Complement-dependent technique. The standard technique used to detect human leukocyte antigen (HLA)-A, -B, -C, -DR, and -DQ antigens has been the microlymphocytotoxicity test. This assay is a complement-dependent cytotoxicity (CDC) in which lymphocytes are used as targets because the HLA antigens are expressed to varying degrees on lymphocytes and a relatively pure suspension of cells can be obtained from anticoagulated peripheral blood. Lymphocytes obtained from lymph nodes or the spleen also may be used. HLA antisera of known specificity are placed in wells on a "Terasaki microdroplet tray." A concentrated suspension of lymphocytes is added to each well. If the target lymphocytes possess the antigen corresponding to the antibody present in the antiserum, the antibody will affix to the cells. Rabbit complement is then added to the wells and, when sufficient antibody is bound to the lymphocyte membranes, complement is activated. Complement activation injures the cell membranes (lympho-cytotoxicity) and increases their permeability. Cell injury is detected by dye exclusion: cells with intact membranes (negative reactions) exclude vital dyes; cells with permeable membranes (positive reactions) take up the dye. Sensitivity of the CDC assay is increased by wash techniques or the use of AHG reagents prior to the addition of complement. Because HLA-DR and -DQ antigens are expressed on B cells and not on resting T cells, typing for these antigens usually requires that the initial lymphocyte preparation be manipulated before testing to yield an enriched B-cell preparation. AHG—antiglobulin-augmented lymphocytotoxicity; RT—room temperature.

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