6.0000
#45-OR
IN VITRO EVALUATION OF HLA-SPECIFIC B CELLS.
Nancy D. Rossiter, Ian Crosby, PhD, Michael Bunce, PhD, Mary S. Leffell, PhD and Andrea A. Zachary, PhD. Baltimore MD, USA, Johns Hopkins University, 21205, Medicine; Oxford United Kingdom, Dynal Biotech and Oxford United Kingdom, Oxford Transplant Centre, Surgery.

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The ability to stimulate, exclusively, HLA-specific B cells , in vitro, would provide opportunities to study B cell regulation and assess historic sensitization to donor antigens. We have used the in vitro CMI® Cylex assay (Cylex, Columbia MD), which assesses activation by measuring ATP release, on CD19+ cells stimulated with PHA, anti-CD40 antibody (Ab) alone, beads coated with purified HLA-A2 molecules (Dynal Biotech Ltd. UK) with or without anti-CD40 Ab, or diluent. Cells were obtained from 3 groups: healthy, unsensitized individuals (G1, n=15), sensitized patients not known to have current or historic A2-specific Ab (G2, n=2), and patients with current or historic anti-A2 Ab (G3, n=5). PHA stimulation yielded a higher mean value in the G1 group (534) than in either the G2 (342) or G3 (315) groups. Stimulation with A2 beads yielded a higher mean value in the G3 group (242.6) than in either G1 (94.7) or G2 (90.5). As expected, using whole blood from patients with A2-specific antibody reduced activation, most likely because of the binding of antibody to the A2 beads. Because mean positive and negative control values varied among the groups, attempts were made to develop algorithms to normalize the data. No algorithm consistently yielded higher values, with A2 bead stimulation, for the G3 group than for G1 and G2 groups, however, collectively, the algorithms indicated greater A2-specific stimulation in the G3 group. These data suggest using HLA-coated beads as stimulators in the Cylex CD19 assay has promise for the ability to assess HLA-specific B cells. However, it may be necessary to standardize cell numbers in order to obtain meaningful comparisons among assays.