Reason and Resolution of High Negative Control Beads in Solid-Phase Immunoassay.

OBJECTIVES: The Luminex technology is the most sensitive diagnostic method for HLA antibody detection and identification. However, the interpretation of immunoassays is commonly affected by the artifact, and non-specific background. Sera from some patients show high negative control bead (NC) value, which makes assessing and interpretation of HLA antibodies difficult. In this study, we evaluated the effect of Adsorb Out reagent, dithiothreitol (DTT), and Ethylenediaminetetraacetic acid (EDTA) on the NC median fluorescence intensity value by comparing treated versus untreated patient sera. In addition, we wanted to identify whether kidney disease and administered medication influenced high NC median fluorescence intensity values by comparing patient versus control results.

MATERIALS AND METHODS: HLA antibody screening was performed on 3500 serum samples. Sera were analyzed using the standard protocol for Luminex antibody screening. Sera with high NC values were preincubated with Adsorb Out, DTT, and EDTA. Screening of these sera was then performed.

RESULTS: We found that 4% of samples showed high NC values. Adsorb Out, DTT, and EDTA decreased the NC values at 723.5 (299.25-1443) versus 85 (34-218; P < .001), at 723.5 (299.25-1443) versus 184 (106-597; P < .001), and at 723.5 (299.25-1443) versus 455 (131-1177; P = .004). These succeeded in bringing back NC values to normal range in 69.2%, 43%, and 30% of treated sera, respectively. In addition, the differences of corticoids, immunosuppressive, and heparin drugs between patients and controls were statistically significant (P < .001, < .001, and = .043). However, presence of kidney disease was not significant between these groups.

CONCLUSIONS: All pretreatments had an important effect in decreasing negative control values, with Adsorb Out having highest efficiency. Serum-specific components could contribute to high negative control bead median fluorescence intensity values. Further studies are needed to determine the adequate pretreatment of patient sera.