Observational study. Consecutive patients from the Rheumatology Department (Hospital Clinic, Barcelona, Spain) meeting American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) 2010 criteria for RA  were enrolled. Clinical, immunological, demographic and US images of patients were collected. Seropositivity was defined as the presence of RF (>50 UI/ml) and/or ACPA (>50 UI/ml) at least twice at any time of the disease course. Probable RA or overlap syndromes were excluded. Before undergoing US, MRI, serological test or synovial biopsies, informed consent was signed. This study was conducted in accordance with the principles of the Declaration of Helsinki.
All sonographic assessments were performed using high-sensitivity US equipment (Acuson Antares®, Siemens AG, Erlangen, Germany) with a linear probe of frequency range from 8 to 14 MHz. Intraarticular infiltrations were not allowed one month previous to the US assessment. US findings were defined according to published OMERACT definitions . An experienced sonographer evaluated 6 joints of each hand (including MCP joints and wrists) for SH and intra-articular PD signals according to EULAR guidelines . All evaluations were scanned on the dorsal aspect using longitudinal midline and transversal planes. PD calibrations and SH/PD assessment have been previously described .
We evaluated the morphology of synovial tissue. Specifically, we looked to identify an extreme proliferative pattern defined as the marked thickness of synovial membrane with PD signal inside the edges of the joint (Figure 1. A). Synovial thickening should bulge over the line linking tops of the periarticular bones and with/without extension to one of the bone diaphysis, similarly to the definition of synovial proliferation grade II or III adopted by Szkudlarek et al . We adopted the term “US proliferative synovitis” (US PS) to define these features. On the contrary, the flat growth of synovial filling the angle between the periarticular bones with PD signal inside the joint, was defined as “flat synovitis”, a concept equivalent to SH grade I (Figure 1.B). Both US patterns included the presence of PD signal.
Four rheumatologists, blinded to clinical data and autoantibody status of patients, scored all the images. Interobserver reliability was evaluated before patients’ inclusion by scoring SH/PD in 50-recorded images of joints from 20 RA patients. Interobserver correlations were all good to excellent (range 0.608-0.831).
Histological and immunohistochemical assessments
All the US-guided synovial biopsies were performed in a case day surgery according to the technique previously described by Kelly et al . Biopsies were taken from selected patients who signed the informed consent. 6-8 synovial biopsies were taken per procedure.
Paraffin embedded slides were prepared from synovial tissues and stained with an automated immunostainer (TechMate 500 Plus; Dako, Cambridge, UK). In short, we immunostained with anti-CD3, CD20, CD117, CD15, CD68, hsp-47 and CD31 antibodies as previously described .
Digital image analysis
The stained slides were scored by digital image analysis by an independent observer. Each stained slide was scored by dividing it in different regions. Within each region, the number of stained cells per area and the percentage of stained cells were measured in at least 20 high-power fields using the AnalySIS®Imaging processing program (Olympus®) as previously described .
Selected patients who signed informed consent were scanned on a 1.5 Tesla system (Siemens Aera, Siemens Medical, Erlangen, Germany) using a dedicated wrist coil. MRI protocol was previously reported .
Images were reviewed on a standard Dicom (Digital Imaging and Communication in Medicine) compliance workstation. Images were scored by two independent radiologists blinded to image time point and patient identity using the Rheumatoid Arthritis Magnetic Resonance Imaging Scoring (RAMRIS) system .
Intraobserver reliability kappa values were 0.82, 0.83 and 0.90 for the RAMRIS semiquantitative scoring of synovitis, bone marrow oedema, and erosions, respectively; interobserver kappa values were 0.69, 0.74 and 0.84 .
Quantification of biomarkers of inflammation/angiogenesis.
Cytokines and angiogenic mediators were analyzed using Quantibody® Human Custom Array (RayBiotech, Norcross, GA, USA)1 . Detection limits for cytokines are displayed on the manufacturer's website . After sample dilution, the effect of RF on the final results was estimated to be around 1% .
Calprotectin serum levels were determined using an ELISA Test Kit [CALPROLAB Calprotectin ELISA (ALP) CALPRO AS, Norway] in accordance with the manufacturer’s protocol .
At baseline and every fourth months patients underwent complete clinical and biological assessment. Treatment change was defined as the change in any conventional synthetic Disease-modifying drugs (csDMARDs) or biological synthetic Disease-modifying drugs (bDMARDS) (but not glucocorticoids) across the observational period.
Epidemiological, clinical and US variables were compared between patients with or without RF or ACPA and with US PS or flat synovitis. Numerical variables were described as mean and standard deviation (SD) and categorical variables as frequencies and percentages. T- student test was used to compare the distribution of numerical variables between groups. Chi-quared test was used to compare categorical variables. To ascertain independent associations between variables we used multivariate analysis. For all tests, p values ≤ 0.05 were considered significant. All analyses were performed using the SPSS 18.00 (SPSS Inc., Chicago, IL, USA).