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#37-OR
DEFINING POSITIVE AND NEGATIVE REACTIONS IN THE SINGLE-HLA-ANTIGEN BEAD ASSAY.
J. Crumpton, R. Vorhaben, CHS, B. Lavingia, CHS, J. Tsai, MS and P. Stastny, MD. Dallas TX, UT Southwestern Med Cntr, Internal Medicine.
Micro particle beads coated with single HLA antigens make it possible to define more precisely the specificity of HLA antibodies in sera of sensitized patients. The results can be used to predict positive donor crossmatches and to estimate the probability of acceptable HLA mismatches. To reliably score the reactions, the median of the fluorescence intensity readings was converted to Equivalent Soluble Fluorescent Molecules (ESFM) using the Quantum R-PE beads and software (Bangs Labs) in order to decrease day-to-day and instrument variability. Next we corrected for antigen density differences from bead to bead and between lots, by testing with W6/32, a monoclonal antibody directed at the HLA class I heavy chain, and determined an antigen density (AD) correction factor, which was adjusted to the 75th percentile of the fluorescence intensities and ranged from 0.809 to 2.314. In all such assays there is some binding of normal sera to the beads. We therefore determined the binding of ten normal sera and used the mean plus three standard deviations to estimate the normal range for each bead. For scoring, the value for each serum was corrected by subtracting the no-antigen bead and AD-corrected ESFM were classified as definitely positive when greater than ten times the mean plus 3SD; definitely negative when less than two times the mean plus 3SD; and intermediate if corrected ESFM values were between two and ten times the mean plus 3SD. This methodology for determining the positive and negative thresholds of single-antigen bead tests provides accurate scoring of results which give comparable scores in multiple assays, correlates with predictions of positive and negative crossmatches and offers a semi-quantitative estimate of antibody strength.