In addition to identifying the HLA antigens expressed by
the recipient, it is also important to determine whether the recipient has any
circulating HLA antibodies, as the presence of donor-specific HLA antibodies at
the time of transplantation may result in hyperacute rejection and loss of the
graft. Testing for HLA antibodies occurs both prior to and at the time of
transplantation, as follows:
1
During transplant
assessment/while on the waiting list – recipient
serum is screened for the presence of HLA antibodies using a number of
techniques with varying sensitivity.
2
At the time of the
transplantation – a cross-match test is performed
to make absolutely sure that the recipient does not have any
donor-reactive antibodies.
Screening Prior To Transplantation
Solid phase assays
ELISA-based assays –
ELISA (enzyme-linked immunosorbent assay) is performed by coating the wells of
a multi-well plate with purified HLA antigens. The recipient’s serum is placed
in these wells, incubated, washed and detected using a labelled anti-human IgG
antibody. This technique is more sensitive than complement dependent
cytotoxicity (CDC) and allows the identification of non-complement-fixing
antibodies.
Flow cytometric/luminex assays–the recipient’s serum is incubated with fluorescent beads that
have been pre-coated with HLA antigens. A secondary anti-human IgG antibody
labelled with a different fluorescent colour is added to identify beads with
anti- body bound, and the sample analysed by flow cytometry. This assay is even
more sensitive than ELISA-based techniques.
Calculated reaction frequency (cRF)
Having defined what HLA-specific antibodies are in the
recipient’s serum, the reaction frequency is calculated. This is the proportion
of a pool of 10, 000 blood group-identical organ donors against which the
recipient has HLA antibodies. A recipient is considered to be highly sensitised
if they have a cRF ≥85%, implying that they will be incompatible with more than
85% of all blood group-identical organ donors.
The cRF has replaced panel reactive antibodies (PRA) as
a measure of sensitisation. PRA was defined as the proportion of an arbitrarily
defined collection of lymphocytes (the panel) that underwent lysis when
recipient sera and rabbit complement were added. Hence the PRA test identifies
only complement-fixing anti-bodies and has low sensitivity.
Screening at the time of transplantation Cross-matching is used to identify the presence of complement-
fixing, donor-reactive HLA-antibodies in the recipient’s serum.
Cytotoxic (CDC) cross-match
A cytotoxic cross-match is performed by incubating the
recipient’s serum with donor T cells (expressing MHC class I antigens) and
donor B cells (expressing both MHC class I and class II antigens) and
complement. These B and T cells are usually obtained from donor lymph nodes or
spleen. If antibodies are present in the recipient’s serum, they will bind to
donor cells, activate complement, and cause lysis of donor cells by CDC. If T
and B cells are lysed, this indicates the presence of class I +/– class II
antibodies. If B cells alone are lysed it is indicative of the presence of MHC
class II antibodies, or a non-HLA binding antibody.
IgM and IgG donor-specific antibodies can be
distinguished by performing the cross-match in the presence or absence of
dithiothreitol (DTT). DTT disaggregates multimeric IgM. Thus, a CDC cross-match
that is positive in the absence of DTT but negative in the presence of DTT
suggests the presence of donor-specific IgM antibodies, which do not represent
a significant risk to the allograft. A positive T cell CDC cross-match
resulting from an IgM anti-body is not a contraindication to transplantation.
In contrast, a positive T cell CDC cross-match due to an IgG antibody precludes
transplantation and, should the transplant proceed, would likely result in
hyperacute rejection.
The importance of a positive B cell CDC cross-match in
the absence of a positive T cell CDC cross-match is less clear and must be interpreted
in the light of HLA antibody screening performed prior to transplantation. If
the recipient is known to have MHC class II antibodies, then a B cell CDC cross-match
is likely due to a complement-fixing class II antibody. Both endothelial cells
and renal tubular cells may express class II antigens, particularly during
inflammation, thus the presence of such antibodies should be considered to be a
contraindication to transplantation. Most class II antibodies are directed
against HLA-DR antigens. HLA-DP and DQ antibodies occur less frequently, and
may be variably pathogenic.
If a recipient is non-sensitised, and has no known
donor-specific antibodies (DSA), then an isolated positive B cell CDC is
unlikely to be due to a class II antibody, but may still indicate the presence
of a pathogenic antibody or autoantibody. B cells express surface monomeric IgM
(their B cell receptor) and also an Fcγ receptor (FcγRIIB), both of which may
bind non-HLA antibodies, which are usually autoantibodies. Historically,
non-HLA antibodies were considered not to be harmful to the graft; however,
there is increasing evidence that they may have a deleterious effect on
long-term graft function and survival.
Flow cytometric cross-match
CDC cross-match testing is effective in identifying the
presence of antibodies that would result in hyperacute rejection, but is not
sufficiently sensitive to identify all DSA. Some IgG isotypes do not fix
complement efficiently (e.g. IgG4) and will therefore not be detected by a CDC
cross-match, but might still damage the graft by activating phagocytes via
FcγRs. Flow cytometric cross- matching overcomes these limitations. It involves
incubating donor lymphocytes and recipient serum in the absence of complement,
and applying a fluorescently labelled secondary anti-human IgG antibody to
identify the presence of IgG bound to lymphocytes by flow cytometry. This
amplification step increases the sensitivity of the test compared with CDC
cross-matching. Cells are also incubated with fluorescently labelled antibodies
recognising B and T cells (e.g. anti-CD19 and CD3 antibodies respectively).
Thus, IgG antibodies binding T and/or B cells can be distinguished.
A positive T cell ‘flow’ cross-match in the presence of
a negative CDC cross-match usually reflects the presence of a lower titre of
MHC class I-binding DSA. Alternatively, it may indicate the presence of a
non-complement-fixing IgG isotype. In such cases, the antibody may not be
sufficient to mediate hyperacute rejection, but can cause early
antibody-mediated rejection (AMR) and would also be considered a
contraindication to transplantation.
The information obtained from antibody screening and the
cross- match allow an assessment of the risk of humoral alloreactivity.
Transplantation without a cross-match
The cross-match is time-consuming and increases cold
ischaemic times. In selected patients it may be safe to proceed to transplantation
without performing a cross-match. Such patients:
ü are receiving their first transplant;
ü have no history of sensitising events, such as blood transfusions or
pregnancies;
ü have no detectable HLA antibodies.
In such patients, the probability of a positive
cross-match is extremely low. The application of this strategy is dependent on
having up-to-date information about the HLA antibody status of recipients, and
thus requires potential recipients to be regularly screened for antibodies,
e.g. once every 3 months.
