All enrolled patients underwent ultrasound-guided synovial tissue biopsy of the knee following the already published protocol.8 (link) Once collected, synovial tissue specimens were fixed in 10% neutral-buffered formalin and embedded in paraffin for histology. Briefly, paraffin-embedded synovial tissue (ST) specimens were sectioned at 3–4 μm. First, sections were stained for H&E as follows: sections were deparaffinised in xylene and rehydrated in a graded ethanol series. Then they were stained in haematoxylin and counterstained in eosin/phloxine. Finally, sections were dehydrated, cleared in xylene and mounted with Bio Mount (Bio-Optica). Other sections were stained for CD68 mouse antihuman monoclonal antibody (clone 514H12) or CD21 mouse antihuman monoclonal antibody (clone 2G9) or CD20 mouse antihuman monoclonal antibody (clone L26) or CD3 mouse antihuman monoclonal antibody (clone LN10) or CD31 mouse antihuman monoclonal antibody (clone 1A10) (all from Leica Biosystem, Newcastle, UK) by immunostainer BOND MAX III (Leica).9
ST slides were stained for collagen using the Masson Trichrome Goldner with light green (Bio-Optica; 04-011802) as follows: slides were washed in distilled water; then six drops of Weigert's iron haematoxylin, provided by the manufacturer, were put on the sections and left to act 10 min. Without washing, the slides were drained, and 10 drops of picric acid alcoholic stable solution were added on the sections. Then, the slides were quickly (3–4 s) washed in distilled water, and 10 drops of ponceau acid fuchsin according to Masson were added on the sections and left to act 4 min. Then, the slides were washed in distilled water, and 10 drops of phosphomolybdic acid solution were added on the sections and left to act 10 min. Without washing, the slides were drained, and 10 drops of Light Green Solution, according to Goldner, were added and left to act 5 min. Then, the slides were washed in distilled water and rapidly dehydrated through ascending alcohols. Reaction was done for 1 min in absolute alcohol. Finally, the slides were cleared in xylene and mounted.
Slides were examined by two independent evaluators using a light microscope (Leica DM 2000), and all tissues were evaluated using a numerical score based on the number of CD68+, CD21+, CD3+, CD20+ and CD31+ cells in the lining and sublining areas of the section (three different fields in each section), with a score of 0 indicating no positive cells; 1 indicating <10% positive cells; 2 indicating 10%–50% positive cells; and 3 indicating >50% positive cells.10 (link) The inter-rater agreement coefficient was assessed for each single immunohistochemistry (IHC) marker (see onlinesupplementary table S1 ).
ST slides were stained for collagen using the Masson Trichrome Goldner with light green (Bio-Optica; 04-011802) as follows: slides were washed in distilled water; then six drops of Weigert's iron haematoxylin, provided by the manufacturer, were put on the sections and left to act 10 min. Without washing, the slides were drained, and 10 drops of picric acid alcoholic stable solution were added on the sections. Then, the slides were quickly (3–4 s) washed in distilled water, and 10 drops of ponceau acid fuchsin according to Masson were added on the sections and left to act 4 min. Then, the slides were washed in distilled water, and 10 drops of phosphomolybdic acid solution were added on the sections and left to act 10 min. Without washing, the slides were drained, and 10 drops of Light Green Solution, according to Goldner, were added and left to act 5 min. Then, the slides were washed in distilled water and rapidly dehydrated through ascending alcohols. Reaction was done for 1 min in absolute alcohol. Finally, the slides were cleared in xylene and mounted.
Slides were examined by two independent evaluators using a light microscope (Leica DM 2000), and all tissues were evaluated using a numerical score based on the number of CD68+, CD21+, CD3+, CD20+ and CD31+ cells in the lining and sublining areas of the section (three different fields in each section), with a score of 0 indicating no positive cells; 1 indicating <10% positive cells; 2 indicating 10%–50% positive cells; and 3 indicating >50% positive cells.10 (link) The inter-rater agreement coefficient was assessed for each single immunohistochemistry (IHC) marker (see online
