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Detecting HLA Antibodies

Detecting HLA Antibodies
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.

Detecting HLA Antibodies

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.