 70.0-003/0130/:8380744.908   953 ./:. /039.88  953  -:9 2470 70.. %88:0 %53 ..8 /11.70/039.-9 41 8:9. -4/ 988:08 7..943 41 .7-0/. 90...908 .8.0/0/9047845-80.0 .9-9 473 74:5   3  (  89:/08 3 .55.77497.9039531.36:08 %080 90.1.07.0.3.430/ 43 .90 5705.90//43478147-4302.88  953   254. 9 . .907 9..70 300/0/  .385.39038  %0  4. 290/ 3:2-07 41 7080../570.039 90 ... .008 08074450714720/.-47..78           ..430 90  .9438  %0  2094/8 070 39. 894.8 %88:0953   :7390.3/ 3 .70 8 0 06:550/ 94 /0.9103/.943.90.0/:38.0 -003 :80/ 94 /0907230 .7.80/95390./943.424489  94:  90.

.55.94708 574/:. 23/0/.008    40.8349.0/-090032.. 850..42207./.90.39.9438 147.0 90 09039 41 54247582 .3/0342./0 .0/4347708971742 -8074494   %0 ...344 94 90 10/ 41 894.88  953   254.:9 3 9480 ./110703.943  43.808 41 5447 .8850349508.89 9039 0.3/ 3 .94170./06:.807 94 .-0 .88  953 0.0452039 .39.903953 1.309080949078:.. 953 .385.7.94708  40.43130/ 94 .908  2.36:0870434-9.4770.0/ :38.36:0147 .39807.80/9490 89:.-47./8 .88 .70 300/0/  .4 03..774 97...0398./070041.90/ /43478 147 -430 2.84 /11.07  /0.4250  .7003 9413/9074370.-47.3/0.3 49078  3 .3 0543039.39-4/ :80/ 147 84.-9 47 5447 05708843 .03941 2.1..3/.8 343 93 80744.309.%0.39038 %0.947 3 953 :370.-9 41 8:9.94317.03906:.39807..07.-47.90/9.425..7094/841%53  %0549039..4..943070-30.$0744.3.0 .84-807.07.8    39.870..-47.9-9 . 10 7080.2 202-078  9 574.-47.:.-0.438090.9434180.94708  %8 20. 749 3 90 .94708.94750714728842095314790 /090.425.70897. 98 .3 2470 .2039 03954247582 #! #013020394190#!908989403.41 -.47894.-47.8 .0398..3/ 22:34039.4258  #0.8 ../0.943 41  90...//943 9080 70. 424489 - 25020393.425.94341.3/ 90 .905705.943 41  254.870/:.36:08  %08090.94708300/0/948.07 .981..908 . 94 70..70.8 8003 .908   $0744.-47.431723 47 701:93 503495./0.78 .3/ .0/ 38902./039...94708.55..0398-09003 .4:/349-07050380/ 898074450714720/...90 708:98 9.-0./11070392094/8-2.-0254.008424489740.0/4.9-9 .80/9532094/8.943.7.:7.. .-0.33.3/ 0/94 8420 ..

#.

.841:831:039.385.989.90/ .3/8. 202-078 41 90 .88  #!  ./:.943.0452039 90700703/.3 43 7.-47.39 25.4030.385.3..:8850..2039.3.-.3/ ".07.:8 . ".3/ 3907 4.0-  5009.789..2  $420   .3/!953 ...7.03. 97.7488 -7/80/ 9 .0 41  -...88.990738 .907.1./.07   ..041#! .8 ..398 1742 30..089. 390 4.94389.3/ ./0243897. .3/70 025.8:306:.:.80/  953 2094/8 3 .99039439490/7.385.2039039!4247582 #!   4250908 41 -3/3 5. 831. 41 4.:7..9478 :80/ 8479 47 043850.39..039708 ./ .700..943.041/0.60320840/48479574-08147#× "× ..00.3/  574-08 070 ..90/ 9 80744.7.3/ #!8 070 /0130/ ../45943 41 #!- 1:79071.93390 9 39073.3.943 41 574-08 /70.943    #0897.9#! .88  030 574-08 .4770.9980.:23.8 2470 .90/43.1.780.39 $9:/   %8 89:/ 840/ 90 89.90 9..008   3 947054798:83%.. 894.07.3..430/ .88  1.9-9 47845  0/ 94 90 /08570..0 %7.7488 -7/8.39. /0130/    ... 574-08 .43897:.9437.4.3.420 9080 574-028 8420 3.3/7..580/-0147090.88.80/ 90 1:3/.425..3 80744  %0 .0.97.703.9.7 97.574-08 3 47/07 94 4.943 41 .9.9 41 #! /0130/  # 2. 3 4. 574-08 290/ 90 .385.19 8:7.. 25479.

4770.8.550/ 806:039. .43943 890 41 %.943 -09003 #! .:/08 90 3:.3/ " 850.  .071706:03.3/ 70897.07. 830 202-7. .049/0 /207 5 .8 574.3 0. /0907230/ .41542475823 :2.3398/20784. # .9438908..908  0850.1907 /0-7/.1.84 -7/80/ 94 4907 0342.7443:.049/08.439.3 4907 70897.0 31472..805.8 9070 .0/0.8574-08-.80/ 43 806:03.39038  -:9 090740309 .3  9.3/ 80744.890:8041-.943  8 90 574-08 .6  3.4:/ -0 .943  94 .03 3 80.943 8908  49 43 .30  %0 70.  #   30419034.0039 .

:.439./907/8.0/.0452039 41 2.80..36:0 70.8343 850.3.834914:3/43479073-498   %0#!2094/8.7-0:83/4033.-3/3..884... 574/:. .8.36:08 .08 93 90 80.0/  -:9 349 -01470 90 708:98 1742 907 :80 . 3 0307.0398  43.5574./ 892:.0 -:9-98 9201:3/..0413:. 70.0708:98147438.806:03.3 !# -.9 43 240.9.//039.8810/3949700.55908890294343 .80/ 90.5/ /0.9 70/:.039:.3/..974547088 070 706:70/ 94 805.049/0 $$ 574-3 !# #! !#1440/-806:03.904708    .3 41 30. ..//943  /:0 94 90 82.0 1742 90 -: 41 90   3 .894574/:. .43/ 043 41 #  " .. 3:2-07 41 .84.0 #!2094/.9438   20703.8.723.9438 %02094/. 0307.33 39073.# .  #!8 070 705.049/0806:03.././/943  90 706:70/ 90 :80 41 # " .##!5.9.943 0320 /08943 .9.0:594/.08 %0//349/70.0780  90 !# 90.90 -09003 .3#.3 -0 /03910/ - . 4..14790.4:/9..709 806:03. 80.0/ 80389.7 -44 7080.990738 %:8 ./4....90/ 4:98/0 9080 0438  3 .4:943..0450/   .2039.9 ..3   .0/ 90 011479 706:70/ 3 8:-806:039 806:03.08  70897.70.39../ . ./.9   701302039 41 98 2094/ .36:08070-03/0..1.045203941-099072094/8147953    !4207.4508 41 90 700.1. 4.90 90 9.8300/0/94 .0084103085072990/90 7.:2-078420.7.3#0.008 !# -.8/08.:.0089.943 !#   !# .3/ 0 00. 70897.  -:9 700/ 43 542475.2508 3437.90/ 542475828 .-0-74.3/ 70./110703990.90/90/0.3/ .36:0  0   !# 806:03.80/2094/82.90 .0 850.9438 94 /8..-0 .43/ 90.0/2:950.7 25.02:2308.3/ 31:03...7.4/3 806:03.943 8908 0307.9/039190 542475.39  98 ..3/ ".03.8 94 :80 90 443:.3545:..079.049/08 94 574-0 949. 443:.55.

3/ $$!  .0 . /1107039 2:9. 43:.9907 .943 89058  0   !# 806:03.0 2094/ 47 .70 5489 .943.9438 0307.:0 ..425.9-9. 90 54247582 8 /03910/ /70.94: .70 90 039 .0/7043410434190 ".3.3/ 806:03.70 /03910/ - 00.049/08 070 -7/80/ 94 .1.3.94341. 443:.43807.894.8 .94: 9070 .0$50.90 850.-00/ .9 .3/ 90 70547907 240.. ! .00 850.088  .88   %0942.308 3 !# 574/:.308932094/.88 3 . .98  %0 .943   39.3 .0 41 443:.9073.08 -09003 90 .43147 2.47 /110703.707054790/    $06:03.9 70:. 34.9..431472.32094/824891706:039.049/08 $$   %0 2.2510/.8 5.3/ 98 /090. 34./4590/94.97454709..0 850. 57207 $$!      .251.79 41 90 !# 574.3.943.. ..7 3907.0 -003 $$  .880  09074/:50. .7.1..3.1.049/0 574-08  .  3 .0 2094/8 147 $$  953 .1.

3 -0 .08/03911.90/ - .-00/ 574-08  094/8 3 90 ./85074/.03983.3..90 .908 706:708 /090.8 4 7084:943  47 0307. /0130/ 850.0 .0 903/0/ 94 :80 0907 .70/806:03.0   7084:943  $$  953 .3 /893:8 .008   7084:943 $$  :8:.0452039 88902 47 .  $$  9073.-0 %0!# $$ 2094/.9..947 . 850.1907-493 .1. 8:-897.. 8:-897.. 343 ...55742.4/0 90 0594508 /090.943  47 0..0 41 953 8 41903 7010770/ 94 ..550/9449074. 0307.1. 706:708 800.39-4/08  %080 8.943 41 8.02:2308.:894280/ 147 0.943 /0.. 806:03.3/#×9.-47..8:80/. .9.. .250  .2080744.908 .74:8574.70/ 542475. .0089. 03.943 41  # 80744. .55.90 3 .030 -:98443.80 .08 .20841.0/:708:83! -4934747807.!×  "×.9 74:58  %8 0.86:.   %0 $$  2094/ .447.8.9-04394908.

47/0 %  ..9..947085705.90/ !# 572078 .0.7.314/ %0:8041.74-49.251.574-0390..425090 4.301470./08.953 147.:9  # .39038.74:54170.3/ 903 90  8 -7/0/9.90  44/ .70/:0945447.08 90 0110.908/11.9 90 8.88.. . 902094/8070:80/94/0130..:.3 .90/:83./390574-08.94190 90.251. .9 0    4794/0391850...88 03087.308 .73 390389  907 .. 2489..250948. 830 850.251.3/90..:80806:03.9..70 4./893:80..94341.70430202-7.-0  74-:89 .8438 %0706:702039147705.90/.#  .-47.07 70.//943 41 9097.84 23208 8..008 .7039  .943 .70.90/ 3940438  40.7041908.8 902507.:9$$ !-7/8.9903943 9:730/ 94 .943 34943 023..3/ " 4725488-0  ! .05424758283.55.3/ /80.80/ .8.-94190-7/8.943 147-49 98.250 .9:70  $420 . 94 ..9:708 - 90 .943 41 .39147 97.3/ .88.308 94 -0 .3.908 90 70:80 41 202-7.943   95390.2489 .943 41 -7/.70..943 /498 41 .88 14780.974:588:.884.943.574507908   %0 $$  90.90574-02.08.-47.0 2.00   %4/./ 3 0.1907 /0-7/.-08 202-7.0453..0 :80/.7.250.0 90 3:2-07 41 /1107039 .008 470.1.9.251.1.:994/090...3/ 574.94708 .385.9 41 .:8 88902 574.3.07..94708 70/:.36:08070 39.4/90:8041%/:0949894.0./3.943 3 4.8 /08930/94-02470...894:994 -0 2470 :7039.3/..07 43.008 41 .9-807440     %4 2094/8 147 90 /090723.209.0/ 94 -0 90 .0083800.1.90/850..4:39070/.4789..:942.70 850. # .3/.850.550/94.94708.8/11.953708:9803.9079.-47.880744.943  -:9 9 .:7..88  4.3.943.0 .894:994-0247025479..-47.0.3041574-08.5.36:0   .708:71.439039439089..090-03019841/0.8      47        .20 039  03.880308.4250-0.55390 2094/8 94 ...88  ...36:0 .8880744.70. 070 ..20 902507.88  0308  39.2510/ 9 800.0/:70 81.9430. %8 574..2243:2 . 70/:.0/ .80 .39.88.943  :79072470  953 147 .1.3/574.8 574.

3/:8374:5850.3.9.14:3/.393-4302.907 2574..8 90 :80 41 . .80.0047..43943890 570803938420.90/ 1742 90 0 .:9.8706:70/    $06:03.9438  94 /089 !# .039 %:84390/0870/.07.3.4380. .83070.0..9 90.1.550/94 "  ".90/   %080 574-028 .88  2094/8  %080 2094/8 070 .5.00/ ..0.. 70. 70..3/8420090744908.330.1.08  $50.0452039 41 $$  .570.-025479.43/ !# #!  47 90 .6 54207.90 147 .00..9.723... .:993 .0.425090 2.8 9025.:70570.2510/994 47 2470 03208 .77497..0/--099077084:943.430/ .39.282.80 282.90/ .9438 9..9  ! 2.5720794304780.84.808 .0/17. 98 2094/ :80/ 90 .08 94 0254 70897.0..420 147  # - .304:8/0894341.   830-..8 097.8 -003 ..943 03/43:.0 74:3/8 41 !##! 1907901789/089434190!#574/:.9439.251011.039:80/390!#70.008 .8 94 043:..3349-0/8.:80 %.4:8 .!72078 $$!   %0 573.049/0 806:03.. 24/10/ !# #! 2094/:8331472.031572075. 90349.9.2/008842092084-8.550/ 30 41 90 1789 3/.9438  0.2510/  0308  #  "   ! 40.039 334.87054790/:8390 !# #! 2094/  70.1.. -.7488-7/.2039.7.3/ :80/ .1.3/2094/841574-0..0/ 57207  -0 2470 011. 3-98 90 5723 41 343 850.57207894. 41 98 2094/ 8 9.0 850.9 .3/8 4.9.943.3 94574.-9 41 8908 3 90 3:.. 572078 3 ..07.1.3/82:9..943 .32.9.3/ !0308.943949070308 %82094/.88 .0 -003 4.78/4.008-:9349349078.7.8.9 0.943 147 /0.-03    !# #!  39.07  82.94303208 .070897..0$50.9.4907439054.80.385.9 8 /0907230/ - 90 :80 41 806:03..0. 80.80.4/ .1.343 850.480940.

078 147 .0450/. 147 953 3/.008 41 .8 70/:.0889.2510/ .39807..:9 94 /090.3/ 94 90 .3/ 706:708  .. 41 90 0.8 .943   :.0 88902 94 .7084:94388902147    %0 574/:.489 .3 903 -0 /090. -0 . 8.4:/-0..4.425020390/0.30147   9078:80/90$$!88902./.089..   .2510/ 574/:. 4 7084:943 953 88902 147    .008 41 90   4.4907 849.47084:943572075.385.9 .425090/9397004:78     ..$$!88902 147 .079.707 3:2-078 41 8.4:/ -0 50714720/ 82:9.9 90 .90/ - .304:8   %8 2094/  90720/ 5494953  .07 ..93 9.088.9 80744..550/94.  %:8 .380..0450/ ..3/ " 953 .  95388902147574.3/ 6:.36:0  4 7084:943 1440/ -  7084:943 . 2094/8 /:0 94 90 4 05708843 41   240.9 - 80744./:.07..47708543/3 .3/ 90 .9 8 30.. 1440/ 9 .0 .20 70.3/ ..908 4743/0907233  :3.425090       #  #  #  # .78390 8.009. .0:80/2:950!# 0 .2508  %8 .8 ...0450/ 14749074.90 90 !# 17.:08 .3/.1.//943 889028070/0.20 74:5 /0830/. 2470 09038.0/ - 389.93 ..572075..08 088 9.7 147 39075709. 850.98 41 2. 94 89..97.3 .707 47 3 ..70 /11.. 8:5502039 94 80744 - 43 953 147 .3 80744.3 .:08 4 /0.-0 .0/. /0..943  $3 41 90 !# 574/:.07...9478.943  30.07:801: 3.3/.3 1.47/3 94 90 1789 708:9  8 90 2094/ 9.008  %0 8.2508  -:9 8 .0 90.0 4:78  9 . 7084:943 06:.40.039 94  6:.39.0 8:71.5.7480 0 00.3 -0 .88  ..9 9072.20398  %0 $$! 2094/ 8 /0.9 90 0 -0 7:3 4307 94 805.974547088   9073.7.9. 41 8:9.8 50714720/ - 7433 .:8 .

08 90 574-0  708:93 3 .7 897.7/ .9 90 03/ 8905 41 0 00.:942.6:03.3/ 70.3/ ..9081742909.70.:80 .0 2470 84 9.90/ - 90 24/10/ 27.0 41 90 !#  ..0 41 0..703495708039309074190 5.008.80 3 1:4708.431472.943397.0780 57207 890  %0 3:.0.049..07 .1.:80/-09074/:501472..97454709.3/ .078.:8..48 .70.:0  %8 ..4..0.03.70/ 00.0 $$!#   %0 $$! 2094/ 8:11078 1742 90 /8. 574547943 41 .7/.3/.9.99073841903/.4/3897.0.708 147 0.089000.90/   //943.70 3:2-078 41 8.:78 3 0.80/ :4708.943 41 90 897.9.00.3 .943 .6 54207.#4. 574-08 0.3/841 .907.709./.3/9025..943 41 90  240..0$50.03 8:-9508 3.0.0  :73 !# $$!  1 90 9.0.3/.80/408349.943 .54950 .-7/809490343.9434190!#574/:.42502039..330..9.974547088.4/3897.3 -0 /090.8.0.0 9.2/0 08  .3/54207. ..-0 147 ..-. .39.03.3/.80#00./3 94 .3 .1.!723 43:. 2..049/0 ..3/ 9:8 0.3/8410.8 90 09074/:50  24.0 908479030/574-0/884.9 41 90 %.:7  1907.. # .0 . .330./:.431472. %8/4:-0897.42-308  974:5:9 9 8500/ .07/0 0390574-0839..38 ..9:73 54.9 /090..80 .41..9  90 57429 41 90 94 /08 708:98 3 90 8:55708843 41 90 70547907 1:4708.0 2094/.:8..3/.$06:03. 41 3:..002.0901700574-0 900320706:708 806:03.943   34.0944..709 41 3907089 8 5708039  90 574-0 850.0 %.7014720/3090744908..3/0/9:8-0 282.3 3..974547088 8 349 8:9.4393:08  %8 574.7...3/8   830 282.088 4.8-0037054790/.3/ . 709.7024.2508 47 147 .9    09074/:5008  :73 90 57207 .3/8. /70.654207.943 3 90 .0. 8308:-899:943.3/  7084:943  %0 2094/ :808 1:47403.42502039.70547907.943 3 343/03.3 90 424/:5008 .3 89.79-0900390574-0.03..90147.8 -09003 90 147.3-0/090../1107039#4.0/ 3 8420 70438 09074/:5008  0.

008 90.....943..008 41 4907  # 4.9.943 :80/ 430 809 41 572078 9 .42243 806:03.9434190.251.990734190 29:70-0908.00806:03.703490 94/090.5503894-0.3.0 06:52039  0.008 -:9. 3 953 147  # .88320398 8:9.008  40.-. 147  # 903 90 -.8-0033:10/  %8897..943 41 /0. 2.70 .98903:. 98904390.94:90../4 3498.943 41 .8832039 41 90 # .80/43806:03.9089070-174294/11070393/.9 .39.93 .0894907#030898574-02.039 .5.425:907 8419.03941.70.1.1.36:0  39.9907 41 920 :39 0307.2510/ 3 90 !#  1 90 3/.-0  9:8 0..8-003.439.251.7-.0790088  9 84:/ -0 43 .890-.3  74:5 850.41 90 3/.549508  %8 503420343 .00 9:8./70../:.90/ $06:03./.0841.  # .:942.0 94 ./394/07:8041$% %0.07 05038.00 :308890.4 ...70 /039.078.8  -:9 1 90 3/.. 970203/4:8-4489 370/:.39 90 82:9. .841$$ 47$$!89.8..07 98 20..344 ./.049/0806:03.90.36:0 .80/ .3 097.3/8    $06:03.30.70 -. .304:8 ..0590/  0.99073410.-03850.8 .80/110703.30:3/0130/.3 806:03.3. 90 29:70  .251.7.5.74882.3/ 0/ 94 /11.008 !# .008  40.90/-90574-0479057207 094/8-.. 0  -09003 !# 574/:.550/94$$ !953   %070..90 4907801742-49...0-0900394.-....3 90.03806:03.0410.48  -0 .9089 574-028 9 98 90.8 :80/390.70 .5574.943850714720/347/079429903:2-0741.84-003..1478420#./:.98 1742 94 /1107039 .70806:03.3/35.9 /090.9.083.0/ 9 90 3974/:.420 94 90 1470  %0 806:03.00 .20.9.:908 3 .9.8 .0. 80708 41 572078 90806:03./:.80/%53 $%   304190/7.39084589.3/ 1:4708.1.990850.5495084:/-05708039 %8  3 9:73  825108 90 8419.3.:7.-041/090. 890/090.3/35.8 94 5071472 57494.830-.-00/ 572078 .8832039 9 706:708 ..43.08706:70/390 .07 - :83 .0. 0   #  #  .8.041$%898./:./.70 349 /039.8 430 41 90 70.-0 .39025..80/806:03.

3/ ..9 94 1472 09074/:5008 %080.20398 -. .-9 41 806:03.943 .6 $ /0 57207 .9:737.998 03/ %08.3/ /70.974547088    349072094/3.049/0 3.870.943 .908  17..803944300320 %.943 .0794390 1:4708.3.70 0.3/.590/94982094/    #010703.3.3/ . 90720/ /4:-0 897.7. 4244:8 7010703.4:/-0950/94 %0 2094/ :808 %.81:4708.039-003.-00/..2510/ :83 572078  43041..98 805.88 #$  39.039 ./..4.80/903:2-0741.90/806:03..943 41 . .-941%.943.42502039.943.3/ $06:03.70.70 89 5708039  090744:8806:03.0780 97.47547.008 343 .9 90 920 41 -070 09 .20 74:5 70.75943 41 #  2489 043  .42-308 .9 806:03.80  %0 8.   41 90    .8479802039.7.0/  %04709.0 8.0/3. 24/1.0 6:.550/982094/9490 #4.039 .6 .-47.2510/ .88   1742 .2510/ .250 8 .250:3/0790898..88 $  8 .990-033341043  %83.3/ 90 0:90/  8 70.3850714720/43.3 .4:/ -0 950/ - 98 2094/  1 ..3/90 :31472 3:.0 3 806:03.0/ . 8 89:/ .98 .550/9490  4. 805.027.431472.:942./03900.0 .8909072489.3.42548943  2510/ 574/:.6..0897.:8  1907 70.0 !# 574/:. 043  .02897 .3.3/ /03910/ .9 8 20/ 9 90 7010703.88 #$   #010703.7 897.80/ 43 907 806:03.90/ :83   .2-:4:8 5.3/ .94708 9 34 570.39.431472..4:8 050703.0 897.947 ./.:8   .707084.80/ 1742 90 0 .2510/ .70 .3/ .3/ 90 574/:.008.090.-47.47/3 94 90 /89.6$ .-00/ /:5008 -03 4-807.039 .431472.7.3.3.38.78 .90/    &80 103.00805.08 41 %.3.3/ 90 !# 574/:.

.0 41 90 .5.3....-47 39038.0/ 94 /090.93 8420/110703.974547088 8 .251.507 9.-041/090.3 $$! :38:9.3.9438 3 43. 24-9 41 830 897.7./4..041 254.3/850.3/0/ 3:.80 5.9:73 54.88  953  .943 0320 .39038 43908:71.43. .0 %0708.02039839890. .0.943.:942.008   3.3/ "   425.9433 .9  # 4.3.907./0/70../0.97454709.39-4/08  '%$ ":.3/ 00.3 -0 /0907230/ - $$!  -:9 09074/:50 ..8 473.42508070841..$30$97.9.:..941 09074/:50.3349 -0 0.0.-4:9 806:03.7949.0 :80/ $$! .-0 147 .7..7 2094/8    $'%$ 408349574.40308 . 343 /03.078.9 94 7084.0907084:943419088902 84207080.908-:83 2434.3.36:0882.3/ ! 542475828  09074/:50 .8 ..29.80/439013/39..3 -0 :80/ 147 9080 4../ -003 /11.00841 #.99000.784341%0.88 8 2470 31472. -:9 706:708 853  !4247582 ./.0/..9. .36:00707054790/ 9.2/0 0 /0503/8349434380 -:9..4/0/90:80417.94343908041 90  17.90/ :73909 2574.3/.8443806:03.2039 94    -.9 5439 2:9.9 31472.0 542475828390:2.3240. .:9 94 /1107039.2510/ 574/:..:0  .9..3/431472.0 2094/ ./ 3 .990738 .8.0.44306:52039 %4 2574. .78  2..94341 "  "  ! .083# 240.90893 0570880/.90/90:8041 70897.36:083%53   %  $0744   $&# $0744.07..3/0/    ( %090.0  %0 $$! .88-:9:808830 897.36:08:801:14790/090.3/ 806:03.7.90  9078 .0.0.02.88 .943!4247582 $$!   %890.303420 -.4.88 .00839474:581440/-$$!94/893:890 .943 94 /.0 8420 5. 3974/:..

3/# .0850.9. 8905  !# $$! !#.2510/-!#  :83572078850.1.049/04390 03/  $$    .0841.0.0/:70.90/  .2508 $06:03.008.008.89079.9 /0503/039 8.1.25083 4307:3  408349706:70 .8.4..5079.1.8.79.1.:7.07.80.99073  893:808 542475828 .943 5.9:8300/0/8 05038.34907 2094/8  0.8.-00/806:03.90.0/    47070....3/!# $$  2094/8    .4:/0.3/ 850./941.1.  850..70 90.8!# 3:..147 90890413907089 !# 574/:.3 570.549508   07850.380744..90/ 6:900.2510/ /0890/970897.3/ 09038.47/394 5.0..!# #! !#.89079.9:708.943 0320940307. #!.3/341 8.3 570.3/ 903806:03..3/ 93 4:78  903/090.0-003 4-807.70897.1.99073.953  0850..8  55.70/0830/9  850.0  ..70 /03910/..80 30.1. 2094/8  !# $$  .3-0 /090.705:710/.943   .0..  ..90/9 # #.3/341  80.9.34907 .0-7/8.95341 9009  .574-08..3!# #! 0.008.:7..2510/.7.36:0  -7/80/94!# 7084:943953 .:7.98.0/ !# $% .3/!#  572078 $$! !72078 $$  .80..-0.884.008 2:89-0343  -7/8.90..0 489850..8.549508   43574.1.943 902507.# .3!#  :83$06:03.953   &306:.90 850.439748.9 9.3/.2510/ ..

3/ 706:702039  8.908 0 # #  .4..3//.8889.9470894809 :59090.1188.2508  47 7010703.0398 .. .9 .-47.3.9435:754808 7084:9439538706:70/  $420 ..42-3.0 .3/ 90 1.3 -0 2.943   47 4 7084:943 953 2.36:0/0503/4390.947 8706:7020398 %0 .. 9.0 4:98/0 9080 70438   89 41 9080 .438/07.385.. 850.-47.3 -0 14:3/ 43 90 %.0/ - .008 43 /1107 3 806:03.0 .8 3 80744  8420 ./0 .094-0 950/ .4.008 ..250 3:2-078  .:80 43 90 -74. :703.3/ 349 .7..1...94708 .80418947.3 .:80..47/3 94 90 .9 70.7. -0 706:70/ /3097.  7084:943 0.-47.0  -0..908 147389.3/ 97.70 :8:.55./  80744.088.385.39.94708 /0503/343 907300/8 2.0  -0 31:03.36:08   .850.9 9.385479 41  8.0 /434930..0 308  ..3..90..041094/  %0:8041.:8041900.0/ 147  .1.943412094/8  0... .943  47 :370.943 147 47.4.3/-:/09 $420.77497.-94106:52039 89.0-003..39.-094.03 394 .90/-4302.-47.4393:.3. 80.

-.80 .9.9 995. /.

.

. :. 0- .

29.

-00/ 9.02897 41 574-0 839088 ..250 .70 .9 5708039 -0 .8-0 147  953  %0 309 10 0.90 147 703.3/ .741.80 .0 070 574-08 .-47.774 97.0  574-.3 903 -0 -7/80/ 9 ..788001:790705.9 90 706:70  47 0.58  ..010-939077084:94303...0/ 94 90 89.344 . 5:7.3843 %8-090708:94190300/83490710/8 147 90.:/39033..943  %0908989... .39.8 41 443:.905.008 43/1107380398:-899:9438 3.049/08 43 .385.8 0.-3 .-084:/.88 47 8.00 0.  9 57080399070.77.88 -:94394.. 20/:2 7084:943 88902 4:/ .08 8:.3 -0 8390880/ 43 8:71.385.9 806:03.943 070. .8  7084:943 94 90 . 84/ 8:55479  .0 84:/ -0 10.708:.4342.4.8 .008 3.5.020398 3 /70.344.420.:942. 97.709 806:03./06:..- -0 706:70/ 147 2.43  %0 :80 41 .943   . .7841...9 #   :9:70  %0 .9-0.94708 94 0.39.3 90. 2574.

3/490.792039  03907 147 0309.  073E3/0  .3 7.80/  953 2094/8  011070 /0  22:344%4/. 0342.07.3/ 894.7J.-. 07707  ..##$    .947  .3/  953 2094/8   33.8 05.08 3  -.3.425.7.  070 //0943  479073 70.9-9 .344      /.8 .0 43  . '44          %53 1742 $0744 94 $06:03.. 330073.04 .3/ 22:340309.-47.

7.39    .. .7:2.7 $'  :590 $  48   4.943 1742 :2-.3903 /897-:943 3 . 3/. :2       995.7 &  !0/30.897. #  &#  #  $%  &   '4  4  :30     $.3 3/: 545:.7.3.9 850.

.

 87 3.

7/.

20/2..

  43:.   5 /1    7002.*.3$ 47003 .049/0574-3 55.94.9438 94953 7..!..-0/  . %53*0/2.