tests have been developed that use a biologic response of lymphocytes to foreign antigens as a measure of histocompatibility.

The MLR test provides an in-vitro model in which lymphocytes can be used both as responders (recipient cells) to foreign HLA-alloantigens and as stimulators (donor cells) carrying those antigens.11 For example, lymphocytes of the recipient are mixed with lymphocytes of the donor, after donor lymphocytes have been treated with mitomycin C or xirradiation to inhibit DNA synthesis. The response of the recipient cells to these treated donor cells in the MLR is measured by the incorporation of radioactive thymidine into the responding cells during cell division. If the responding cells have been stimulated, large amounts of labeled thymidine are incorporated into the newly synthesized DNA. This incorporation can be quantitated in a liquid scintillation counting device. Appropriate controls and replicate samples must be tested. The MLR reaction recognizes the antigenic differences on stimulator cells; therefore, cells bearing the same antigens as the responding cell will cause no stimulation. This indicates that the stimulator and the responder cells have the same HLA-D type.

HLA Antibody Detection Techniques

Detection and identification techniques of HLA antibodies are similar to those for RBC antibodies. The unknown serum is tested against a panel of cells or soluble antigen of known HLA phenotype. Targets from a large panel of donors must be selected if antibodies to all HLA specificities are to be detected. A panel of at least 30 carefully selected targets is required for initial screening in the determination of panel reactive antibody (PRA), and a panel of at least 60 cells is required for accurate antibody identification.

Microlymphocytotoxicity
The method depends on the purpose of the screen. When seeking alloantisera as typing reagents, it is essential to screen using the method for the typing procedure, the standard CDC being the most common. When screening recipient serum samples, a more sensitive techniqueAmos-modified, extended incubation, or antihuman globulinshould be employed. The Amos-modified technique introduces a wash step after the initial serum-cell incubation. The wash step removes anticomplementary activity: aggregated immunoglobulin in the serum that can activate complement, making it unavailable for binding on the cell membranes. Standard CDC tests rarely detect 100 percent of the antigenbinding specificities of cross-reactive antibodies.45,46 Sensitivity of the CDC test can be greatly enhanced by the addition of an antihuman globulin reagent following serum and cell incubation.45 The addition of goat antihuman kappa chain increases the likelihood of complement binding and subsequent cell injury, especially in circumstances in which the amount of HLA-antibody binding is below the threshold of detection in the standard technique. The antihuman globulin test functions like a complement-independent technique with respect to HLA alloantibodies. If antibodies to class II molecules (DR and DQ) are to be identified, then separated or labeled B-lymphocyte suspensions of known phenotype must be used. Serum can be screened using freshly prepared lymphocytes or lymphocytes frozen in bulk or in trays. Lymphocytes frozen in trays have the advantage of rapid

preparation, enabling serum to be screened in just a few hours. The lymphocytotoxicity assay has several disadvantages. First, viable T and B lymphocytes are essential for an accurate assessment of the presence of antibody. Many laboratories routinely use frozen cells to maintain a consistent, representative panel of antigens. The process of freezing renders lymphocytes more fragile and susceptible to cell lysis, resulting in false positives. A second problem is the necessity to maintain a reliable and consistent antigen panel that reflects the ethnic composition of the patient population; HLA antigen frequencies are known to vary among different races. The requirement for activation of complement in the cytotoxicity assay results in a third disadvantage, the inability to detect noncomplementfixing antibodies. Differentiation between HLAspecific and nonHLA-specific antibodies and between IgG and IgM antibodies is not possible with the standard cytotoxicity assays. Two new techniques have been introduced as alternatives to the lymphocytoxicity assay for the detection of anti-HLA antibodies. These techniques overcome many of the pitfalls of the standard cytotoxicity assays. They employ ELISA and flow cytometry.

ELISA
The ELISA test uses purified HLA antigens