Antiphospholipid syndrome (APS) is a systemic autoimmune disease characterized by thrombosis and/or pregnancy complications. Diagnosis and classification of APS involve testing for anticardiolipin (aCL) and anti-β2-glycoprotein I (anti-β2GPI) antibodies. For many years, these antibodies have been measured using an in-house enzyme-linked immunosorbent assay (ELISA) at the Laboratory of Immunology, University Medical Centre Ljubljana. However, to comply with the new European regulation (Regulation (EU) 2017/746), which mandates the use of commercially available IVDR kits, the laboratory is transitioning to the automated chemiluminescent immunoassay QUANTA Flash. Additionally, the diagnostic scope will expand to include anti-β2GPI antibodies against domain I (aDI).
Before introducing the new method into routine diagnostics, it must be analytically and clinically evaluated, and the results must be compared to those obtained using the in-house ELISA. This master's thesis aimed to perform this evaluation and comparison. We incorporated the recently published 2023 ACR/EULAR APS classification criteria, which emphasize the importance of setting clinically relevant thresholds. In the analytical part, we demonstrated that QUANTA Flash achieves adequate repeatability and intermediate precision (CV 2.00 %-8.16 %) for aCL and anti-β2GPI IgG, IgM and IgA classes, as well as for aDI. Furthermore, our results show linearity within the observed range for all tests except aCL IgA. By calculating the 99th percentile of the results from 139 clinically healthy individuals, we were unable to verify the manufacturer's cutoffs for the criteria aCL and anti-β2GPI antibodies IgG and IgM. The calculated cutoffs ranged from 33.5 CU to 47.4 CU. However, we successfully verified the manufacturer's reference values for the non-criteria antibodies aCL and anti-β2GPI IgA, as well as for aDI. We also determined the thresholds for the criteria antibodies using ROC curve analysis on a group of 159 patients with APS, individuals showing clinical signs of the disease, and patients with other autoimmune disorders. Comparable diagnostic performance to the in-house ELISA was achieved at thresholds of 20 CU for low positive, 40 CU for moderate positive, and 80 CU for high positive results. Cohen's coefficients indicated moderate to substantial qualitative agreement between the two methods, with total agreement ranging from 76.10% to 91.19%. Additionally, we demonstrated a significant correlation between the results of both methods. In this work, we successfully verified the new method, showing it provides results comparable to the in-house ELISA, and introduced the new 2023 ACR/EULAR APS classification criteria for future observational clinical studies and trials.
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