7.2
TITLE: ANTIBODY SCREENING WITH MULTIPLEXED POLYSTYRENE BEADS COATED WITH HLA CLASS I ANTIGENS

J. Crumpton, B. Lavingia, R. Vorhaben, P. Stastny.

Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX

Sensitization of transplant recipients is an important determinant of transplant outcome and has been traditionally analyzed by cytotoxicity with antiglobulin enhancement (CDC–AHG). Binding assays with immobilized purified HLA antigens appear to be more specific and more sensitive. In the present experiments we have compared results obtained by CDC–AHG, with an enzyme–linked immunoassay (ELISA) method (GTI) and with a binding assay on multiplexed polystyrene beads (MPB) analyzed by flow cytometry (One Lambda/Luminex). We studied 94 sera obtained from 72 kidney recipients and 22 patients being prepared for other organ transplants. Out of the 94, 33 were regraft patients. Tests performed with the three methods on 40 sera showed that ELISA was considered positive in 23, MPB in 21 and CDC–AHG in 16 of the 40 sera. Overall, the mean PRA obtained with 94 sera was 37.3% ± 0.345 for MPB and 28% ± 0.304 for CDC–AHG. The relative sensitivity of the assays was also compared by determining the endpoint obtained with serial dilutions of a potent alloantiserum, CA. The endpoint titer by CDC–AHG was found at a dilution of 1:320, for ELISA at 1:5120 and for MPB at 1:5120. The MPB method provides high throughput, simplicity and reproducibility. Specificity of HLA antibodies could be determined and were similar to those obtained by CDC–AHG in 23/36 (56%) of sera in which specific anti–HLA antibodies were recognized. Because of the ease of performance, accuracy and sensitivity, the MPB method has largely replaced the other techniques in our laboratory.