Comparison of EDTA versus DTT treatment in overcoming the prozone effect in a Luminex-based HLA antibody assay

Abstract

Aim One of the limitations of solid-phase HLA antibody assays is the false low/negative reading in a subset of samples with high titer/strength HLA antibodies, a phenomenon known as the prozone effect. The goal of this study was to compare the efficacy of ethylenediaminetetraacetic acid (EDTA) vs. dithiothreitol (DTT) treatment of serum samples in overcoming the prozone effect in solid-phase HLA antibody assays on the Luminex platform. Methods A total of 21 serum samples were tested by single-antigen bead assays (One Lambda), including 14 previously identified prozone samples (3 class I, 11 class II), 6 previously identified non-prozone samples with HLA antibodies (3 each for class I and II), and 1 negative sample. Serum was treated either by adding EDTA to a final concentration of 5 mM, or by incubating with 5 mM DTT for 30 min at 37 °C. To evaluate the efficacy of prozone effect reversal on HLA single antigen beads assay, fourfold serial dilutions, from neat to 1:256, were tested for each positive sample, using PBS as a diluent. Results Prozone effects were reduced in all 14 samples by either EDTA or DTT treatment. Results revealed sharp increases of MFI values in treated serum samples at neat, compared to non-treated samples. However, EDTA and DTT treatment were not equally efficient in overcoming the prozone effect. EDTA had nearly 100% reversal of prozone effect in all the tested samples, estimated by the ratio of MFI at neat to peak MFI at any given dilution. In contrast, the efficacy by DTT treatment ranged from 47% to 100%. The difference between the treatments was particularly notable in samples with strong prozone effects, defined by peak MFI values beyond a 1:16 dilution with untreated samples. Consequently, EDTA-treated serum samples showed virtually no further MFI increase when diluted, while some DTT-treated serum samples still had MFI increases when diluted, an indicator of residual prozone effect. For the negative control sample as well as non-prozone samples, comparable results were obtained from EDTA, DTT and non-treated serum. Conclusions Our study indicates that EDTA is more efficient in reversing the prozone effect, compared to DTT treatment. Furthermore, the stability of EDTA and the simplicity and robustness of serum treatment provide additional advantages over DTT for routine clinical testing.