HLA Typing

The human leucocyte antigen (HLA) loci are a part of the genetic region known as the major histocompatibility complex (MHC). This  system, encoded by two highly polymorphic gene families located within a 3600 kb region located at chromosome 6p21.3, plays a critical role in regulating the immune response.HLA region on hromosome 6p21.3. Adapted from Mehers & Gillespie, 2008.  The ‘classical’ HLA genes are the most polymorphic in the human genome, with a large number of allelic variants at each locus. Allelic variation is maintained at the population level due to the survival advantage conferred, with marked inter-individual differences in immune responses to foreign antigens. As a consequence of its role in immune regulation, the HLA system also constitutes an immunological barrier which must be avoided or otherwise overcome in clinical transplantation. In the last twenty years there has been an exponential growth in the application of DNA technology to the field of histocompatibility and immunogenetics. Histocompatibility between the patient and donor is a prerequisite for the success of haematopoietic stem cell transplantation.

In haematopoietic stem cell transplantation allele-level typing needs to evaluate compatibility for the HLA-A,B,C Class I and DRB1 and DQB1 Class II loci in the average transplant program because it is well established that mismatches at certain HLA loci between donor-recipients are closely linked to the risk of graft versus host disease. Resolution at an antigen level in solid organ transplantation is currently sufficient for HLA-A,B and DR antigens and it could be achieved by serological or molecular biology techniques. In solid organ transplantation the definition of antibodies in the recipient to HLA antigens is more important and it was performed primarily by serological technique and more recently by solid phase immunoassays that are more sensitive and specific.

DETERMINING HLA TYPE

Methods for determining individual HLA polymorphisms or ‘HLA typing’ have evolved enormously since the discovery of the human major histocompatibility complex and have developed in parallel with, and contributed to, the unravelling of the genetic complexity of this region, such that over 2000 alleles of the classical HLA class I (A, B and C) and class II (DR, DQ and DP) loci are now known. Methods for HLA typing are either based on detection of genetic variation in the expressed HLA molecules (serological typing), or now almost universally, at DNA sequence level (DNA typing).

Serological HLA typing

HLA typing methods were originally based on the detection of expressed HLA molecules on the surface of separated T cells (HLA class I) and B cells (HLA class II) using panels of antisera, usually obtained from multiparous women in a complement dependent cytotoxicity test. Such ‘serological’ HLA typing suffers from a number of drawbacks. Live lymphocytes are required, and lymphocyte counts can be low in some transplant patients. Panels of antisera must be maintained, although commercial kits are now available. Finally, the typing resolution obtainable from serological methods is low. While good serology may provide a level of resolution adequate for renal transplant HLA typing, it is inadequate for stem cell transplant matching and therefore has been largely superseded by DNA-based typing in clinical HLA laboratories. However, serological typing still has a useful role as an adjunct to DNA-based typing, for example to determine whether a particular HLA allele is actually expressed at the cell surface. A number of such non-expressed ‘null’ HLA alleles are now known.

DNA-based HLA typing 

DNA-based typing methods offer a number of advantages over serological typing methods. Live lymphocytes are not required and DNA is easily extracted from any nucleated cell, although peripheral blood lymphocytes are the usual source. DNA is easily stored, allowing repeat sample testing when required. A number of different DNA-based HLA typing methods are in everyday use in clinical HLA typing laboratories, all of which are based on PCR amplification of target sequences in the HLA genes under investigation. PCR primers and oligonucleotide probes can be designed and validated in-house, or purchased commercially. As such, unlike antisera, they are a renewable resource.

PCR with sequence specific primers (PCR-SSP)

One commonly applied approach is to use panels of ‘sequencespecific primers’ which amplify particular HLA alleles or allele groups. The presence or absence of a particular allele is determined by the presence or absence of DNA amplification by a particular primer pair, as determined by agarose gel electrophoresis.SSP HLA Typing This method, termed PCR-SSP, is rapid and ideally suited to deceased donor typing. The method is usually used at ‘low resolution’ to detect allele groups, but secondary panels can be used to achieve allele level typing. However, allelic typing is cumbersome using this approach. Due to logistical considerations the method is unsuited to typing large numbers of samples.

PCR with sequence specific oligonucleotide probes (PCR-SSOP)

Another commonly employed approach is to detect HLA polymorphisms in locus-specific PCR products using short oligonucleotideDNA‘ probes’ in a hybridisation assay. In its original form, PCR-SSOP typing is most appropriate for typing large numbers of samples in batches, since multiple oligonucleotide probes are required per locus. A recent variant of this technique utilises probes coupled to fluorescently labelled microbeads in a flow cytometric assay using X-Map technology (Luminex), and this approach is now being used increasingly for clinical HLA typing. In this format, the method is suitable for typing small or mediumnumbers of samples.

Sequence-based typing

Sequence-based typing (SBT) can also be used to achieve allelic level HLA typing as required for stem cell transplantation programmes. SBT is also required for investigation and confirmation of new allelic sequences.
A number of other methods are in use some clinical histocompatibility & immunogenetics laboratories, although none of these are widely used.

ANTIBODY IDENTIFICATION

The deleterious effect of antibodies in renal transplant hyperacute rejection is well documented. However, the deleterious role of antibodies in all forms of solid graft rejection has become more apparent over the last few years, driven by the development of new assays, such as detection of intragraft C4d deposition.
In addition, new assays have been developed, allowing the identification of previously undetectable antibody specificities.
Antibody detection methods also need to be as sensitive as the crossmatch technique employed.

HLA antibody screening by flow cytometry/X-map techniques

Currently considered to be the gold standard for antibody screening and identification, these assays rely on either soluble or recombined HLA molecules bound to polystyrene particles. The X-Map (Luminex) assay relies on two different recording systems. The first is a laser whichd etects differences in 100 individual polystyrene particles, each stained with varying amounts of two different
fluorochromes, giving a unique but reproducible gated position. Each bead set is then bound to either a mixture of HLA antigens or a single HLA antigen. Beads are then mixed with patient serum to
allow antibody binding. A second anti-human antibody linked to a reporter molecule is then added to the reaction mixture, and the reporter fluorescence measured with another laser, giving a semiquantitative level of antibodies in the patient sample. beadDetection
Luminex and flow cytometric assays can be used for both antibody screening or identification in different formats. Both complement fixing and non-complement fixing antibodies can be detected by flow cytometric screening techniques. Luminex assays are able to independently identify HLA class II specificities in the presence of HLA class I specificities. Single antigen Luminex tests or flow beads can also be used to determine specific antigen reactions in high panel reactive samples, allowing determination of acceptable HLA mismatches and increasing chances of transplant.
New antibody identification techniques have allowed determination of HLA antibodies in transplant patients with failing grafts in all forms of solid organ transplantation. Furthermore, many previously unexplainable flow cytometric positive crossmatches can be explained by antibody identification using flow based antibody identification techniques.

LABScreen reagents are powered by Luminex xMAP technology, a microbead platform used to deliver multiplex antibody assays. This antigen-bead based assay allows for a precise determination of antibody profiles against HLA and MICA. AntiBodyDetectionThe proven reliability of LABScreen’s consistency, high sensitivity and robustness for PRA screening has gained rapid momentum in the transplant community. The LABScreen product line consists of color-coded microbeads coated with purified HLA Class I, Class II and MICA antigens. The beads are analyzed using Luminex xMAP multiplex technolog.

 

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