EDTA Treatment of Serum Unmasks Complement-Mediated Prozone Inhibition in Human Leukocyte Antigen Antibody Testing

Abstract

Objectives: Luminex-based single-antigen bead human leukocyte antigen (HLA) antibody testing is widely used to define HLA antibodies for transplant compatibility. False-negative results can occur with complement-mediated prozone inhibition. This study assessed the effect of EDTA on the assay background reactivity and fluctuations in antibody mean fluorescent intensity. Methods: Serum specimens were retrospectively tested using Luminex-based single-antigen beads with and without EDTA. Treated and untreated serum samples were compared by two measures: changes in background reactivity and changes in HLA antibody strength after EDTA treatment. Results: Ten pretransplant and 48 posttransplant specimens were identified: lung (22), heart (10), kidney (21), heart/lung (two), pancreas (one), small bowel (one), and liver (one). After EDTA treatment, weak antibodies (below 2,000 mean florescent intensity) demonstrated the largest fluctuations. Newly identified HLA antibodies were seen in 16% (8/49) of class I and 26% (15/57) of class II beads. EDTA treatment did not result in false-negative reactions compared with untreated serum. Conclusions: EDTA serum pretreatment mitigated complement-mediated prozone inhibition and improved accurate HLA antibody detection. The background reactivity and the false-negative rate of the assay appear unchanged.