Flow cytometric detection of HLA antibodies using a spectrum of microbeads

We describe here the use of HLA antigen coated beads for specificity and class determination of HLA antibodies by flow cytometry. The HLA specificity of antibodies was determined by use of beads containing eight levels of fluorescence. HLA antigens isolated from eight cultured cells were coated onto these beads so that each bead was the equivalent of one cell. By using four sets of eight beads, an equivalent of 32 cells could be examined in four test tubes. A total of 76 class I and 25 class II specificities could be determined by the 32 class I bead-panel and 32 class II bead-panel used, respectively. We noted no cross-reactivity of reactions between class I and II. The sensitivity of the test was shown to be higher than that of the standard cytotoxicity by dilution experiments and detection of additional cross-reacting antigens. By use of these coated beads, we achieved improved standardized detection of HLA antibodies. Antigen-coated beads have several advantages over the use of spleens or lymphocytes. (a) A highly selected panel of antigens can be routinely used. (b) Class I and class II antibodies can be readily distinguished from each other, even when they are present as mixtures in one serum. (c) Non-HLA antibodies are not detected because the beads do not have any other antigens than HLA on them. (d) The quantity of antigens coated on beads is more uniform than that found in cells from different individuals. (e) Beads are more convenient for storage and daily use.