**3.1. Lymphocytotoxicity (serological testing)**

In this serological test, lymphocytes are added to sera, which may or may not have antibodies directed to HLA or other cell surface antigens [13]. 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. If there is a cell bound antibody that is able to fix complement, the complement pathwayis activated causing 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 antigens and ease of isolation compared to most other tissue. The most important use of this test is to detect specific donorreactive antibodies present in a potential recipient prior to transplantation.

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 have led to the introduction of DNA-based methods.

#### **3.2. Mixed Lymphocyte Culture (MLC)**

When lymphocytes from two individuals are cultured together, each cell population is able to recognize the "foreign" HLA class II antigens of the other. As a response to these differences, the lymphocytes transform into blast cells, with associated DNA synthesis. Radio-labelled 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 HLA-D antigens. The "HLA-D" defined in this way is actually a combination of HLA-DR, DQ and DP.

An important use of the MLC is in its use as a "cellular crossmatch" prior to transplantation especially bone marrow. By testing the prospective donor and recipient, an in-vitro transplant model is established which is an extremely significant indicator of possible rejection or Graft-Versus-Host reaction.
