#2646 Precision verification and method comparison of anti-MPO anti-hsPR3 immunoassay on Alegria2 instrument
Authors: Djerek Lovorka , Crnogorac Matija , Živković Ksenija Kukuruzović , Strizić Ana , Horvatic Ivica , Stančin Nevenka 
Published: May 2024

Background and Aims

Antibodies to myeloperoxidase (anti-MPO) and proteinase 3 (anti-PR3) are recognized as the only clinically relevant antineutrophil cytoplasmic antibodies (ANCA) specificities so far. ANCA testing can be used in different clinical settings, especially in situations that require rapid diagnosis like renal-pulmonary syndrome that require result within 24 hours.

The aim of this study was to perform analytical verification of the new automated single test ELISA based method on Alegria2 instrument to test if the reagents and analyser were acceptable for the routine use.

Method

For precision assessment we tested two concentration levels of commercial control material in triplicate for five days in a row. We used EASI acceptable criteria for precision for anti-MPO as well as for anti-hsPR3.

For method comparison we included 50 patients with history or suspicion of vasculitis. Anti-MPO and anti-PR3 were measured using 5 different reagents: Anti-Myeloperoxidase ELISA (IgG) and anti-PR3-hn-hr ELISA (IgG) on Euroimmun Analyzer I-2P (EUROIMMUN AG, Lubeck, Germany), MPO and PR3 II chemiluminiscent immunoassay (CLIA) on IDS iSYS (IDS ISYS, Pouilly en Auxois, France), QUANTA Flash® MPO and QUANTA Flash® PR3 CLIA on BIO-FLASH® (Inova Diagnostics Inc, San Diego, USA), Anti-MPO and anti-PR3 hs on Alegria2 ELISA based method (Orgentec, Mainz, Germany), and Anti-MPO and anti-PR3 ELISA (Orgentec, Mainz, Germany).

Results were categorized as positive or negative and we calculated kappa statistics as well as intraclass correlation coefficient. Complete statistical analysis was done using MedCalc statistical software (MedCalc version 14.8.1, Ostend, Belgium).

Results

Within-laboratory precision (CV%) for anti-MPO for normal and pathological controls were 5.73% and 15.14% respectively. For anti-PR3 hs within-laboratory precision (CV%) for normal and pathological controls were 5.46% and 18.89% respectively.

Intraclass correlation coefficient showed excellent degree of consistency in average measures (0.9778 (95% CI: 0.9665-0.9862)) for anti-MPO and good reliability on single ratings (0.8981 (95% CI: 0.8522-0.9344). For anti PR3 intraclass correlation coefficient showed excellent degree of consistency in average measures (0.9257 (95% CI: 0.8873-0.9539)) but only moderate consistency in single ratings (0.7136 (95% CI: 0.6115-0.8053)). Separate Cohen`s kappa testing revealed almost perfect agreement for most reagent combination for anti- MPO (kappa from 0.825 to 0.958). For anti-PR3 we found moderate agreement between QUANTA Flash® PR3 and PR3 II IDS iSYS (kappa 0.520) but mostly substantial agreement (kappa from 0.629 to 0.865). We also found almost perfect agreement between anti-PR3 hs on Alegria2 and QUANTA Flash® PR3 (kappa = 0.901).

Conclusion

Our results showed a rather small relative variability and met the precision criteria defined by EASI initiative. Satisfactory agreement between methods is very reassuring but due to the lack of standardisation in autoimmune diagnostics imposes that information about methodological differences must be clearly stated on laboratory report. According to our results the new method for anti-MPO and anti-PR3 proved to be suitable for the routine use.