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Recently, a review on the clinical relevance of human epithelial (HEp)-2 indirect immunofluorescence assay (IIFA) patterns was published by the International Consensus on ANA Patterns (ICAP) consortium.1 The clinical relevance was primarily defined within the context of the clinical manifestations, that is, the suspected disease, and included recommendations for follow-up or reflex testing. It is anticipated that if a solid-phase test identifies the disease-specific target autoantigen, the clinical relevance can be further refined. The analyses by Vulsteke et al 2 elegantly illustrate that the positive predictive value (PPV) of the HEp-2 IIFA pattern can be further improved by taking into account the titre of the respective patterns. Given the value and importance of the large dataset (n=9851) obtained in this routine laboratory practice without any selection, except for studying only monospecific nuclear patterns, and the extensive analyses, it is unfortunate that the results have been condensed into a letter. In addition to the patient cohort from the University Hospitals Leuven, a smaller cohort (n=529) was analysed. In this second cohort from the OLV Hospital Aalst, sera with a nuclear speckled pattern (n=88) were included; only four sera were obtained from patients with a systemic autoimmune rheumatic disease (SARD). Evidently, only the large dataset warranted reliable associations between the combination of HEp-2 IIFA pattern and titre on the one hand and the final diagnosis on the other. With respect to the ICAP consensus on the clinical relevance of HEp-2 IIFA patterns, there are three deviations from this consensus as reported in the study of Vulsteke et al . First, the genetically modified HEp-2000 substrate was used, whereas this substrate is currently not included in the ICAP consensus …

The large study of 600 cases from 40 countries of the Covid-Global Rheumatology Alliance has shown that the use of conventional disease-modifying antirheumatic drug, alone or in combination with biologics/Janus Kinase inhibitors, and tumour necrosis factor inhibitor was associated with a reduced odds of hospitalisation.1 Schulze-Koops et al described two fatal outcomes in patients with rheumatoid arthritis treated with rituximab and focuses on careful vigilance on immunosuppression in the treatment of immune-mediated rheumatic diseases.2 To reinforce this observation, we report the case of a female patient aged about 60 years old, with a history of polymyositis and Sjogren’s syndrome. Her history was significant for previous cancer: thyroid carcinoma in 1995, phylloid tumour of the right breast in 2010 and carcinoid of the annexes in 2011. In January 2020, the patient had problem of fatigue, myalgia, together with rise in creatinekinase (CK) and Aldolase with positive anti-Mi-2 antibody, while on stable maintenance treatment with methotrexate 12.5 mg/weekly and prednisone 10 mg/day, rituximab 1 g 2 weeks apart was employed since 2017 with further cycles on the basis of B …

We read the interesting epidemiological study on 600 patients with rheumatic diseases hospitalised for COVID-19 infection (COVID-19 Global Rheumatology Alliance Registry) published by Gianfrancesco et al .1 Data analysis showed a slightly increased risk of hospitalisation for prednisone doses of ≥10 mg, while biological disease-modifying anti-rheumatic drug (b-DMARD)/targeted synthetic disease-modifying anti-rheumatic drug (ts-DMARD) monotherapy just prior to COVID-19 diagnosis appeared protective, in particular, the tumour necrosis factor targeting agents, as result of a subsequent subanalysis. The epidemiological impact of COVID-19 in patients with rheumatic diseases being treated with b-DMARD has been the subject of several reports by Italian groups. In the first report, only 4 cases were confirmed through rhinopharyngeal swabs out of 320 observed cases.2 In another study assessed in the emergency period in Lombardy through visit or phone contact, only 3 cases through rhinopharyngeal swabs out of 520 cases were confirmed.3 Another study of 859 patients in another rheumatology centre in Tuscany, which applied a phone contact methodology, identified only 2 patients suffering from COVID-19 pneumonia.4 As part of the Tuscany population serological screening, the Incidence COVID-19-Rheumatic Disease–Biologics study has been planned. …

We read with great interest the new European League against Rheumatism/American College of Rheumatology (EULAR/ACR) classification criteria for systemic lupus erythematosus (SLE) by Aringer et al ,1 that recommend a positive antinuclear antibody (ANA) test as an entry criterion for SLE classification. According to these criteria, ANA testing has to be performed by a highly sensitive screening method before initiating the cascade work algorithm. This is a strategic move compared with the previous criteria by the Systemic Lupus International Collaborating Clinics (SLICC)2 in which ANA was considered just one of the immunological items together with the more specific antibodies against Sm and dsDNA. With this …

We have read the interesting research by Bonelli et al on the role of rituximab in vaccination for SARS-CoV-2. In the data on five patients, two had repopulated B cells and had antibodies to SARS-CoV-2 RBD after vaccination.1 In our experience, we evaluated a group of patients with rheumatoid arthritis who had received the last infusion of rituximab 6 months earlier (group A, four patients), a group of patients who had received the last dose of rituximab 9 months earlier (group B, five patients) and finally a group of patients who had received rituximab 12 months earlier (group C, five patients). All patients received two doses of SARS-CoV-2 mRNA BNT162b2 vaccine 21 days apart. Patients underwent evaluation of the lymphocyte …

Background:anti-NXP2 antibodies is considered a serological marker of dermatomyositis (DM), with calcinosis, severe myositis and, in some series, cancer. Historically, these associations have been detected with immunoprecipitation (IP), but in the last few years commercial lineblot (LB) assay have been released.Objectives:to analyze the clinical features associated to anti-NXP2 antibodies, including the onset of concomitant cancers, both with LB and homemade IPMethods:clinical and serological data from medical charts of 213 patients with a diagnosis of inflammatory miosidites without anti-NXP2 (NXP2-), followed-up by two third-level Centers, and 61 anti-NXP2+ patients from 10 Rheumatological centers were analyzed. Anti-myositis specific (MSA) and anti-myositis associated antibodies (MAA) were detected in single centers by LB (Euroimmun Autoimmune Inflammatory Myopathies 16 antigens). Anti-NXP2 was confirmed by protein and RNA IP, as previously described (1)Results:clinical diagnosis of anti-NXP2+ positive with LB were 42 DM, 11 PM, inclusion body myositis (IBM) 4, necrotizing myositis and overlap (OM) 1 each. Anti-NXP2+ showed a lower age at onset (pIP did not confirmed anti-NXP2 antibodies in 18 sera: in 4 cases at least one MSA/MAA was identified by IP; these 18 patients did not show differences when compared with 213 anti-NXP2-.Conclusion:Protein IP confirmed anti-NXP2 antibodies in 63% of LB+ sera. Double positive cases showed more typical DM features and rarely occurred in IIM not DM. Anti-NXP2 positivity by LB should be confirmed by other methods in order to correctly diagnose and characterize IIM patients.References:[1]Arthritis Res Ther 2012,30;14:R97Acknowledgments:Forum Italiano per la Ricerca Malattie Autoimmuni (FIRMA)Disclosure of Interests:Micaela Fredi: None declared, Ilaria Cavazzana: None declared, Angela Ceribelli: None declared, Maria Grazia Lazzaroni: None declared, Simone Barsotti: None declared, Maurizio Benucci: None declared, Lorenzo Cavagna: None declared, Ludovico De Stefano: None declared, Andrea Doria Consultant of: GSK, Pfizer, Abbvie, Novartis, Ely Lilly, Speakers bureau: UCB pharma, GSK, Pfizer, Janssen, Abbvie, Novartis, Ely Lilly, BMS, Giacomo Emmi: None declared, Marco Fornaro: None declared, Federica Furini: None declared, Roberto Gerli: None declared, Maria Grazia Giudizi: None declared, Marcello Govoni: None declared, Anna Ghirardello: None declared, Luca Iaccarino Speakers bureau: GSK, Pfizer, Janssen, Novartis, Florenzo Iannone Consultant of: Speaker and consulting fees from AbbVie, Eli Lilly, Novartis, Pfizer, Roche, Sanofi, UCB, MSD, Speakers bureau: Speaker and consulting fees from AbbVie, Eli Lilly, Novartis, Pfizer, Roche, Sanofi, UCB, MSD, Maria Infantino: None declared, Alessandro Mathieu: None declared, Emiliano Marasco: None declared, Paola Migliorini: None declared, Boaz Palterer: None declared, paola parronchi: None declared, Matteo Piga: None declared, Federico Pratesi: None declared, Antonella Radice: None declared, Carlo Selmi: None declared, Valeria Riccieri: None declared, Marilin Tampoia: None declared, Giovanni Zanframundo: None declared, Angela Tincani: None declared, Franco Franceschini: None declared

Background:Anti-3-hydroxy-3-methylglutaryl-coenzime A reductase (HMGCR) myopathy is a new entity, which has been clearly associated to statin use, even if it can be diagnosed in patients without a history of exposure to statin or even in the childhood (1).Objectives:The aim of the study is to describe the efficacy of a triple therapy regimen consisting in high-doses of intravenous immunoglobulins (IVIG), methotrexate (MTX), and glucocorticoids (GC) in 16 patients with Anti-HMGCR myopathy enrolled in 6 specialized centres.Methods:A total of 16 patients with anti-HMGCR myopathy (7 females; 9 males) were collected. Mean (±standard deviation) age at the onset of disease was 72.4±10.3 years old. All patients were diagnosed having anti-HMGCR myopathy [anti-HMGCR antibodies were measured by chemiluminescence assay (BioFlash, Inova, CA)] (2). Median follow-up was 29.5 months (interquartile range: 15.75-60 months). Anti-HMGCR antibodies were available in the follow-up in 8/16 patients.Results:Thirteen out of 16 patients (81.3%) had been exposed to statin (1/13 to red rice), 3/16 (18.7%) were not exposed. As induction therapy, 11/16 patients have been treated with triple therapy (high-dose IVIG, MTX and GC), 2/16 with double therapy (high-dose IVIG and GC), 2/16 have been treated with GC alone, the patient exposed to red rice resolved only with red rice suspension. Clinical remission and normalization of CPK values within month +24 were obtained in all the patients. All the patients were in remission at the last follow-up. Gradual improvement started soon from the first month, and among the 13 patients treated with an aggressive immunosuppresssive therapy including IVIG (13/13), GC (13/13) and methotrexate (11/13), 9/13 normalized the CPK value within 6 months. Clinical and laboratory response was accompanied by significant decrease or normalization of the anti-HMGCR antibody titer. All the patients were either not taking GC (56.3%), or were taking low doses of GC (43.7%) at the last follow-up. Four patients had stopped GC within 6 months. No serious side effects were recorded. After persistent remission, a maintenance immunosuppressive therapy was then administered. Only 3 relapses in 3 different cases were recorded, all of them during drug-free remission in long-term follow-up. Reinduction was again effective in all.Conclusion:Anti-HMGCR myopathy is a rare and serious myopathy which usually affects older people during statin treatment. After statin suspension, a rapid and sustained remission can be achieved by induction with a triple aggressive therapy consisting in medium-to high doses of GC, high-dose IVIG, and MTX (3). GC should be tapered as soon as possible. Relapse appears infrequent during maintenance treatment. Monitoring anti-HMGCR antibody titer may be clinically relevant.References:[1]AL Mammen et al. N Engl J Med. 2016;374:664-9[2]Musset L et al. Autoimmun Rev. 2016;15:983-93.[3]Aggarwal A et al. Scand J Rheumatol. 2019; 1-7.Acknowledgments:We thank MD Francesca Grosso and MD Valentina Mecheri from the University of Florence, MD Angela Zuppa and MD Chiara De Michelis, from San Martino Hospital, Genova, for their valued collaboration in data collectionDisclosure of Interests:Elena Treppo: None declared, Maria Infantino: None declared, Maurizio Benucci: None declared, Viviana Ravagnani: None declared, Boaz Palterer: None declared, Marina Grandis: None declared, Martina Fabris: None declared, Paola Tomietto: None declared, Mariangela Manfredi: None declared, Arianna Sonaglia: None declared, Maria Grazia Giudizi: None declared, Francesca Ligobbi: None declared, Daniele Cammelli: None declared, Paola Parronchi: None declared, Salvatore De Vita Consultant of: Roche, GSK, Speakers bureau: Roche, GSK, Novartis, Luca Quartuccio Consultant of: Abbvie, Bristol, Speakers bureau: Abbvie, Pfizer

We have read with great interest the recent article from Espinosa-Ortega et al 1 and the commenting letter by Mahler et al 2 on the reliability of line immunoassay (LIA) versus immunoprecipitation (IP) in the detection of myositis-specific autoantibodies (MSAs) for the diagnosis of idiopathic inflammatory myopathies (IIM). Even if the search for MSA by dot immunoassay (DIA) or LIA has been used for over a decade and represents a ‘non-criteria’ test to assist clinicians in IIM diagnosis, MSAs have been included in classification criteria only for a couple of years.3 However, initial studies on the clinical utility of the DIA/LIA methods in diagnosing IIM were conducted on a restricted panel of MSA and mainly on selected IIM patients.4 …

The recent article by Pisetsky et al 1 showing data derived from a comparison of different antinuclear antibody (ANA) assays in a cohort of patients with established systemic lupus erythematosus highlighted the critical issue of ANA detection. With great interest, we read the correspondence by van Hoovels et al 2 describing variation in ANA detection by automated indirect immunofluorescence (IIF) analysis and the following letter from Mahler and Auza on the strong need for ANA testing standardisation.3 Nowadays, the IIF on human epithelial cells (HEp-2) is considered the gold standard for ANA testing, but biological and non-biological issues limiting the IIF test are not currently adequately clarified in study literature.4 5 In fact, despite the fact that the IIF method on HEp-2 cells has existed for about 40 years, there are little data on this topic, and studies are mainly focused on selected patient groups, rather than samples from ‘real …

With great interest, we read the letters that have commented on the recent European League Against Rheumatism/American College of Rheumatology classification criteria for idiopathic inflammatory myopathies1 (IIMs) and ensuing discussions.2 3 This highlights how harmonising myositis-specific antibodies (MSA) testing methodologies is currently a hot topic of debate among laboratory specialists, clinicians and manufacturers. I would particularly like to share our own immunology laboratory experience by expanding on the letter entitled ‘Detection of myositis-specific antibodies: additional notes’ by M Mahler and M Fritzler4 with which we wholly agree. Newer technologies, such as line immunoassays (LIA) and dot blots (DB), which provide greater sensitivity and rapid serological diagnoses, have standardisation and specificity drawbacks, but the tight connection between autoantibodies and clinical phenotypes underline a great need …

We would like to underline the fundamental role of the assay method used to detect anti-drug antibodies. In Emery et al's article1 a very high percentage of patients show anti-etanercept (ETN) antibodies, 13.1%, which is much higher compared with main literature data, reporting generally less than 5% incidence.2–5 This apparent discrepancy can be explained by the different technical findings of laboratory methods employed in the works. Emery et al employs electrochemiluminescence method (ECL), while most of …

Background The gold standard for ANA screening is indirect immunofluorescence (IIF) on HEp-2 cells. Automation of IIF image reading can provide a reliable basis for cost-effective serological diagnostics. Recentely there has been an increase demand of computer-aided diagnosis (CAD) tools to support clinicians in the field of IIF. The novel digital imaging reading approach can help us to overcome the reader subjectivity, allowing a permanent archival record of the results and CAD use. We described a CAD system (SLIM-system) for ANAIIF images reading (1). Objectives To assess inter-observer variability using digital images reading approach instead of manual visual approach. To compare accuracy of expert readers of three different laboratories to automated (CAD) classification. Methods Three laboratories (named A, B, C) expert in IIF field were involved in the multicenter project which recruited 305 consecutive serum samples submitted to laboratory for routine ANA testing. Every image has been classified from experienced physicians. We acquired 915 images using HEp-2 cell slides (BION-DASIT) at 1:80 screening dilution as recommended by the current guidelines. Each expert took 3 different images per sample with an acquisition unit consisting of the fluorescence microscope coupled with a LED light source and a digital camera. The images with a resolution of 1388x1038pixels, color depth 24 bits were stored in bitmap format. We used 40-fold magnification. Digital images have been reclassified from two blinded experts into three intensity classes: positive, negative, weak positive. Thus we compared obtained data with CAD classification. Data have been analyzed in a pairwise manner. Agreement have been performed by Cohen9s kappa test Results Fig1 shows results. We obtained 99.6%, 69.48% and 29.55% of sensitivity on positive, negative and weak positive images of respectively comparing A and B; 92.74%, 60.49% 49.24% comparing C and A. We evaluated accuracy and specificity of visual reading: 71.65% and 69.48% respectively comparing group B-A; 67.39% and 60.49% comparing group C-A. A-B, B-C and A-C agreement were 0.551 (±0.0237), 0.69 (±0.02) and 0.509 (±0.023) respectively The SLIM-system image classification showed a sensitivity of 92.03%, 94.94%, and 43.41% on positive, negative and weak positive, respectively Comparing SLIM-system results to average experts9 classification we obtained: accuracy 86.87%vs69.52%, specificity 94.94%vs64.98%. Conclusions In literature there are no studies on IIF interobserver reading variability by digital images reading approach. We have compared interobserver variability among 3 laboratories experienced in ANA field. The operating principle of new automated systems is acquiring, storing, and analyzing of digital images of stained IIF slides, and is needed to standardize image acquisition. SLIM-system data analysis showed a higher sensitivity on negative and weak positive images classification compared to the manual visual approach. The new CAD systems can eliminate the need for a darkroom and separate image acquisition from image interpretation, besides have the potential to improve the quality and utility of the ANA HEp-2 assay. References Rigon A et al Autoimmun Rev. 2011 Aug;10(10):647-52. Disclosure of Interest None declared

Background Ro-52 is an interferon (IFN)-inducible protein of the tripartite motif (TRIM) family. Antibodies against Ro-52 have been described in patients with different autoimmune diseases such as systemic lupus erythematosus (SLE) and Sjogren’s syndrome (SS), which are often associated with anti-Ro-60 antibodies. However, in inflammatory myositis, anti-Ro-52 antibodies are frequently present without anti-Ro-60 antibodies. Ro-52 autoantigen is extraordinarily antigenic and its autoantibodies are directed against both linear and conformational epitopes. Objectives The first aim of the study was to evaluate antibodies against the full length of the Ro-52 antigen (Ro-52) and its five fragments, and their association with serological and clinical data. The second aim was to assess the prevalence of anti-full-length Ro-52 and its five fragments in different systemic autoimmune rheumatic diseases (SARDs). Methods Serum samples were obtained from 78 SARDs patients and 68 controls (50 healthy donors and 18 patients with other autoimmune diseases). Ro-52 antibodies were measured using four different Methods: Bioplex2200 Multiplex-Biorad (Laboratories Inc. Hercules, CA, USA), ANA Profile3-Blot, Profile Autoimmune Liver Diseases-Blot, and Myositis Profile3-Blot EUROIMMUN AG. (EUROIMMUN AG, Lubeck, Germany). Serum was considered positive for anti-Ro-52 when at least one of these methods revealed their presence. Full-length Ro-52 antigen was expressed in insect cells using the baculovirus system, and five recombinant Ro-52 antigen fragments [Ro-52-1, Ro-52-2, Ro-52-3, Ro-52-4 (partly overlapping Ro-52-1 and -2) and Ro-52-5 (partly overlapping Ro-52-2 and -3)] expressed in E. coli, were coated onto nitrocellulose membranes in a line immunoassay system. Sera were incubated in accordance with the manufacturers standard protocol (30 min serum, 30 min anti-human IgG/alkaline phosphatase, 10 min NBT/BCIP substrate). Prevalence and reaction intensities were automatically evaluated using the commercial computer programme EUROLineScan from EUROIMMUN AG. Results All of the samples obtained from the SARDs patients were positive for antibodies against the full-length Ro52 and fragment 2, except those obtained from the myositis patients. None of the sera samples of the patients and controls were positive for anti-Ro-52-3. Moreover, all of the control samples were negative for the full-length Ro52 and all of the fragments. Myositis group (n=3) gave an isolated but very weak reaction with the full length Ro-52 and displayed no reaction with the fragments. In the SARDs patients, the fragment prevalence rates were Ro-52-1= 4/82 (4.9%), Ro-52-2= 79/82 (96.3%), Ro-52-3= 0/82 (0%), Ro-52-4= 6/82 (7.3%) and Ro-52-5= 22/82 (26.8%). Conclusions The main epitope is localised on fragment 2, and all diseases have antibodies that target this fragment. Antibody positivity for full-length Ro52 and negativity for all five fragments suggests a diagnosis of myositis. As all of the samples were positive for fragment 2, and only some for fragments 5 or 4, it seems that the target epitope is localized in the middle of fragment 2 or in the area between fragments 4 and 5. Further studies involving a large number of patients are suggested to confirm our findings. Disclosure of Interest None Declared

Background Anti-TNF drugs have changed the clinical course of rheumatoid arthritis (RA), but the survival rate and the resistance to the therapy showed that about 30% of RA patients fail to respond to these drugs. Objectives The aim of our study was to evaluate the correlation between the development of anti-drug antibodies and specific IgG-4 antibodies against TNF inhibitors and the resistance to this therapy in RA patients. Methods The retrospective study involved 130 established RA patients [98 females and 32 males, mean age 56,7±12,3 years, disease duration 6,3±1.2] who fail to respond to conventional DMARDs treated with anti-TNF agents [32 pts with INF, 58 pts. with ETN, 40 pts with ADA] with a baseline DAS28 range between 3.2-5.6. After 6 months of treatment according to DAS28 values the patients were classified in remission for DAS28 3.2. The patients were tested for serum anti-drugs antibodies (Anti TNF-α Blocker ELISA kit Immundiagnostik AG, Bensheim - Listarfish, Milan, Italy) and IgG4 specific antibodies against TNF- inhibitors (ImmunoCAP® Assay fluoroenzyme immuno assay Kit for ImmunoCAP250, Phadia, Uppsala, Sweden). Results After a mean of 6 months in IFN group: 15.65% of the patients were in remission (R ), 31.2 % in LDA and 53.12% NRs; in ETN group 22.41% of the patients in R, 41.37% in LDA, 36.20% NRs, in ADA group 30% of the patients in R, 25% in LDA and 45% NRs. In the INF group the anti-drug antibodies were detected in 21. 87% of the patients and specific IgG-4 antibodies in18%, in ETN group the anti-drug antibodies were detected in 10.34% and specific IgG-4 antibodies in 8.67%, in ADA group the anti-drugs antibodies in 25% and specific IgG-4 antibodies in 5%. The presence of anti-drug antibodies in INF e ADA group correlate with the high percentage of NRs (R:0.032 ;R:0.039). No correlation with the specific IgG-4 antibodies against TNF and clinical response was observed. Conclusions In patients with RA the presence of all anti-drug antibodies but not the specific IgG-4 antibodies against TNF could influence the activity of TNFblockers in particular in INF and ADA group. This study also confirmed the low immunogenecity of ETN. Disclosure of Interest None Declared