BACKGROUNDFecal calprotectin is a valuable non-invasive marker for intestinal inflammation and contributes to the selection of patients with suspected inflammatory bowel disease (IBD) for endoscopy. The aim of this study was to evaluate the performance of three automated immunoassays for fecal calprotectin (FC), fecal lactoferrin (FL) and fecal alpha-1-antitrypsin (A1AT) for diagnosis and follow-up of IBD, to investigate if automated analysis of this biomarker profile may further improve the diagnostic process, and to compare them to manual ELISA tests from different manufacturers.METHODSStool samples from 72 patients with Crohn's disease (42), ulcerative colitis (17), irritable bowel syndrome (5), other gastrointestinal inflammation (8), and 72 healthy controls were analyzed for FC, FL, and A1AT on the automated Alegria® system (ORGENTEC Diagnostika, Germany). The results were verified by commercially available manual ELISA tests and discrepancies were further analyzed by immunoblotting. The fecal test results were correlated with the patients' endoscopic findings and with disease activity.RESULTSThe automated assays FC and FL for Alegria® detected endoscopically active intestinal inflammation with a sensitivity and specificity of 74% and 87% (FC) and 62% and 87% (FL), respectively, and efficiently discriminated IBD from non-IBD samples in the patient cohort. Healthy controls tested negative in all assays. The results of the automated biomarker assays significantly correlated to those of the manual ELISAs. Alegria® results for FC were confirmed by immunoblotting in 7 discrepant samples. Levels of A1AT out of the normal range were detectable in a substantial number of IBD and irritable bowel syndrome (IBS) samples: 50% Crohn's disease, 35% ulcerative colitis, and 60% IBS, reflecting the disease-related changes of intestinal permeability in these patients. In IBD patients, elevated levels of A1AT correlated with relapsing disease.CONCLUSIONSMeasurement of FC concentrations with Alegria® is a convenient, promising, and useful tool for improving laboratory diagnostic accuracy and accelerating the diagnostic process and helps to identify those patients in whom endoscopy may be avoided. Automated analysis of a comprehensive profile of fecal biomarkers with Alegria®, including A1AT, provides further substantial benefit for laboratory diagnostics of IBD by improving stratification of patients for treatment and care.

Xavier Bossuyt*, Jan Damoiseaux, Niels Rasmussen, Pieter van Paassen, Bernard Hellmich, Bo Baslund, Daniel Blockmans, Pieter Vermeersch, Marcos Lopez-Hoyos, Martine Vercammen, Elisa Barret, Friederike Hammar, Ulrich Leinfelder, Michael Mahler, Nina Olschowka, Dirk Roggenbuck, Wolfgang Schlumberger, Roger Walker, Johan Rönnelid, Jan-Willem Cohen Tervaert, Elena Csernok and Walter Fierz for (i) the European Federation of Laboratory Medicine (EFLM) Task and Finish Group “Autoimmunity Testing,” (ii) the European Autoimmune Standardization Initiative (EASI) and the (iii) European Consensus Finding Study Group on autoantibodies (ECFSG)

Antineutrophil cytoplasmic antibodies (ANCAs) are important laboratory markers to support the diagnosis of ANCA-associated vasculitis. A 2017 international consensus states that high-quality immunoassays for proteinase-3 (PR3)-ANCA and myeloperoxidase (MPO)-ANCA can be used as the primary screening method for patients suspected of having granulomatosis with polyangiitis or microscopic polyangiitis.1 Efforts have been undertaken to standardise PR3-ANCA and MPO-ANCA measurements. More than 10 years ago, PR3-ANCA and MPO-ANCA reference standards have been prepared (from single patients) under the umbrella of the International Union of Immunological Societies (IUIS) and are available through the Autoantibody Standardization Committee (www.AutoAb.org). Several companies are using these reference standards (assigned value: 100 IU/mL) to calibrate their assays, such as Svar Life Science (ELISA), Thermo Fisher Scientific (fluoroenzyme immunoassay (FEIA)), Medipan (CytoBead assay) and Inova Diagnostics (chemiluminescence assay (CLIA)). The Institute for Reference Materials and Measurements (IRMM) recently released certified reference materials for MPO-ANCA (ERM-DA476/IFCC) and for PR3-ANCA (ERM-DA483/IFCC).2 3 Both reference materials are based on plasmapheresis samples from single patients. We evaluated whether using the IRMM-certified reference materials aligns results for PR3-ANCA and MPO-ANCA across assays. A random selection of 98 serum samples covering low, medium and high PR3-ANCA or MPO-ANCA levels from the Leuven and the Maastricht cohort included in the international EUVAS study4 were distributed to five manufacturers, together with the PR3-ANCA and …

Background: Serum autoantibodies are measured in RA patients for diagnostic and prognostic purposes. Antibodies against citrullinated peptides are directed against citrulline that results from post-transla- tional modification (PTM) of arginine residues. Antibodies against another PTM residue, homocitrulline, have recently been described in RA patients’ sera. Lysine acetylation is another important PTM. Proteins carrying this modification regulate metabolic pathways and host protein acetylation is a mechanism through which the microbiome affects its host. Recent sequencing of the intestinal microbiome revealed a high prevalence of a specific bacterium, Prevotellacopri , inpatients with early RA suggesting a pathogenic function. This function may be exerted through protein acetylation. To date there have been no studies of immunity against acetylated proteins in RA. Objective: To investigate antibody reactivity against acetylated, citrullinated and homocitrullinated vimentin peptides in patients with early inflammatory arthritis. Methods: Microtitre plates were coated with vimentin derived peptides identical in length and composition except at one amino acid that was changed to introduce a citrullinated, carbamylated or acetylated residue. By using a peptide of known sequence and introducing one modification at a time, we were able to determine specific antibody reactivities. Sera of 268 treatment naive patients with early inflammatory arthritis and symptoms (cid:3) 3 month’s duration were tested. Patients were assigned to outcome categories at 18 month follow up. Anti-CCP antibody levels were determined using a commercial anti-CCP2 antibody assay (ELiA Background: Early diagnosis of RA improves outcomes but is challenging, particularly among ACPA-negative individuals. Previously we identified an IL-6-mediated CD4 þ T cell transcriptional signature, enriched for signal transduction and activation of transcription-3 (STAT3) target genes, which had discriminatory value for this purpose. In the present work we sought independent replication of those findings, the development of a more readily applicable diagnostic assay and insight into mechanisms of disease induction. Methods: Among 210 early arthritis patients attending the Newcastle Early Arthritis Clinic (NEAC) who were naive to immunomodulatory treatment, serum cytokine levels were ascertained by immuno-assay. Constitutive and IL-6-induced expression of phosphorylated STAT1 and 3 (pSTAT1/3) were determined in paired fresh circulating lymphocytes using flow cytometry. Finally, contemporaneous CD4 þ T cell gene expression was measured in highly purified, fresh CD4 þ T cells by hybridizing high-integrity RNA isolates to Illumina Human HT12 BeadChips, and employing appropriate microarray normalization algorithms. Patients were followed up for > 12 months and diagnostic outcomes confirmed. Analyses included non-parametric ANOVA (Dunn’s post-hoc group comparisons), Spearman’s rank correlation, multiple regression and hierarchical clustering. Results: Consistent with previous observations, serum IL-6 levels were significantly higher in early ACPA-negative RA patients ( n ¼ 29) than in Background: RA occurs when genetically predisposed individuals are exposed to environmental factors. The dominant RA environmental risk factor in European populations is smoking. Drinking alcohol appears to protect against RA in Europeans. The relevance of these risk factors to non-European populations is uncertain. We undertook a case–control study to evaluate the associations between smoking, drinking and RA in Black African and Black Caribbean ethnicity individuals. Methods: We evaluated 180 RA cases enrolled to the GENetics of RA in individuals of African ancestry (GENRA) study. All met the 1987/2010 ACR RA classification criteria and were of self-reported black or black Caribbean Patients were recruited from South London Rheumatology Smoking and alcohol intake data were captured by The study was ethically approved; all participants provided consent. Controls were evaluated from the 2004 Health England (focussing on the health of Ethnic minorities). 1812 controls of black African or black Caribbean ethnicity a history of arthritis, rheumatism fibrositis were Smoking and alcohol intake data were cases. Associations (ever vs . never), . case-control logistic for age and case-control (cid:4) pack-years) RA risk never-smokers/ pack-years). Results: In black individuals, smoking risk Ever-smoking rates lower in cases than controls adjusted odds ratio (OR) . black African ethnicity individuals. Our findings suggest that environ- mental risk factors for RA differ between European and African populations. They also indicate that research into RA risk factors needs undertaking separately in African and Caribbean ethnicity individuals. Disclosure statement: is an Research UK Clinical Research Fellowship NIHR Lectureship. All authors