Tissue Typing
Introduction
Key elements
Key elements for successful transplantation are:
ability to correctly identify tissue types of recipients and donors
prediction of graft rejection (host versus graft)
in the case of a bone marrow transplant, prediction of graft-versushost disease (GvHD).
Antigens of the MHC system are defined and reviewed by the WHO to ensure there is a common approach to nomenclature and typing.
Ideal match is an identical twin: few individuals requiring transplantation have such a donor.
Most transplants are from matched unrelated donors (MUDs) or parents/siblings with a close but not identical match.
In the case of a parental donor, this will usually only be a haploidentical match (half-identical) unless there is consanguinity in the family, in which case the match may be better.
Not all transplantation is affected identically by HLA matching.
For renal and bone marrow transplantation, the better the match, the better the graft function and the fewer the complications.
For liver transplantation, HLA matching is not beneficial.
For cardiac transplantation, HLA matching is impractical because of time constraints and door limitations.
Matching for living related donors is less critical than for cadaveric transplants.
If a poorly matched solid organ is transplanted, the recipient may require considerable immunosuppressive therapy to prevent rejection, increasing risks of secondary malignancies and opportunist infections.
Historical approach
Requirement for a high-resolution match in BMT and HSCT has led to the generation of highly specific techniques capable of identifying very minor changes in histocompatibility antigens.
Tissue typing used to be undertaken by two techniques.
HLA class I antigens were identified (and in many cases defined) by serology, using sera derived from multiparous women who often develop anti-HLA antibodies.
As this technique does not identify all class II antigens, cellular tests had to be used.
Current approach
Molecular biological techniques are now used.
Where antigens have previously been defined serologically, it is now clear from molecular genotyping that some antigens classed as completely distinct by serology are more closely related to each other than some specificities thought, on serological grounds, to be part of a closely related family (‘splits’ of an antigen).
Nomenclature has been changed to reflect the differences.
Molecular typing is critical in deciding whether a donor is a good match for a given recipient, as two different HLA-B antigens, defined
serologically, may differ by only one amino acid, and therefore represent a better match than two splits of the same antigen which may differ by five or more amino acids.
Process
In transplant matching there are two main steps:
tissue types of recipient (and donor) must be established
cross-match stage, in which the suitability of the proposed match is tested.
For renal transplantation, the donor will be tissue typed and blood grouped (ABO and rhesus (Rh)) and the recipient screened at regular intervals for the presence of anti-HLA antibodies.
If there are potential living related donors, these will be tissue typed and blood grouped and then the recipient’s sera will be tested against donor cells for anti-donor antibodies.
A good donor will be ABO compatible, with the best match of HLA antigens, and the recipient will lack anti-donor antibodies (preformed antibodies are a cause of hyperacute rejection).
If there is no suitable living related donor, the patient will be listed to receive a cadaver organ, with their clinical and immunological details stored on a central register.
This allows best use of cadaver organs across the country, as cadaver organs can be given to the best-matched recipients who are likely to derive most benefit.
The cadaver organ will have been ABO/Rh and HLA typed and recipients chosen on the best match if they have no pre-formed antibodies against the identified HLA antigens.
Immediately before transplant takes place, a fresh sample of the patient’s serum will be cross-matched against donor lymphocytes to check that no new antibodies have developed.
Normally, to ensure that there are sufficient donor lymphocytes to cross-match (as this may have to be done several times against different potential recipients), the spleen is removed to provide a source of cells.
Patients who have had previous grafts are often highly sensitized, and have high levels of antibodies, which can cause difficulty in identifying suitable donors.
Matching procedures: detecting pre-formed circulating antibodies
Cross-match allows detection of antibodies in the recipient that may affect graft viability.
It is used in the case of solid organs, where the patient’s serum is tested against donor cells.
Antibodies of interest are mainly IgG anti-HLA class I and anti-HLA class II antibodies.
IgM antibodies are often (but not always) considered to be autoantibodies whicht may cause false-positive responses that are not deemed significant to the outcome of the transplant unless there has been a recent sensitizing event.
Microlymphocytotoxicity
Recipient’s serum is incubated with donor cells in the presence of complement and the wells are scored for cytotoxicity.
To control for autoantibodies, the donor’s cells are also tested.
To distinguish anti-HLA class I and anti-HLA class II antibodies, T cells and B cells are run separately since B cells express higher levels of HLA class I antigens.
Positive IgG anti-T-cell antibody is generally regarded as a contraindication to transplantation because of risk of hyperacute rejection and increased incidence of early vascular rejection.
B-cell reactivity may occur in the absence of a positive T-cell match.
IgM antibodies can be detected by performing the assay in the presence of dithiothreitol or dithioerythritol to disrupt pentameric IgM.
If a positive cross-match becomes negative in the presence of these agents, an IgM antibody is likely.
Performing tests at 37°C helps eliminate cold-reactive antibodies that are often non-specific.
ELISA
Purified HLA class I or class II antigens are coated on to microtitre wells and the patient’s serum is added.
Bound antibody is identified using labelled anti-human IgG ± IgM antibodies.
Less sensitive and specific than flow cytometry but more suitable for testing large numbers of samples.
Flow cytometry
Flow cytometry can be used to sensitively and specifically identify antibodies to HLA class I and II.
Donor lymphocytes are incubated with the patient serum’s and then washed.
They are then incubated with fluoresceinated anti-human IgG or IgM and anti-CD3 or anti-CD19 antibodies conjugated to a different fluorochrome.
Analysed on flow cytometer using dual-colour fluorescence.
Magnetic bead technique
Newer and quicker method using magnetic beads coated with specific class I and class II antigens (Luminex® technology).
Beads are incubated with recipient serum and then fluoresceinated anti-human IgG or IgM, separated with a magnet, and analysed in flow cytometer.
Positive reactions may be diluted to assess titre of antibodies.
Allows distinction between IgG and IgM antibodies.
Identifies both complement-fixing and non-complement-fixing antibodies.
Complement-fixing antibodies are more likely to have a major deleterious effect.
Increased sensitivity picks up weaker antibodies, making positive results harder to interpret.
Excludes non-HLA antibodies, which may cause false-positive lymphocytotoxic cross-matches but do not affect graft suitability.
Monitoring
Patients on waiting lists for renal transplants will be screened at intervals by microcytotoxicity and other antibody screening techniques, which should be as sensitive as the cross-matching technique against panels of pre-typed cells to identify the presence of any anti-HLA antibodies.
This speeds up the cross-matching, as cadaver grafts which lack the antigens recognized can be selected.
Donor-specific cross-matching has limited relevance to liver transplants since they are relatively resistant to humoral rejection.
Liver allograft may even protect a subsequent kidney transplant from hyperacute rejection.
Mixed lymphocyte reaction (MLR)
MLR is an in vitro reaction to analyse recipient T-cell response to foreign HLA and non-HLA molecules predicting the likelihood of T-cell-mediated graft rejection, which may not be predicted from HLA typing alone. This has been used in the assessment of potential bone marrow donors but is now no longer in routine use.
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