Chapter 9 Answers to discussion questions

9.1 Explain why HLA polymorphism is important to the human species.

Human leucocyte antigen polymorphism is pathogen driven and HLA class I and II molecules have evolved to present peptides from a wide variety of different pathogens to the immune system. At a population level it is important that there are many different HLA alleles so that there should be some individuals able to respond to a pathogen and live to maintain the survival of the species.

 

9.2 Outline the methods available for HLA typing.

The first HLA typing methods were serological, based on the detection of cell surface HLA molecules by specific antibodies. Serological typing is still used, as many commercial companies utilize monoclonal antibodies directed against different HLA specificities. The principle of the typing technique, termed the lymphocytoxicity test (LCT) or complement dependent cytotoxicity (CDC), is to incubate the lymphocytes with the appropriate antisera. Then rabbit complement is added and after further incubation the reaction is stopped and the reactions read. Where there has been a specific antigen:antibody reaction, the complement would have disrupted the cell membrane allowing the entry of dyes that can be visualized microscopically. DNA-based HLA typing was introduced to overcome the limitations of serological HLA class II typing, essentially the problems of obtaining good, specific antisera. DNA-based typing for HLA class II molecules was introduced before HLA class I typing. The main methods used are: sequence specific primer (PCRSSP), which requires the design of short sequences of DNA or primers specific for a particular HLA allele or groups of alleles that can serve as templates for DNA synthesis when that particular allele is present. Sequence specific oligonucleotide probes (PCR-SSOP), which require the design of strands of DNA that bind in the region of the HLA allele that is specific for that allele or allele group. The probe can be labelled with a fluorescent or radioactive tag so that specific binding can be detected. Sequence based typing (PCR-SBT) involves determining the nucleotide sequence of the HLA alleles of an individual. The sequencing is usually restricted to the region of greatest polymorphism, the peptide binding cleft; in HLA class I exons 2 and 3 code for this region of the molecule and in HLA class II molecules, exon 2.

 

9.3 Outline the methods available for the detection of HLA antibodies.

The LCT technique, as described for HLA typing, can be used for antibody screening but in this process the panel of well-characterized sera is replaced by a panel of HLA typed donor lymphocytes, employed to detect antibody of unknown specificity. This procedure is still used because it is simple, reproducible, and relatively inexpensive, but cannot detect non-complement fixing antibodies that are also clinically significant, and is probably the least sensitive technique available. False positive reaction can occur when testing patients receiving antibody therapy such as antithymocyte globulin (ATG), or anti-CD3 therapy. ELISA is also a relatively simple technique, with an objective readout and is suitable for testing large batches of samples. This technique can indicate the presence of HLA specific antibody as it is a not affected by non-HLA antibodies such as ATG. Patient serum or plasma is added to the test lymphocytes and after incubation, to allow antibody antigen binding to take place, excess serum is washed off and an antihuman immunoglobulin conjugated to an enzyme such as horse radish peroxidise is added. This binds to any HLA antibody-antigen complex and when the enzyme substrate is added, the breakdown of the substrate is linked to a colour change which can be read in an ELISA reader. The flow cytometry method uses the same principle of the LCT and ELISA methods; a panel of HLA typed lymphocytes or purified HLA molecules immobilized on beads is mixed with patient sera or plasma and incubated to allow antibody binding to take place. The detection of bound antibody is achieved using a fluorescently labelled anti-human Ig antibody. In the flow cytometer the cell suspension is passed through a laser, which excites the fluorescent tag on the cell surface and the light emitted, at a specific wavelength governed by fluorescent label, is detected and measured by the machine. Luminex technology is the most widely used technique for both HLA specific antibody detection and definition of specificity. In this system fluorochrome-dyed polystyrene beads are coated with specific HLA antigens which are then used to detect the antibodies in the serum or plasma. Antibody detection using Luminex has been shown to be more sensitive than either CDC or ELISA, although the clinical benefit of this increased sensitivity is not yet clearly defined, particularly in the transplant setting.

 

9.4 What level of HLA matching is required for major solid organ and stem cell transplants?

In the UK kidneys are normally allocated on the basis of blood group and HLA type where priority is given to donor recipient pairs where there is no (0) mismatch at HLA-A, B, and DRB1, that is, 000. Paediatric patients are given first priority, then the level of sensitization is taken into account. Other factors that are also considered in the allocation algorithm are: waiting time, HLA match and age combined, donor-recipient age difference, location of the patient relative to the donor, HLA-DRB1 and HLA-B homozygosity, and blood group match. For pancreatic transplants blood group matching is a priority over HLA matching. Heart and lung: HLA matching, particularly for HLA-DRB1, has a beneficial effect on graft survival but blood group and organ size are the most important factors for allocation of organ. Livers are allocated on the basis of blood group compatibility and matching of O group donors for O group patients where possible. Human leucocyte antigen matching plays no role in the allocation of livers for transplantation. For adult unrelated stem transplantation the matching requirements are more stringent, as haemopoietic stem cell transplantation involves transfusing immunocompetent cells into an immunosuppressed recipient and any HLA mismatches could be recognized as foreign by the donor lymphocytes and lead to graft versus host disease (GVHD) which can be fatal. Most centres would aim to match for both alleles at the HLA-A, B, C, DRB1, and DQB1 loci, so they describe a fully matched donor as a 10 out of 10 match, and would aim to achieve at least a 9 out of 10 match. HLA-DP is taken into account by some transplant centres where there is a choice between equally matched donors.