Serum levels of soluble urokinase plasminogen activator receptor in juvenile idiopathic arthritis: a single-center Swedish case-control study

Study populations

We included 51 children (32 girls and 19 boys), classified with JIA according to the International League of Associations for Rheumatology (ILAR) criteria [1]. Besides JIA subtypes, data regarding age, gender and antirheumatic treatments were recorded and the patients were carefully followed clinically over 3 years. Presence of joint swelling, tenderness or motion-induced joint pain without traumatic origin were evaluated according to the ACR definition of disease activity [4]. Radiography (X-ray) investigations of affected joints were performed as part of clinical routine in 47 (92%) patients. The X-rays were always evaluated at the same unit (Radiology Unit, Vrinnevi Hospital in Norrköping), but not necessarily by the same radiologist.

The control group comprised 50 sex- and age-matched children (31 girls and 19 boys) who lived in the same region as the patients, median age 13.3 years (range 2–18) and were recruited among friends (n = 36) and siblings (n = 14) to the patients. None of the controls had signs of arthritis or had a history of rheumatic disease, and neither of them had asthma, renal disease or hypertension. They were sampled once and not investigated by radiography.

Peripheral venous blood sampling was performed in patients and controls at inclusion. After centrifugation at 2000 g for 10 min, sera were stored at − 70 °C until analysis of suPAR.

suPAR analysis

For suPAR analysis, enzyme-linked immunosorbent assay (suPARnostic® AUTO Flex ELISA, ViroGates, Birkerod, Denmark) was performed according to manufacturer’s instructions [11]. Briefly, serum samples diluted 1:10 and peroxidase-conjugated anti-suPAR was mixed and then incubated in an anti-suPAR pre-coated 96-well plate. After 1 h incubation, tetramethylbenzidine substrate was added and the enzymatic reaction was stopped after 20 min by adding 2 N sulfuric acid and read at 450 nm (plate reader Sunrise, Tecan, Männedorf, Switzerland; software Magellan version 7.1, Tecan). All samples were run in duplicates.

A large Danish study of 5 538 adult individuals showed a mean serum concentration of suPAR of 3.51 ng/mL for men and 3.90 ng/mL for women [16]. The highest value among the controls herein (> 3.6 ng/mL) was used as cut-off; this corresponded to the 98th percentile among the healthy controls.

ESR and CRP

Erythrocyte sedimentation rate (ESR; mm/h) and C-reactive protein (CRP; mg/L) were analyzed according to routine methods at the Department of Clinical Chemistry, Vrinnevi Hospital, Norrköping. CRP was analyzed by turbidimetry (Advia® 1800, Siemens Healthcare Diagnostics, Terrytown, NY, USA). Concentrations of CRP < 10 mg/L were reported as 5 mg/L.

ANA

ANA were detected by indirect immunofluorescence (IF) microscopy using slides with fixed HEp-2 cells (Immunoconcepts, Sacramento, CA, USA) and fluorescein-isothiocyanate (FITC) conjugated γ-chain-specific anti-human IgG as detection antibody (Agilent, Glostrup, Denmark), as previously described [17]. The chosen cut-off for ANA corresponds to the 95th percentile among healthy blood donors (n = 300; 50% females, 50% males) in line with the international recommendations [18].

In addition, ANA-subspecificities (including SSA/Ro52 and SSA/R060, SSB/La, Sm, Sm/RNP, U1RNP, dsDNA, CENPB, Jo-1, Scl-70, PmScl, PCNA, histone and Ribosomal P were analyzed in all samples using addressable laser bead immunoassay (ALBIA) and the FIDIS™ Connective profile, Solonium software ver. 1.7.1.0 (Theradiag, Croissy-Beaubourg, France) at the Clinical immunology laboratory at University Hospital in Linköping [11].

Anti-CCP and RF

Anti-CCP antibodies were analyzed, according to the manufacturer’s instructions, by fluorescence enzyme immunoassay (FEIA) on a Phadia 250 instrument with second generation cyclic citrullinated peptides (CCP) as antigen (EliA, Thermo Fischer AB, Uppsala, Sweden). RF was analyzed with nephelometric technique (Beckman Coulter, Inc., CA, USA) at the Department of Clinical Chemistry, University Hospital in Linköping.

Statistics

Normal distribution was evaluated by using Shapiro-Wilk test and Kolmogorov-Smirnov test. Mann Whitney’s U-test was used to compare suPAR levels between JIA and controls as well as between oligoarticular, polyarticular, JPS, enthesitis-related arthritis, undifferentiated and controls. Possible correlations between suPAR and sex, age, disease duration or disease activity at the sampling occasion was evaluated by Pearson’s correlation. Pearson’s correlation was also used to analyze the levels of suPAR versus presence of RF and anti-CCP. Fisher’s exact test was used to analyze differences between high suPAR (> 3.6 ng/mL) among subjects with JIA compared to controls. Fisher’s exact test was also used to analyze possible associations between erosions and high suPAR levels (> 3.6 ng/mL), or RF positivity or anti-CCP positivity. P-values < 0.05 were considered statistically significant. Statistical analyses were performed using GraphPad Prism version 9 (GraphPad software Inc, La Jolla, CA, USA).

Ethics approval

Oral and written informed consents were obtained from all patients and controls. The study was conducted according to the Declaration of Helsinki and the study protocol was approved by the Regional Ethics Boards regarding SLE (Linköping M70 − 07/2007).

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