Standardisation of ACPA tests: evaluation of a new candidate reference preparation

Abstract

Introduction: Commercial assays measuring antibodies to citrullinated protein/peptide (ACPA) show poor quantitative agreement. The diagnostic industry has never adopted the International Union of Immunological Societies-Centers for Disease Control and Prevention (IUIS-CDC) ACPA reference standard. Recently, the National Institute for Biological Standards and Control (NIBSC) prepared a new candidate ACPA standard (18/204). We evaluated both reference materials using different commercially available ACPA assays.

Materials and methods: This is an international study in which the NIBSC candidate ACPA standard and the IUIS-CDC ACPA reference material were analysed together with 398 diagnostic samples from individuals with rheumatoid arthritis (RA) and in 1073 individuals who did not have RA using nine commercial ACPA assays.

Results: For both reference materials and samples from individuals with RA and individuals who did not have RA, there were large differences in quantitative ACPA results between assays. For most assays, values for the IUIS-CDC standard were lower than values for NIBSC 18/204 and the IUIS-CDC/NIBSC ratio was comparable for several, but not all assays. When NIBSC 18/204 was used as a calibrator, an improvement in alignment of ACPA results across several of the evaluated assays was obtained. Moreover, NIBSC 18/204 could align clinical interpretation for some but not all assays.

Conclusion: Adoption of an international standard for ACPA determination is highly desirable. The candidate NIBSC 18/204 standard improved the standardisation and alignment of most ACPA assays and might therefore be recommended to be used as reference in commercial assays.

Keywords: NIBSC; anti-citrullinated protein antibody; reference material; standardization.

Figure 1

Quantification of candidate NIBSC 18/204 ACPA reference material (A) and IUIS-CDC ACPA reference material (B). The reference materials were reconstituted according to the guidelines, aliquoted, stored frozen (−20°C) on analysis and tested in 19 different runs with every ACPA assay. (A, B) Box-whisker plots of the results obtained. Boxes represent median and IQR, whiskers represent lowest and highest measurement excluding ‘outside’ values (ie, larger than the upper quartile plus 1.5 times the IQR; highlighted in red). The manufacturer’s cut-offs are marked as red bars. The y-axis represents the manufacturer-specific units. (C) Box-whisker plots of the CDC ACPA reference material recalculated taken the reactivity of the candidate NIBSC 18/204 ACPA standard arbitrarily as 100 units. ACPA, antibodies to citrullinated protein/peptide; CDC, Centers for Disease Control and Prevention; IUIS, International Union of Immunological Societies; NIBSC, National Institute for Biological Standards and Control.

Figure 2

Correlations of individual values of ACPA measured by nine different immunoassays. The immunoassays included were from Thermo Fisher (TF, 1), Roche (R, 2) Svar life science (SV, 3), IDS (I, 4), Orgentec (O, 5), Abbott (A, 6), Euroimmun (E, 7), BioRad (B, 8) and Siemens (S, 9). The samples were from patients with RA (n=398) and (disease) controls (n=1073) obtained in 11 European hospitals. The NIBSC 18/204 candidate ACPA reference preparation and dilutions thereof (0/4-1/4-2/4-3/4-4/4) are represented by triangles. Spearman’s rank correlation coefficients (rs) are shown in the insert on the graph. Detailed statistical data on Spearman’s correlation and Bland-Altman are given in online supplemental table 5. ACPA, antibodies to citrullinated protein/peptide; IDS, immunodiagnostic systems; NIBSC, National Institute for Biological Standards and Control.

Figure 3

ROC curve analysis and likelihood ratios for NIBSC 18/204. Left hand pane: ROC for nine different ACPA assays with indication of the sensitivity and ‘1-specificity’ of the result associated with a 1:4 dilution of NIBSC 18/204 (red filled circle surrounded by black line). Right handpanel: likelihood ratio of a test result interval with as centre the result of the candidate NIBSC standard. The interval was chosen such that the number of data points with results higher than the result of NIBSC 18/204 equaled the number of data points with results that were lower than the NIBSC 18/204. The intervals were as follows: thermo Fisher: 9–148 U/mL, Roche: 134–387 U/mL, Svar: 28.6–200 U/mL, IDS: 7.9–677 AU/mL, Orgentec: 3.2–16.3 U/mL, Abbott: 7.3–65.1 U/mL, Euroimmun: 9.7–59.9 U/mL, Siemens: 5.3–69.9 RU/mL. For BioRad, no likelihood ratio was calculated as many results had values exceeding the upper limit. ACPA, antibodies to citrullinated protein/peptide; CCP, cyclic citrullinated synthetic peptides; IDS, immunodiagnostic systems; NIBSC, National Institute for Biological Standards and Control; ROC, receiver operating characteristics.