B-124 Evaluation of Anti-Citrullinated Peptide Antibody (ACPA) Result Normalization as Defined in the 2010 Rheumatoid Arthritis Classification Criteria Recommended by American College of Rheumatology/Alliance of Associations for Rheumatology (ACR/EULAR)

Background Anti-citrullinated peptide antibodies (ACPA) (tested as anti-cyclic citrullinated peptide [CCP] antibodies) are frequently ordered in the evaluation of patients with suspected rheumatoid arthritis (RA) and considered to be specific biomarkers for RA. The 2010 American College of Rheumatology/European Alliance of Associations for Rheumatology (ACR/EULAR) classification criteria for RA include both ACPA and rheumatoid factor in their scoring system, despite limited standardization of commercially available anti-CCP antibody tests. In an attempt to overcome this limitation, the classification criteria recommends using multiples of the assay-specific upper limit of normal (ULN) to normalize results and standardize classification. This study evaluates the correlation between normalized results from different assays and the concordance in result classification according to the ACR/EULAR criteria. Methods A total of 265 samples with ACPA ordered as part of routine clinical care were tested on four commercial anti-CCP antibody assays, including QUANTA Lite CCP3 IgG ELISA (Werfen) (Negative: <20 Units), QUANTA Flash CCP3 chemiluminescence immunoassay (Werfen) (Negative: <20 Chemiluminescent Units), EUROIMMUN Anti-CCP ELISA (IgG) (EUROIMMUN) (Negative: <=5 Relative Units/mL), and Elecsys anti-CCP electrochemiluminescence immunoassay (Roche) (Negative: <17.0 Units/mL). The QUANTA Lite and EUROIMMUN ELISA were performed on the Agility platform (Dynex), QUANTA Flash was performed on BioFlash platform (Werfen), and Elecsys anti-CCP was performed on Cobas e601 platform (Roche). All testing was performed according to manufacturer’s instructions. Results from each assay were converted to the multiples of assay-specific ULN as the normalized results. Regression analysis was performed, and the coefficient of determination (R²) was calculated to assess the correlation between paired assays. The concordance in result classification across assays was also investigated using both assay specific cut-offs and the ACR/EULAR criteria-defined cut-offs: Negative (<=1xULN), Low positive (>1xULN and <3xULN), High positive (>=3xULN). Results Using assay-specific cut-offs, 63 samples were negative on all assays. 202 samples were positive on at least one assay, of which 79 (39%) were positive across all assays. The overall concordance of qualitative result was 54% ([63+79]/265). After result normalization using multiples of assay-specific ULNs, the highest correlation was observed between the EUROIMMUN ELISA and Elecsys anti-CCP (R²=0.83), despite differences in methodology and manufacturer. The lowest correlation was observed between QUANTA Lite and QUANTA Flash (R²=0.49), even though both are from the same manufacturer although methodologies differ. When applying the criteria-defined cut-offs, 64 samples were negative on all assays. 130 samples were classified as low positive on at least one assay; however, only 3 (2%) samples were classified as low positive across all assays. 103 samples were classified as high positive on at least one assay, of which 54 (52%) were classified as high positive across all assays. With result normalization, the overall concordance in result classification across all assays using the criteria-defined cut-offs were 46% ([64+3+54]/265). Conclusion Despite normalization using multiplies of assay-specific ULN values, the inter-assay correlation remains suboptimal, and the concordance in result classification is limited. These findings suggest that the anti-CCP antibody results from different assays may not be interchangeable even after normalization as recommended by current criteria, potentially impacting disease classification.