I am requesting: amber111 to write this.

Read the article below and answer the two questions below:

1 1/2 pages each – separate by topic

Topic 1. Analyze the current challenges with organ transplantation related to genetic variability of MHC.

Topic 2 . Describe the challenges of human leukocyte antigen (HLA) matching and identifying sensitized individuals.

Sensitization to non-self human leukocyte antigens (HLA) occurs through three
main routes: blood transfusion, pregnancy, and transplantation. This can be
illustrated by the demographics of patients on the transplant list. Recent audit
data from UK Transplant (2006) shows that 23% (1416/6019) of the patients
awaiting renal transplantation are sensitized. Significantly more female than
male patients have HLA antibodies (33% vs. 17%, respectively; PP

An analysis of waiting time to transplant in the United Kingdom showed that
waiting time increases with increasing levels of sensitization. For unsensitized
patients registered between July 1998 and December 2005 the median waiting time
to transplant was 788+/-26 days, whereas for patients with an antibody reaction
frequency of 61% to 84% the median waiting time was 1696+/-213 days and for
highly sensitized patients 2232+/-773 days.

There are different approaches that can be taken to transplant sensitized
patients. Organ sharing schemes provide significant advantages to sensitized
patients where crossmatch-negative organs may be found through access to a large
donor pool. In the UK national organ sharing scheme, the identification of
suitable donors for sensitized patients has been facilitated by a requirement
for accurate definition of a patient’s antibody profile (2). Histocompatibility
and Immunogenetics laboratories have different strategies for alloantibody
detection and specificity definition in patients awaiting transplantation, but
all strategies culminate in the definition of HLA antigens that are unacceptable
in a prospective donor (3, 4). The unacceptable HLA antigens are then registered
on the National Transplant Database so that a patient will not be considered for
donor expressing those HLA antigens. In effect, for every donor, the national
computer performs a “virtual crossmatch” against every blood group-eligible
patient on the transplant list. Comprehensive antibody definition is critical
because all deceased heartbeating donor kidneys are allocated by the national
algorithm and shipped to patients predicted to have a negative crossmatch. Once
shipped, it is important that the donor kidney can be transplanted into the
designated patient. The virtual crossmatch approach is widely used in organ
allocation and transplantation (5, 6).

This approach requires laboratories to have procedures in place for regular
receipt and testing of patient sera for the presence of antibodies and when
applicable, for specificity definition (7). Standards and guidelines produced by
the European Federation of Immunogenetics, the British Transplantation Society,
and the British Society for Histocompatibility and Immunogenetics stipulate at
least quarterly testing.

There are now a range of techniques available for the detection and definition
of HLA specific antibodies and to interpret the results from the various assays
it is important to understand the different principles underlying the assays as
well as their relative strengths and weaknesses (8-10). The following techniques
are discussed: complement-dependent cytotoxicity (CDC), flow cytometry and solid
phase assays including Enzyme-linked immunosorbent assay (ELISA), flow cytometry
using antigen coated microparticles, and Luminex. These techniques can variously
be applied to antibody identification and donor-recipient crossmatching.
Antibody Identification
Complement Dependent Cytotoxicity

The long established CDC assay is still widely used (11). In the context of
antibody screening, the patient serum under investigation is tested against a
panel of lymphocytes from HLA typed individuals. If the serum contains
antibodies specific for the HLA antigens on the lymphocytes then, on addition of
rabbit complement, cell death will occur which can be visualized by staining
followed by microscopic evaluation (Fig. 1). If a random panel is employed, for
example, local blood donors, then the frequency of positive reactions (%PRA)
gives an indication of the percentage of local organ donors with whom the
patient would be crossmatch positive. To determine the HLA specificity of the
antibody it is more informative to use a selected panel of lymphocytes that do
not express common combinations of HLA antigens, for example, HLA-A2 with -B44,
and that include rarer HLA phenotypes. When selected panels are used, the
calculated %PRA simply reflects the HLA composition of the panel and is of
little value in predicting the frequency of positive crossmatches. In addition,
variation in the %PRA between samples may simply reflect differences in panel
composition rather than a change in the humoral sensitization of the patient.

Disadvantages of the CDC assay have been widely discussed. It is subjective,
cumbersome, relies on rigorous batch control of the complement used and will
only detect complement-fixing antibodies unless a secondary anti-human
immunoglobulin reagent is added to the test. This is important because there is
evidence that noncomplement fixing antibodies are relevant for transplant
outcome. The interpretation of a CDC assay is complicated because non-graft
damaging IgM antibodies directed against non-HLA antigens (autoreactive
antibodies) are detected as well as alloantibodies. In the absence of HLA
antibodies, autoreactive antibodies can be distinguished from HLA antibodies
because they are frequently unreactive with peripheral blood lymphocytes from
patients with chronic lymphocytic leukemia (12, 13). Nevertheless the interpretation
of these antibody reactivity patterns is complex, as patients frequently have
HLA antibodies and autoreactive antibodies.

For these reasons, many laboratories are now moving away from cell based to
solid phase assays that offer the advantages of greater specificity and
sensitivity. However, although a CDC assay is still used for donor and recipient
crossmatching it has a place in antibody screening to indicate how a serum might
react in the crossmatch test. Cell-independent methods have recently been
described that use solid phase assays and flow-cytometric detection to identify
the presence of HLA antibody triggered complement activation (14, 15). These
have the advantage of detecting complement-fixing, HLA specific antibodies,
known to pose a high risk for graft rejection, without a requirement for viable
lymphocytes.
Flow Cytometry

To increase the sensitivity of antibody detection, flow cytometry based
methodology was introduced more than 20 years ago (16). Screening using
individual cell panels was too cumbersome so cell pools were used, for example,
Epstein Barr Virus transformed lymphoblastoid cell lines (LCL), chronic
lymphocytic leukemia cells, and peripheral blood lymphocytes (17, 18). In
general, the assays were designed to detect IgG antibodies so the problem of
detecting IgM autoantibodies was avoided. However, some patients do have IgG
autoantibodies which are detected in these assays. The use of pooled cells
identified the presence of antibodies and because the level of fluorescence was
proportional to the amount of bound antibody the results could be interpreted as
%PRA. However, further testing was required to determine antibody specificity.

With the introduction of solid phase assays for antibody testing, the use of
flow cytometry with cell pools has been largely superseded.
Solid Phase Assays

Solid phase methodologies use solubilized or recombinant HLA class I or class II
molecules coated on to a solid matrix combined with automated optical detection
methods. These assays offer considerably increased sensitivity and specificity.
Enzyme-Linked Immunosorbent Assay

ELISA was the first type of solid phase assay to be developed for HLA antibody
specificity definition with PRA-STAT being introduced more than 10 years ago
(19). This is no longer available but has been replaced by other commercially
available tests for both antibody detection and definition (20-22). HLA
molecules are bound to each well of an ELISA plate. For antibody detection these
are purified from a pool of platelets from a large number of donors (for the
detection of antibodies to HLA class I specificities) or from a pool of LCL (for
the detection of antibodies to HLA class II specificities). For specificity
definition, each well is coated with purified HLA class I or class II molecules
from one individual.

After the addition of patient serum, antibodies directed against HLA molecules
in the well will bind. They are detected by the addition of a secondary
antibody, an enzyme-conjugated anti-human immunoglobulin that induces a color
change on addition of the enzyme substrate, detected by measuring optical
density (Fig. 2). This is therefore an objective, semi-quantitative assay that
can to a certain extent be automated. It is usually designed to detect
immunoglobulin (Ig) G antibodies but can be used to detect other Ig isotypes. It
has the advantage over CDC of being more sensitive and detecting noncomplement
fixing antibodies that have been shown to be relevant to transplant outcome. The
assay is designed to detect only HLA specific antibodies and is generally not
prone to the false positive results caused by IgM autoantibodies in a CDC assay.
However, nonspecific binding of other immunoglobulins can occur with some
patients with autoimmune disorders and patients with cardiolipin antibodies.
Flow Cytometry

Flow cytometry based solid phase assays involve the use of microparticles coated
with purified HLA class I or class II antigens (FlowPRA, One Lambda) (23, 24).
HLA class I or class II antigens from individual LCL are coated on to individual
microparticles beads. For the purpose of antibody detection, microparticles
coated with different antigens are pooled. A fluorescence-conjugated secondary
antibody directed against human immunoglobulin is then added to detect antibody
binding using a flow cytometer (Fig. 3a). As for ELISA, these are semi-quantitative
assays that are designed to detect HLA specific antibodies. Comparison of the
fluorescence signal for the test as compared with the negative control gives a
percentage result that can be considered as the %PRA. However, for patients with
a low level of antibodies to a single specificity the percentage reactivity may
not reach the positive threshold. These can be identified as small secondary
peaks on the fluorescence histogram. It is therefore important that the peak
architecture is reviewed by an experienced individual (Fig. 3b).

For specificity testing, antigens from different LCL are coated on to beads with
differing fluorescent properties so that the beads and hence the antigens to
which antibodies have bound can be identified (24) (Fig. 3c). As for ELISA, the
tests are usually designed to detect IgG but can also be set up to detect other
immunoglobulin isotypes.
Luminex

Luminex technology also uses pools of HLA class I or class II antigen-coated
microparticles. These are colored with a combination of two dyes and for each
set of beads the dyes are in different proportions so that the bead sets can be
distinguished (Fig. 4a). Up to 100 beads can be combined in a single test. HLA
specific antibody binding to the microparticles is detected using a R-Phycoerythrin-conjugated
anti-human Ig. Fluorescence is measured using a flow analyzer within which the
lasers excite the internal dyes that identify each microparticle and also the
reporter dye captured during the assay. As each set of beads can be distinguished,
it can be determined which HLA antigens have bound antibody (Fig. 4b).

The original assays used beads coated with HLA antigens derived from individual
cells but a major advance has been the construction of HLA class I and II
recombinant single antigens from transfected cell lines. This has allowed the
production of microparticles coated with single HLA antigens for accurate
assignment of antibody specificity. These provide higher resolution and
increased sensitivity as compared with microparticles coated with multiple
antigens. This technology enables identification of antibody specificities in
highly reactive sera (25) and hence can be applied to monitoring patient
antibody profiles in relation to particular specificities such as following
antibody removal pretransplantation (26) and identification of donor specific
antibodies posttransplantation (27). However, the level of antibody binding and
consequent fluorescence intensity will be dependent on the binding affinity of
the antibody and the antigen density (28). Binding and dissociation constants of
an antibody with its target are known to vary between antibodies and there is
some anecdotal evidence that antigen levels vary between sets of microparticles.
Therefore fluorescence intensity can only be directly compared for one
individual’s antibodies in different serum samples binding to microparticles
from the same lot or alternatively by normalizing the data for the quantity of
antigen per bead. Taking this into consideration it has been demonstrated that
the strength of fluorescence on the microparticles can be directly related to
antibody titer (28).

Another advantage of single antigen bead technology is that the data can be used
to define epitopes accurately and hence understand patterns of antibody
reactivity (29).
Patient Sensitization Profile

To transplant sensitized patients successfully, all of the results obtained from
sequential antibody screening and specification analysis are combined with
information about the potential sensitizing events to produce a patient
sensitization profile. Information about sensitizing events will include the
timing and number of blood transfusions, the HLA type of a previous transplant
and for parous women, where possible, the HLA type of the partner. The
sequential antibody analysis will result in an understanding of the timing of
appearance and disappearance of HLA antibodies, their specificity and, depending
on the technology used, the antibody class. Inclusion of an autologous
crossmatch as part of the immunological work up of the patient can assist in the
analysis of antibody screening data and in the interpretation of the final
crossmatch.

The patient sensitization history is used to specify HLA antigens in a donor
that are considered unacceptable. These include specificities to which the
patient has preexisting antibodies, but depending on the policy of the
Transplant Unit, may include HLA mismatches from a previous transplant,
particularly where historic serum samples are not available and mismatched
paternal specificities in parous women. In certain circumstances Units may wish
to avoid certain HLA antigens in a transplant. For example for pediatric
patients where a live donor transplant may be considered in the future, it is
prudent to avoid the mismatched antigens in the live donor and therefore list
these as unacceptable for a deceased donor transplant.

Knowledge of the sensitization profile can provide clinicians and patients with
information about the likelihood of a suitable deceased donor becoming
available. Antibody specificities can be used to calculate a patient’s %
antibody reactivity against an HLA-typed donor pool (30, 31). In the United
Kingdom, the patient’s HLA type and unacceptable HLA specificities are run
against an HLA typed pool of 10,000 deceased donors to produce a “matchability
score” that reflects the number of well matched blood group identical donors for
that patient in the UK donor pool (2, 3). This can assist in patient management
and is informative in considering the options of deceased or live donor
transplantation and in planning transplantation for children.
Transplanting Sensitized Patients

Highly sensitized patients are given priority in the allocation algorithm for
well matched transplants (HLA-A, B, DR mismatch grade of 000) to increase the
chances of transplant for these patients (2, 32). If a patient’s antibody
profile has been completely specified then highly sensitized patients are also
eligible for mismatched transplants because there is then a minimal risk of a
positive crossmatch. Extending this principle, some centers will now proceed to
transplantation without a prospective crossmatch when the patient’s HLA
antibodies have been comprehensively defined (33).

The definition of antibody specificities is also integral to transplanting
sensitized patients through the highly successful Eurotransplant Acceptable
Mismatch program (6).

The combination of advances in antibody detection and specification and
increased options for clinical management means that a crossmatch result can be
viewed as presenting the risk of progressing to transplant (3, 34, 35).

The lowest risk for sensitized patients is to receive a transplant, either from
a deceased or living donor, where there are no incompatibilities between the
patient’s preexisting antibodies and the HLA antigens expressed on the donor.
For patients with an incompatible living donor this can be achieved by entry
into living donor exchange programs (36-38). In these programs accurate
definition of unacceptable HLA specificities is critical in ensuring that donor
and pairs identified progress to transplant.

Transplantation in the presence of donor specific antibody presents a higher
risk and therefore understanding the specificity of the antibody causing the
positive crossmatch result is critical in assessing the risk for a particular
patient. The risk of hyperacute rejection depends not only on the presence of
donor specific antibodies but also the titer. Low titer antibodies present at
the time of transplantation are less likely to result in hyperacute rejection
but indicate that the recipient is at risk of early antibody mediated rejection
(35). Low titer antibodies will only be detected by the more sensitive solid
phase assays so an understanding of the techniques used enables assessment of
the risk posed by the antibodies and hence selection of appropriate therapeutic
protocols. A case has been reported of acute antibody-mediated rejection of a
kidney transplant because of pregnancy-induced sensitization to donor HLA
mismatches. Pretransplant, the donor specific antibodies could only be detected
by Luminex based techniques although by the fourth day posttransplant the
cytotoxic crossmatch was positive (39).

Antibody removal is a further option for proceeding to transplant in sensitized
patients (40-42). There are now a number of successful programs where inclusion
of patients in the program is informed by antibody titer and specificity data
and the antibody removal process, transplant and posttransplant management are
underpinned by antibody monitoring data.

The current range of technologies available to laboratories provide the means to
specify a patient’s antibody profile and support transplant programs in
determining the most appropriate pathway to transplantation for individual
patients.