Response to: 'Pitfalls of antinuclear antibody detection in systemic lupus erythematosus: the positive experience of a national multi-center study' by Pregnalato et alWe appreciate the comments by Pregnolato et al 1 on our original paper 2 and subsequent correspondence in the journal. 3 4 The data presented are very interesting and suggest that, under certain circumstances, results of antinuclear antibody (ANA) testing with samples from patients with established systemic lupus erythematosus can show both a high frequency of positivity and high concordance among testing laboratories.Such agreement may result from the nature of the samples themselves, including the titre of the antibodies and the array of specificities present, the kits used for the assays and the experience and training of the observers.While we would acknowledge that ANA assays can perform very well, our experience as well as that in the literature indicates that variability occurs significantly in a way that complicates screening of patients for clinical trials.Notwithstanding the demonstrations of the kind reported by Pregnolato et al, we think that it is important to try to understand assay variability and improve serological determinations for both routine care as well as clinical research.

BACKGROUND:Patients with suspected idiopathic inflammatory myopathies (IIM) are commonly tested for the presence of anti-nuclear antibodies (ANA) by indirect immunofluorescence (IIF) on HEp-2 cell substrates. However, ANA-IIF false negative tests may occur in IIM because some antigens, such as Jo1 and Ro52, may be scarcely expressed on HEp-2 cells. In addition, cytoplasmic staining is often not appropriately investigated by a specific antibody assay, leading to decreased clinical sensitivity of the ANA test. We evaluated the diagnostic impact of different strategies using different combination of myositis-related autoantibody tests.METHODS:Sera from 51 patients with an established diagnosis of IIM were tested for ANA by IIF on HEp-2 cells and for myositis-specific antibodies (MSA) and myositis-associated antibodies (MAA) by lineblot methods.RESULTS:Forty-four/51 (86.3%) samples tested positive with at least one of the three methods and seven were negative with all methods. Of the 44 positive samples, 9 (20.5%) tested negative for the ANA-IIF test and positive for MAA/MSA. Anti-Ro52 were the most prevalent autoantibodies in IIM patients (21/51; 41%), frequently associated with anti-Jo1 antibodies (13/21; 62%). 13 (16%) anti-Ro52 and anti-Jo1 negative samples were reactive to MSA.CONCLUSIONS:Our findings suggest that when IIM is clinically suspected, the optimal diagnostic algorithm is to associate the ANA-IIF screening test with a specific test for anti-Ro52 and anti-Jo1 antibodies. Should all these tests be negative, serological tests for MSA are recommended.

Objective Increasing evidence indicates systemic inflammation as a new potential cause of acquired long QT syndrome (LQTS), via cytokine-mediated changes in cardiomyocyte ion channels. Torsade de pointes (TdP) is a life-threatening polymorphic ventricular tachycardia occurring in patients with LQTS, usually when multiple QT-prolonging factors are simultaneously present. Since classical risk factors cannot fully explain TdP events in a number of patients, we hypothesised that systemic inflammation may represent a currently overlooked risk factor contributing to TdP development in the general population. Methods Forty consecutive patients who experienced TdP (TdP cohort) were consecutively enrolled and circulating levels of C-reactive protein (CRP) and proinflammatory cytokines (interleukin-6 (IL-6), tumour necrosis factor alpha (TNF alpha), interleukin-1 (IL-1)) were compared with patients with active rheumatoid arthritis (RA), comorbidity or healthy controls. An additional 46 patients with different inflammatory conditions (acute infections, n=31; immune-mediated diseases, n=12; others, n=3) and elevated CRP (inflammatory cohort) were prospectively enrolled, and corrected QT (QTc) and cytokine levels were measured during active disease and after a CRP decrease of > 75% subsequent to therapy. Results In the TdP cohort, 80% of patients showed elevated CRP levels (median: similar to 3 mg/dL), with a definite inflammatory disease identifiable in 18/40 cases (acute infections, n=12; immune-mediated diseases, n=5; others, n=1). In these subjects, IL-6, but not TNFa and IL-1, was similar to 15-20 times higher than in controls, and comparable to RA patients. In the inflammatory cohort, where QTc prolongation was common (mean values: 456.6 +/- 30.9 ms), CRP reduction was associated with IL-6 level decrease and significant QTc shortening (-22.3 ms). Conclusion The data are first to show that systemic inflammation via elevated IL-6 levels may represent a novel QT-prolonging risk factor contributing to TdP occurrence in the presence of other classical risk factors. If confirmed, this could open new avenues in antiarrhythmic therapy.

Background— In patients with autoimmune disease, anti-Ro/SSA antibodies (anti-Ro/SSA) are responsible for a novel autoimmune-associated long-QT syndrome by targeting the hERG potassium channel and inhibiting the related current ( I Kr ). Because anti-Ro/SSA are also present in a significant proportion of healthy subjects and may be associated with torsades de pointes (TdP) arrhythmia, we tested the hypothesis that anti-Ro/SSA may represent a silent risk factor in patients developing TdP. Methods and Results— Twenty-five consecutive patients who experienced TdP were prospectively collected independent of ongoing therapies and concomitant diseases. Anti-Ro/SSA were detected by fluoroenzyme immunoassay, immuno–Western blotting, and line-blot immunoassay. Purified IgGs from anti-Ro/SSA-positive and anti-Ro/SSA-negative patients were tested on I Kr using HEK293 cells stably expressing the hERG channel. As expected, in TdP patients, many known corrected QT interval–prolonging risk factors were simultaneously present, including hypokalemia that was the most common (52%). Anti-Ro/SSA were present in 60% of the subjects, mostly the anti-Ro/SSA-52-kD subtype detected by immuno–Western blotting only. A history of autoimmune disease was found in only 2 of anti-Ro/SSA-positive patients. Experimental data demonstrated that purified anti-Ro/SSA-positive IgGs significantly inhibited I Kr and cross reacted with hERG-channel proteins. Moreover, anti-Ro/SSA-positive sera exhibited high reactivity with a peptide corresponding to the hERG-channel pore-forming region. Conclusions— Anti-Ro/SSA may represent a clinically silent novel risk factor for TdP development via an autoimmune-mediated electrophysiological interference with the hERG channel. We propose that TdP patients may benefit from specific anti-Ro/SSA testing even in the absence of autoimmune diseases as immunomodulating therapies may be effective in shortening corrected QT interval and reducing TdP recurrence risk.

Mounting evidence indicates that in chronic inflammatory arthritis (CIA), QTc prolongation is frequent and correlates with systemic inflammatory activation. Notably, basic studies demonstrated that inflammatory cytokines induce profound changes in potassium and calcium channels resulting in a prolonging effect on cardiomyocyte action potential duration, thus on the QT interval on the electrocardiogram. Moreover, it has been demonstrated that in rheumatoid arthritis (RA) patients, the risk of sudden cardiac death is significantly increased when compared to non-RA subjects. Conversely, to date no data are available about torsades de pointes (TdP) prevalence in CIA, and the few cases reported considered CIA only an incidental concomitant disease, not contributing factor to TdP development. We report three patients with active CIA developing marked QTc prolongation, in two cases complicated with TdP degenerating to cardiac arrest. In these patients, a blood sample was obtained within 24 h from TdP/marked QTc prolongation occurrence, and levels of IL-6, TNFα, and IL-1 were evaluated. In all three cases, IL-6 was markedly elevated, ~10 to 100 times more than reference values. Moreover, one patient also showed high circulating levels of TNFα and IL-1. In conclusion, active CIA may represent a currently overlooked QT-prolonging risk factor, potentially contributing in the presence of other “classical” risk factors to TdP occurrence. In particular, a relevant role may be played by elevated circulating IL-6 levels via direct electrophysiological effects on the heart. This fact should be carefully kept in mind, particularly when recognizable risk factors are already present and/or the addition of QT-prolonging drugs is required.

Reflex tests are widely used in clinical laboratories, for example, to diagnose thyroid disorders or in the follow-up of prostate cancer. Reflex tests for antinuclear antibodies (ANA) have recently gained attention as a way to improve appropriateness in the immunological diagnosis of autoimmune rheumatic diseases and avoid waste of resources. However, the ANA-reflex test is not as simple as other consolidated reflex tests (the TSH-reflex tests or the PSA-reflex tests) because of the intrinsic complexity of the ANA test performed by the indirect immunofluorescence method on cellular substrates. The wide heterogeneity of the ANA patterns, which need correct interpretation, and the subsequent choice of the most appropriate confirmatory test (ANA subserology), which depend on the pattern feature and on clinical information, hinder any informatics automation, and require the pathologist’s intervention. In this review, the Study Group on Autoimmune Diseases of the Italian Society of Clinical Pathology and Laboratory Medicine provides some indications on the configuration of the ANA-reflex test, using two different approaches depending on whether clinical information is available or not. We further give some suggestions on how to report results of the ANA-reflex test.

1 Laboratorio di Immunopatologia e Allergologia, Azienda Ospedaliero Universitaria di Udine, p.le S. Maria della Misericordia 15, 33100 Udine, Italia 2 Laboratorio di Patologia Clinica, Ospedale San Antonio, Tolmezzo, Italia 3 Dipartimento Scienze Mediche, Chirurgiche e Neuroscienze, Sezione di Reumatologia Siena, Siena, Italia 4 Laboratorio di Patologia Clinica, Ospedale S. Maria degli Angeli, Pordenone, Italia 5 Allergologia e Immunologia Clinica, Ospedale S. Maria degli Angeli, Pordenone, Italia 6 Patologia Clinica I, A.O Policlinico Consorziale, Bari, Italia 7 Dipartimento Biotecnologie Mediche, Universita degli Studi di Siena, Siena, Italia 8 UO Microbiologia e Virologia, Ospedale San Carlo Borromeo, Milano, Italia 9 U.O.C Medicina di Laboratorio, Dipartimento di Patologia Clinica, Ospedale di Treviso, Treviso, Italia 10 Laboratorio Analisi Chimico Cliniche, Ospedale Papa Giovanni XXIII, Bergamo, Italia 11 Laboratorio Analisi, Ospedale Buccheri La Ferla, Palermo, Italia 12 Laboratorio Immunologia Allergologia, Dipartimento di Patologia Clinica, Ospedale San Giovanni di Dio, Firenze, Italia 13 Laboratorio di Analisi, U.O. Patologia Clinica, O. Civico e riflessivo, che comprende un ruolo attivo del patologo sulla base di informazioni cliniche, dati demografici e risultati precedenti [1]. Nella diagnostica autoimmune si utilizzano da tempo algoritmi a cascata in particolare nella ricerca degli anticorpi anti-nucleo (ANA). Il test ANA-riflesso proprio perche non comporta automatismi informatici, deve piu propriamente essere definito “ANA-riflessivo”. Questo si differenzia infatti dai test “reflex” fino ad oggi utilizzati in laboratorio sia concettualmente che dal punto di vista organizzativo. Il test TSH-reflex, ad esempio, prevede l’esecuzione a cascata di alcuni test inseriti in un algoritmo ben definito, in base al risultato della determinazione del TSH senza che gli operatori intervengano con aspetti decisionali. E’ evidente che il test ANA-riflessivo risulta piu complesso del TSH-reflex o del test PSA-reflex per diversi motivi: il primo, e principale, e che il test ANA, a differenza del TSH o del PSA, e un test con un valore predittivo estremamente basso; il secondo, non meno importante, e che l’ANA e un test di primo livello per l’inquadramento non di una singola malattia, ma di svariate patologie reumatiche autoimmuni (LES, connettivite mista, connettivite indifferenziata, sindrome di Sjogren, sclerodermia, artrite reumatoide, etc.), e anche di epatopatie autoimmuni; il terzo, e oltremodo peculiare, e che l’ANA

Ro52 is an interferon-inducible protein of the tripartite motif family. Antibodies against Ro52 have been described in patients with different autoimmune diseases, such as systemic lupus erythematosus and Sjögren’s syndrome, that are often associated with anti-Ro60 antibodies. The Ro52 autoantigen is extraordinarily immunogenic, and its autoantibodies are directed against both linear and conformational epitopes. The aim of this study was to evaluate the prevalence of antibodies to the five Ro52 domains, as well as to Ro52 176– to 196–amino acid (aa) and 200–239-aa peptides, in different systemic autoimmune rheumatic diseases (SARDs). We also aimed to verify whether antibodies to a single domain or domain association could increase their diagnostic specificity for any SARD.

Il microarray, spesso paragonato alla polymerase chain reaction e alle tecniche del DNA ricombinante, è tra le metodiche di biologia molecolare quella di più forte impatto scientifico. Nonostante la tecnica si basi su principi noti della biochimica e della biologia molecolare, come la specificità di legame di sequenze nucleotidiche complementari, a un certo momento del processo evolutivo tecnologico è stato possibile effettuare analisi su grande scala avvalendosi di tecniche miniaturizzate; è stata questa la vera conquista che ha portato gli scienziati a fare un grande “balzo avanti”. Esistono varie classi di microarray, tutte di grande interesse sia nel campo della ricerca di base sia nella diagnostica medica. In certi settori della medicina la tecnica è entrata nella diagnostica di routine, mentre in altri rappresenta solo una speranza in attesa di validazione. Esistono problematiche diverse per ciascuna tipologia di microarray; in generale, il disegno sperimentale, l’elaborazione del campione, la normalizzazione dei dati e infine l’analisi e l’interpretazione costituiscono i passaggi fondamentali per produrre risultati con tecnologia microarray. Oggi le tecniche di biologia molecolare permettono l’analisi contemporanea di migliaia di geni, proteine o metaboliti e hanno cambiato la dimensione della ricerca biologica, facendo passare i ricercatori dallo studio dei singoli geni o proteine allo studio sistematico di tutti i geni e proteine. Risulta, quindi, di particolare interesse per la medicina molecolare poter disporre di una visione globale dei processi biologici, aprendo così la strada per migliaia di potenziali applicazioni. Infatti, la disponibilità di tecniche che permettono questo approccio “globale” offre, per esempio, la possibilità di confrontare due diverse situazioni biologiche contemporaneamente come, per esempio, lo stato di salute nei confronti della presenza di malattia o un trattamento farmacologico rispetto all’assenza di trattamento ecc., oppure esaminare i processi biologici complessi, le vie metaboliche e le interazioni tra biomolecolecole.

Il Gruppo di Studio in Autoimmunologia (GdS-AI) della Società Italiana di Patologia Clinica e Medicina di Laboratorio (SIPMeL), dopo 14 anni dalla ultima edizione, ha ritenuto opportuno revisionare e integrare le “linee guida per l’impiego di test per autoanticorpi nucleo-citoplasmatici nella diagnosi e nel monitoraggio delle malattie autoimmuni sistemiche”, con l’aggiunta di nuove tematiche, la modifica di precedenti raccomandazioni e la rivalutazione di aspetti controversi del precedente documento.

A large body of evidence links the presence of circulating anti-Ro/SSA antibodies (anti-Ro/SSAs) in a mother with the risk of her newborn developing neonatal lupus, a paradigmatic model of transplacental passively acquired autoimmunity whose main cardiac manifestation is congenital complete atrioventricular block (congenital-III°AVB).1 Although the precise underlying mechanisms of the block have not been fully elucidated, many experimental studies have demonstrated the ability of anti-Ro/SSA to biochemically cross-react with L-type and T-type calcium channels and thus significantly inhibit the related currents, both of which play a key role in the slow action potential of the conduction system cardiomyocytes.

OBJECTIVE:To evaluate two new diagnostic methods for the identification of anti-DFS70 antibodies in samples showing a DFS70-staining pattern by indirect immunofluorescence (IIF). METHODS:We studied 731 patients: 576 were collected consecutively among those that in the ANA test on HEp-2 cells had produced a DFS70 fluorescence pattern and 155 were a consecutive series of patients sent by referring physicians for routine ANA testing. As controls we studied 50 patients with autoimmune diseases and 120 patients with active infectious disease. All 731 sera were assayed for anti-DFS70 antibodies by a specific chemoluminescence assay (CLIA); 70 randomly selected IIF-positive sera and 35 samples from patients with autoimmune diseases were studied by inhibition tests using the HEp-2 Select method. RESULTS:Assays performed with the CLIA-DFS70 method were positive in 30.4% of the samples presenting a DFS70 pattern by IIF, in 1.3% of the routine ANA sera, in 1.6% of the infectious sera and in none of the 50 autoimmune controls. However, as the IIF-DFS70 positive group included 106 patients with systemic autoimmune rheumatic diseases (SARD), 11 of which were DFS70 positive by CLIA, the prevalence of DFS70 antibodies in SARD was 7.5%. The ANA test performed after the use of HEp-2 Select showed an inhibition in 95.7% of the sera. No change in fluorescence intensity and pattern morphology between the native sera and the same sera tested with the solution containing the DFS70 antigen was observed in the 35 samples from patients with autoimmune diseases. CONCLUSIONS:To avoid misinterpretation of ANA pattern and consequent diagnostic errors, confirmation of the DFS70-IIF pattern by CLIA or other specific methods is mandatory before reporting the presence of anti-DFS70 antibodies. The HEp-2 Select test in most cases eliminates the interference by anti-DFS70 antibodies and avoids the possible reporting of false positive results.

Il Gruppo di Studio in Autoimmunologia della SIMeL ha svolto un’indagine per rilevare le caratteristiche strutturali, le tecnologie impiegate, i volumi di attività diagnostica e gli aspetti di governo clinico dei Laboratori italiani di autoimmunologia nell’anno 2012.

Background In recent years the diagnostic approach to rheumatoid arthritis (RA) significantly changed. Among new serological assays, an ELISA test detecting antibodies directed against recombinant mutated citrullinated vimentin (anti-MCV), a protein widely expressed in the synovium, has been introduced to improve diagnosis. Anti-MCV appear slightly more or as sensitive as anti-cyclic citrullinated protein antibodies (anti-CCP), but less specific. However, its additional diagnostic value with respect to anti-CCP both in early and established RA is controversial. Objectives Evaluate the diagnostic performance of anti-MCV assay in RA and to compare it to a standard anti-CCP test and the recently developed assay targeting viral citrullinated peptide 2 (VCP2) (1,2) in a large cohort of RA patients and healthy and disease controls. Methods Sera (n=512) from long-standing RA patients (n=285) and healthy and disease controls (n=227) were randomly collected. Anti-CCP were measured by the routine method used in each Center. As 4 different assays were used, anti-CCP values were normalized and expressed as a ratio. Anti-VCP IgG were detected by the 2nd generation method (Astra Diagnostici, Milan, Italy) and anti-MCV by the available ELISA kit (Orgentec Diagnostika GmbH, Mainz, Germany). The receiver-operating characteristic (ROC) curve was calculated to determine the optimal cutoff values for each antibody assay. Results At the calculated cutoff value of 55 U/mL, anti-MCV resulted to have a sensitivity of 59% and a specificity of 92%. In comparison, anti-CCP (calculated cutoff of 5 U/mL) and anti-VCP2 (calculated cutoff of 21 AU) displayed a sensitivity of 77% and 61% and a specificity of 96% and 95%, respectively. Of interest, a high percentage of healthy subjects as well as Epstain Barr and hepatitis C virus-infected patients resulted anti-MCV positive at the manufacturer recommended cutoff value of 20 U/mL. Conclusions In our large cohort of RA patients, at the calculated cutoff of 55 U/mL, anti-MCV demonstrated lower sensitivity than anti-CCP and VCP2 test, thus not allowing to confirm previously published data. Moreover, the high rate of detection in infectious diseases limits its diagnostic value in undifferentiated arthritis. On the basis of our results, an international validated anti-MCV cutoff value to standardize the results is surely needed. References Bizzaro N et al., J Clin Pathol 2011;64:1139 Pratesi F et al., Clin Exp Immunol 2011;164:337 Disclosure of Interest None Declared

INTRODUCTION:The diagnostic, predictive and prognostic role of anti-cyclic citrullinated peptide (CCP) antibodies in rheumatoid arthritis (RA) patients is widely accepted. Moreover, detection of these antibodies in subjects presenting with undifferentiated arthritis (UA) is associated with a significant risk to develop the disease. On the other hand, clinical and prognostic significance of evaluating anti-CCP levels in subjects with inflammatory arthritis at disease onset has not been fully clarified. The goal of this prospective study is to analyze the value and prognostic significance of anti-CCP titer quantification in UA subjects.METHODS:Serial anti-CCP assays were measured in 192 consecutive patients presenting with UA lasting less than 12 weeks. Clinical and serological data and arthritis outcome were evaluated every 6 months until two years of follow-up.RESULTS:Anti-CCP positivity, at both low and high titer, and arthritis of hand joints significantly predicted RA at two years, risk increasing in subjects with high anti-CCP titers at baseline. Moreover, time to RA diagnosis was shorter in patients with high anti-CCP2 titers at enrollment with respect to those with low antibody concentration.CONCLUSIONS:Presence of anti-CCP antibodies, at both low and high concentration, is significantly associated with RA development in subjects with recent onset UA. However, time interval from the onset of the first symptoms to the fulfilment of the classification criteria appears to be directly related to the initial anti-CCP level.