Background/Purpose: The OMERACT soluble biomarker sub-committee has published validation criteria related to truth, discrimination and feasibility for biomarkers reflecting structural damage¹. The large majority of biomarker assays assessed in RA have not undergone such validation, particularly the key performance criteria considered essential prior to clinical validation studies². Extracellular 14-3-3 eta is 1) a synovial-derived novel mediator of inflammation/damage with serum levels being differentially expressed in early and established RA compared with controls, 2) modifiable with TNF therapy and 3) independently associated with joint damage in RA and PsA. With 14-3-3 eta fulfilling several OMERACT criteria, this study aimed to focus on aspects of feasibility and discrimination by testing assay reproducibility, reliability, biomarker stability, and sources of variability.

Methods: The 14-3-3 eta ELISA was evaluated for intra- and inter-assay reproducibility by running 20 duplicate measurements on 3 samples within a single assay and over 4 days by 4 operators. Possible interferents [hemoglobin, lipids, bilirubin, albumin, RF, erythrocytes, Aspirin, MTX, and anti-TNFs] were spiked into serum and 14-3-3 eta % recovery was determined. Biomarker stability was examined in 1) 3 samples over 3 freeze-thaw cycles and 2) in 6 samples up to 2 years of storage at -80^oC. Age and gender effects were assessed on 100 healthy controls, 50 males and 50 females [median age 55.0 and 57.5 years]. The effect of menopause was evaluated in the 50 females, 20 under and 30 over the age of 51. Correlations were used to evaluate the relationship of age and 14-3-3 eta concentration. 2-tailed t-tests and Mann-Whitney U-tests were performed to examine mean and median differences between genders and menopausal status.

Results: The intra- and inter assay coefficients of variation (CV%) were less than 10% [range (R)=6.0-9.2%]. Interference testing delivered a 106% median 14-3-3 eta recovery [R=100-115%] across the analytes tested demonstrating 14-3-3 eta quantification is not confounded by common RA patient serum substances. Results from sample stability testing indicate that serum 14-3-3 eta is stable over 3 freeze-thaw cycles with the median CV% being 109% [R=92-129%]. Long-term storage studies show that samples negative for 14-3-3 eta remain negative while those with levels above the upper limit of quantification of >20ng/ml have substantially equivalent levels. Stability of 14-3-3 eta was further confirmed using samples with levels in the linear range of the assay; median CV% was 104% of the original values [R=89-128%]. There was no correlation between age and 14-3-3 eta. Median 14-3-3 eta serum concentrations in healthy males and females did not differ significantly nor were there any significant differences in females aged over and under 51 years.

Conclusion: This 14-3-3 eta ELISA fulfills several key performance criteria considered essential by OMERACT. Quantification of 14-3-3 eta using this assay is reproducible and the biomarker is highly stable with no confounding of age, gender or menopause.

References: 1. Maksymowych WP et al. J Rheumatol. 2009 Aug;36(8):1785. 2. Maksymowych WP et al. J Rheumatol. 2009 Aug;36(8):1792.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/validation-of-prognostic-biomarkers-for-ra-testing-of-14-3-3-eta-according-to-the-omeract-soluble-biomarker-criteria/