322 disease, including intra-abdominal fistulae, perforations, and abscesses. Here, at baseline, the presence of perianal (P) disease was classified as a separate entity from the Montreal disease behavior classification. Classifications were based on clinical data and objectively confirmed by retrospectively available endoscopies (images, reports), histopathological reports (e.g., from bowel resections), and radiologic information (e.g., MRI or CT images). Disease behavior was recorded as the patients’most severe phenotype (where B1<B2<B3) at the time of sampling. Prospective (follow-up) outcomes and definitions Patients were followed-up from baseline until the most recent date of contact (either outpatient visit, clinical visit, endoscopic investigation, or telephone appointment) with their treating gastroenterologist (as of May 4th, 2021). During follow-up, the progression or recurrence of stricturing disease, penetrating disease, and the occurrence of surgical interventions was recorded. Stricturing disease by radiographic assessment was defined as an intestinal wall thickness > 4 mm, or the presence of luminal narrowing or a pre-stenotic dilation.26,27 Penetrating disease was defined as a chronic tract of granulation tissue between two epithelial-lined surfaces, which was evidently described in endoscopic, radiologic or physical examination reports.10 No differentiation was made between fistula type (i.e., perianal, enterocutaneous, or rectovaginal). Surgical interventions consisted of all CD-related surgeries, which were classified by indication as described in surgical reports. Indications consisted of stenosis- or fistula-related surgeries or intestinal resections due to therapeutic failure. Progression or recurrence of stricturing and/or penetrating disease was defined as either the development of new stricturing/penetrating disease (progression) or as the recurrence of active stricturing (recurring symptomatic stenosis after a period of remission or after intervention, i.e. balloon dilation or surgery) or penetrating disease (recurring fistulizing disease after a period of remission or after intervention). These definitions were based on either endoscopic evidence for progression or recurrence (based on physician’s assessment, retrieved from endoscopy and physical examination reports) or radiologic evidence (i.e., by magnetic resonance imaging [MRI] or rectal endosonography). Biomarker assays All biomarkers analyzed in this study are listed in Table 1. Neo-epitope fragments of extracellular matrix synthesis and degradation were measured using protein fingerprint assays with solidphase competitive enzyme-linked immunosorbent assays (ELISAs). Assays were based on either colorimetry or chemiluminescence. Ninety-six (96) well plates pre-coated with streptavidin (Roche Diagnostics, CAT no. 119-40-279, Hvidovre, Denmark) were coated with biotinylated peptides corresponding to each biomarker for 30 minutes at 20°C. Samples were diluted in assay buffer (50 mM PBS-BTB 8 g/L NaCl, pH 7.4). Subsequently, samples were incubated with horseradish peroxidase-conjugated target-specific monoclonal antibodies for 1 hour at 20°C or for 20 hours at 4°C, depending on the specific assay, and shaken at 300 rpm. Each incubation step was followed by washing of the plates with washing buffer (25 mM TRIZMA, 50 mM NaCl, Chapter 10
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