Verification, implementation and harmonization of automated chemiluminescent immunoassays for MPO- and PR3-ANCA detection.

OBJECTIVES: Antineutrophil cytoplasmic antibody (ANCA) testing assists clinicians diagnose ANCA-associated vasculitis (AAV). We aimed to verify and harmonize chemiluminescent immunoassays for the detection of myeloperoxidase (MPO)- and proteinase 3 (PR3)-ANCA. METHODS: An in-house ELISA, a capture ELISA, and a chemiluminescent assay QUANTA Flash on a BIO-FLASH analyzer were used to detect MPO- and PR3-ANCA in sera from 39 patients with AAV, 55 patients with various non-AAV, and 66 patients with connective tissue diseases. The results of the assays were evaluated, and their clinical performance was assessed. The precision and linearity of the QUANTA Flash assays were determined, and likelihood ratios (LRs) for AAV at diagnosis were calculated. RESULTS: The precision and linearity of the QUANTA Flash assays were confirmed. Overall agreement between 97.5 and 98.8 % and Cohen's kappa coefficients between 0.861 and 0.947 were observed for the results of the QUANTA Flash assays and ELISAs. The diagnostic sensitivity, specificity, and ROC analysis of the assays for AAV were statistically similar (in-house ELISA 89.7 %, 95.0 %, and 0.937; capture ELISA 92.3 %, 98.3 %, and 0.939; and QUANTA Flash 89.7 %, 95.9 %, and 0.972). For the QUANTA Flash assay results, the interval-specific LRs for AAV at diagnosis were: 0-8 CU had LR 0.08, 8-29 CU had LR 1.03, 29-121 CU had LR 7.76, 121-191 CU had LR 12.4, and >191 CU had LR ∞. CONCLUSIONS: The QUANTA Flash MPO and PR3 assays provide precise and consistent results and have comparable clinical utility for AAV. The calculated LRs were consistent with published LRs, confirming the utility of LRs for harmonization of ANCA results.

Insights into the immunological description of cryoglobulins with regard to detection and characterization in Slovenian rheumatological patients.

The detection of cryoglobulins (CG) used to diagnose cryoglobulinemic vasculitis requires strict adherence to protocol, with emphasis on the preanalytical part. Our main objectives were to introduce a more sensitive and specific protocol for the detection of CG and to characterize CG in Slovenian patients diagnosed with cryoglobulinemic vasculitis, other vasculitides, connective tissue diseases or non-rheumatic diseases examined at the Department of Rheumatology (University Medical Centre Ljubljana). Samples were routinely analyzed for the presence of CG with the protocol using the Folin-Ciocalteu reagent. In the newly introduced protocol, the type of CG was determined by immunofixation on visually observed positive samples and the concentration of CG in the cryoprecipitate and rheumatoid factor (RF) activity were measured by nephelometry. RF, C3c and C4 were measured in patients` serum and a decision tree analysis was performed using all results. The agreement between negative and positive results between the two protocols was 86%. Of the 258 patient samples tested, we found 56 patients (21.7%) with positive CG (37.5% - type II, 62.5% - type III). The RF activity was observed in 21.4% of CG positive subjects. The median concentration of type II CG was significantly higher than that of type III CG (67.4 mg/L vs. 45.0 mg/L, p = 0.037). Patients with type II had lower C4 concentrations and higher RF compared to patients with type III CG. In the decision tree, C4 was the strongest predictor of cryoglobulinemia in patients. With the newly implemented protocol, we were able to improve the detection and quantification of CG in the samples of our rheumatology patients and report the results to adequately support clinicians.

Neutralizing effects of anti-infliximab antibodies on synergistically-stimulated human coronary artery endothelial cells.

BACKGROUND AND AIMS: Patients with rheumatic diseases have an increased risk of atherosclerosis with up-regulated serum amyloid A (SAA), tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß), which were reported to activate human coronary artery endothelial cells (HCAEC). We aimed to investigate the effects of TNF-α inhibitor infliximab and anti-infliximab antibodies on the TNF-α/IL-1ß/SAA activated HCAEC. METHODS: HCAEC were incubated with TNF-α, IL-1ß, SAA, infliximab, anti-infliximab antibodies and their combinations. The protein levels of pro- and anti-atherogenic analytes were measured in supernatants using ELISA and multiplex assays, while mRNA expression was determined by RT-PCR. Anti-infliximab antibodies were purified from sera samples by affinity chromatography. RESULTS: IL-6, IL-8, GM-CSF and GRO-α were synergistically up-regulated in triple stimulation with TNF-α, IL-1ß and SAA, while their levels in solely SAA- or TNF-α-stimulated HCAEC did not increase. IL-1Ra, IL-1α, VCAM-1, MCP-1, IL-10 and IL-17A were increased, but no synergistic responses were observed in triple stimulation. Infliximab was effective in lowering the synergistic effect of IL-6, IL-8, GM-CSF and GRO-α in triple stimulation, while anti-infliximab antibodies restored the levels. The changes were confirmed at the mRNA expression level for IL-6, IL-8 and GM-CSF. CONCLUSIONS: Triple stimulation with TNF-α, IL-1ß and SAA synergistically elevated IL-6, IL-8, GM-CSF and GRO-α release in supernatants of HCAEC, with infliximab substantially inhibiting their levels. An isolated, enriched fraction of polyclonal anti-infliximab antibodies was capable of neutralizing infliximab, in the presence of TNF-α/IL-1ß/SAA. The long-term presence of anti-infliximab antibodies in the circulation of patients with chronic rheumatic diseases is potentially important for promoting the atherosclerotic process.