Background The soluble biomarker sub-committee of OMERACT has published validation criteria for biomarkers reflecting structural damage end-points that focus on assay performance characteristics to address the impact of potential confounders. The 14-3-3eta (η) test is currently available for clinical use as a laboratory developed test (LDT) in the United States (US) and in Europe and Canada as an in-vitro diagnostic (IVD) test ELISA with European (CE) marking and Health Canada (HC) approval.
Objectives The purpose of this study was two-fold; to demonstrate that (1) 14-3-3η clinical results on the LDT in the US are substantially equivalent to those reported on the 14-3-3η ELISA IVD and (2) rheumatoid factor (RF) does not interfere with the quantitation of 14-3-3η.
Methods Serum 14-3-3η levels were measured with the US LDT in 40 samples which were then measured on a blinded basis on the CE/HC approved 14-3-3η IVD. A Spearman correlation was run to determine the level of agreement of the clinical readouts of the two assays and the number that were positive and negative by both assays was determined. The potential effects of RF interference were evaluated on the CE/HC 14-3-3η IVD using the following three methods: (1) in nine RA samples with high 14-3-3η (8.2-1653 ng/ml) and high RF (91-574 IU/ml) concentrations, the variability in reported 14-3-3η concentrations was evaluated across serial dilutions; (2) using four serum samples with known 14-3-3η and RF levels, samples were mixed 1:1 with a negative control to examine the 14-3-3η % recovery between the expected and measured 14-3-3η concentrations; and (3) five 14-3-3η negative patient serum samples that had high RF concentrations (150-479 IU/ml) were serially diluted to determine if any of the negative samples became 14-3-3η positive. For each of these methods, three or four serial dilutions were performed 1:1 ensuring that the first sample dilution read within the linear range of the assay.
Results Of the 40 samples tested 37 were in agreement; 25 samples on the US LDT and 26 on the CE/HC 14-3-3η IVD were 14-3-3η negative while 15 and 14 were positive, respectively. A Spearman correlation revealed high agreement between the 14-3-3η titre results reported by both assays, r=0.94, p<0.00001. Linear dilution studies in 9 rheumatoid arthritis (RA) samples showed that the quantitation of 14-3-3η was highly reproducible in the presence of high RF concentrations with a 6.8% mean variation. Mixing of high RF and 14-3-3η samples with negative control serum revealed that the overall recovery was 96% (88% - 105%) which is within the OMERACT acceptable range of 10%. All of the high RF, 14-3-3η negative samples, remained 14-3-3η negative along all serial dilutions further indicating that RF concentrations did not confound 14-3-3η readouts.
Conclusions 14-3-3η values reported in the United States using the laboratory developed test are substantially equivalent to those using the CE/HC 14-3-3η IVD. RF does not impact the quantification of 14-3-3η.
Disclosure of Interest S. Naides Employee of: Quest Diagnostics LLC, O. Zhukov Employee of: Quest Diagnostics LLC, J. Popov Employee of: Quest Diagnostics LLC, R. Abolhosn Employee of: Quest Diagnostics LLC, K. Lam Employee of: Augurex Life Sciences Corp, Y. Gui Employee of: Augurex Life Sciences Corp, W. Maksymowych Consultant for: Augurex Life Sciences Corp, A. Marotta Employee of: Augurex Life Sciences Corp