Model au 400

SF samples were obtained during knee joint aspiration from 10 anti-CCP-negative (median anti-CCP: 3.6 U/ml [interquartile range: 2–11.4 U/ml]) and 32 anti-CCP-positive (878 U/ml; [136–1353] U/ml) RA patients fulfilling the American College of Rheumatology criteria [38 (link)] and characterised with respect to disease-activity, as assessed by DAS28. The majority of the SF samples were also used in a recent study [12 (link)]. All SF samples were centrifuged at 1900 g for 10 min to remove cells and debris and stored at -80°C until use. Plasma from all patients but four was isolated from peripheral venous blood from the same patients at the time of arthrocentesis. The study was approved by the local ethics committee of the Institute of Rheumatology in Prague (No. 3294/2012), Czech Republic, and written informed consent was obtained from all patients.
Serum anti-CCP antibodies and IgM-rheumatoid factor were determined by standard ELISA kits (TestLine Clinical Diagnostics, Brunn, Czech Republic). Serum CRP was measured using an immuno-turbidimetric technique with an Olympus biochemical analyser, model AU 400 (Olympus, Tokyo, Japan) and SF leukocytes were counted using the Iris IQ200 (Beckman Coulter, CA, USA) analyzer.

Blood samples were collected from all the patients when they underwent therapeutic arthrocentesis of the knee or not more than 5 days after arthrocentesis. Paired samples were immediately centrifuged, and both the serum and synovial fluid were stored at −80°C until analysed. Before analysis, the Hylase-Dessau treatment, including heating of the synovial fluids for 30 min at 37°C, was performed. Commercially available ELISA kit (Adipogen Inc., Seoul, Korea) with the detection limit of 32 pg/mL and with the assay range 0.063 ng/mL–4 ng/mL was used to analyse the levels of PGRN in the serum and in the synovial fluid. Absorbance was detected by the Sunrise ELISA reader (Tecan, Salzburg, Austria) with 450 nm as the primary wavelength. CRP levels were determined via an immunoturbidimetric technique using an Olympus biochemical analyser (model AU 400, Japan). Analysis of serum levels of anticitrullinated protein/peptide autoantibodies (ACPA) and IgM rheumatoid factor (IgM-RF) was done with the standard ELISA kits (Test Line s.r.o., Czech Republic).