Tissue Typing Education Primer

Part 4 of 6

Move to Part 1(Intro), Part 2(Antigens), 3(Genetics), 5(Relevance),6(References)

METHODS OF TESTING FOR HLA ANTIGENS

Lymphocytotoxicity
In the lymphocytotoxicity test, lymphocytes are added to sera which may or may not have antibodies directed to HLA antigens.
If the serum contains an antibody specific to an HLA (Class I or Class II) antigen on the lymphocytes, the antibody will bind to this HLA antigen. Complement is then added. The complement binds only to positive cells (ie where the antibody has bound) and in doing so, causes membrane damage. The damaged cells are not completely lysed but suffer sufficient membrane damage to allow uptake of vital stains such as eosin or fluorescent stains such as Ethidium Bromide. Microscopic identification of the stained cells, indicates the presence of a specific HLA antibody.

The cells used for the test are lymphocytes because of their excellent expression of HLA and ease of isolation compared to most other tissue.

The most important use of this test is to detect specific donor-reactive antibodies present in a potential recipient prior to transplantation.

Historically, this test has long been used to type for HLA Class I and Class II antigens, using antisera of known specificity. However, the problems of cross-reactivity and non-availability of certain antibodies has led to the introduction of DNA based methods. Currently, many laboratories have changed to molecular genetic methods for HLA Class typing.

Mixed Lymphocyte Culture (MLC)
When lymphocytes from two individuals are cultured together, each cell population is able to recognise the "foreign" HLA­class II antigens of the other. As a response to these differences, the lymphocytes transform into blast cells, with associated DNA synthesis. Radio­labelled thymidine, added to the culture, will be used in this DNA synthesis. Therefore, radioactive uptake is a measure of DNA synthesis and the difference between the HLA Class II types of the two people.

This technique can be refined by treating the lymphocytes from one of the individuals to prevent cell division, for example by irradiation. It is thus possible to measure the response of T lymphocytes from one individual to a range of foreign lymphocytes. It has thus proved possible by using the mixed lymphocyte culture (MLC) test to use T lymphocytes to define what were previously called HLA­D antigens. The"HLA­D" defined in this way is actually a combination of HLA­DR,DQ and ­DP.

An important use of the MLC is in it's use as a "cellular crossmatch" prior to transplantation ­ especially bone marrow. By testing the prospective donor and recipient, an in­vitro transplant model is established which is an extremely significant indicator of possible rejection or Graft­Versus­Host reaction.


Molecular Genetic Techniques
RFLP
Restriction Fragment Length Polymorphism (RFLP) methods rely on the ability of certain enzymes to recognise exact DNA nucleotide sequences and to cut the DNA at each of these points. Thus the frequency of a particular sequence will determine the lengths of DNA produced by cutting with a particular enzyme.

The DNA for one HLA (Class II) antigen, eg DR15, will have these particular enzyme cutting sites (or "restriction sites") at different positions to another antigen, eg DR17. So the lengths of DNA seen when DR15 is cut by a particular enzyme, are characteristic of DR15 and different to the sizes of the fragments seen when DR17 is cut by the same enzyme.

Polymerase Chain Reaction.
The Polymerase Chain Reaction(PCR) is a recently developed and revolutionary new system for investigating the DNA nucleotide sequence of a particular region of interest in any individual. Very small amounts of DNA can be used as a starting point such that it is theoretically possible to tissue type using a single hair root. Sequencing DNA has been transformed from a long and laborious exercise to a technique that is essentially automatable in the not too distant future.

The first step in this technique is to obtain DNA from the nuclei of an individual. The double stranded DNA is then denatured by heat into single stranded DNA. Oligonucleotide primer sequences are then chosen to flank a region of interest. The oligo- nucleotide primer is a short segment of complementary DNA which will associate with the single stranded DNA to act as a starting point for reconstruction of double stranded DNA at that site.

If the oligonucleotide is chosen to be close to a region of special interest like a hypervariable region of HLA­DRB then the part of the DNA, and only that part, will become double stranded DNA when DNA polymerase and deoxy-ribonucleotide triphosphates are added. From one copy of DNA it is thus possible to make two.
Those two copies can then, in turn, be denatured, reassociate with primers and produce four copies. This cycle can then be repeated until there is sufficient of the selected portion of DNA to isolate on a gel and then sequence or type.

There are a number of PCR based methods in use. For example:-

Sequence Specific Priming (SSP)
In this test, the oligonucleotide primers used to start the PCR have sequences complimentary to known sequences which are characteristic to certain HLA specificities.

The primers which are specific to HLA-DR15, for example, will not be able to instigate the PCR for HLA-DR17. Typing is done by using a set of different PCR's, each with primers specific for different HLA antigens.

Sequence Specific Oligonucleotide (SSO) Typing
By this method, the DNA for a whole region (eg the HLA DR gene region) is amplified in the PCR. The amplified DNA is then tested by adding labelled (eg Radioactive) oligonucleotide probes, which are complementary for DNA sequences, characteristic for certain HLA antigens. These probes will then "type" for the presence of specific DNA sequences of HLA genes.

Move to Part 1(Intro), 2(Antigens), 3(Genetics), 5(Relevance), 6(References)