Recipient lymphocytotoxic HLA antibodies to donor antigens have been associated with accelerated graft rejection and with

poor response to platelet transfusion. Antibody in donor plasma to recipient leukocytes has been associated with severe pulmonary infiltrates and respiratory distress following transfusion. Thus, the clinical management of patients, pre- and post-transplant, includes screening for and determination of the specificity of anti-HLA class I and II antibodies that may be present. Crossmatching involves serologic and cellular procedures. Serologic crossmatching is performed by cytotoxicity and flow cytometric techniques.39 Enzyme-linked immunoadsorbent crossmatch assays (ELISA) are in development. Lymphocyte-defined compatibility is determined by the mixed-lymphocyte reaction or one of its modifications. Over the past 10 years, practical techniques have been developed to characterize gene structure and specific alleles. The techniques of molecular genetics have revolutionized molecular biology. The three most common molecular assays employed in the clinical laboratory are sequence-specific oligonucleotides (SSO), sequence-specific primers (SSP), and sequence-based typing (SBT).

HLA Antigen Detection

The agglutination methods initially used to define the HLA complex have been succeeded by a precise microlymphocytotoxicity test.40 Cytotoxicity techniques require only 1 to 2 _L of serum and are sensitive and reproducible. Acid-citrated dextrose or phenol-free heparinized blood is used for testing. A purified lymphocyte suspension is prepared by layering whole blood on a Ficoll-Hypaque gradient and centrifuging. Residual RBCs and granulocytes are forced to the bottom of the gradient, and platelets remain in the supernatant. Lymphocytes collect at the gradients interface and can be harvested, washed, and adjusted to appropriate test concentrations (Fig. 2210). HLA-A, HLA-B, and HLA-C typing is performed on this lymphocyte suspension. HLA-DR typing requires a purified B-lymphocyte suspension. B-lymphocyte suspensions were generally prepared in two ways: 1. Nylon wool separation41,42 2. Fluorescent labeling43 The nylon wool separation of B lymphocytes was based on the observation that B cells adhere preferentially to nylon wool, from which they can be eluted, whereas T lymphocytes do not adhere to wool. Fluorescent labeling involved the incubation of lymphocytes with fluorescein isothiocyanatelabeled antiimmunoglobulin. B lymphocytes developed distinct fluorescent caps because of the binding of the labeled anti-immunoglobulin to the cell surface immunoglobulin found on B cells and not on T cells. A major advantage of the fluorescent labeling technique was that it did not involve the physical separation of T and B cells, in contrast to nylon wool separation. Techniques currently used are immunomagnetic beads to positively select (target cells rosetted on beads) lymphocyte subpopulations for use in HLA typing, both for class I and II antigens.44 These techniques provide rapid isolation of cells with a high degree of purity and use immunofluorescence lymphocytotoxicity. The technique for isolation of either T or

B cells depends on the MoAb for bead coating. HLA testing is performed in 60-well or 72-well microtiter trays. Antiserum test trays are prepared by dispensing 1 _L of serum into the bottom of each well, which contains mineral oil. Mineral oil is used to prevent evaporation of antisera during test incubations. Antiserum trays are frozen at _70_C until just before use. Upon use, they are removed from the freezer and thawed for 3 to 5 minutes. Every laboratory performing