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Classification criteria of autoimmune rheumatic diseases are an important means to define homogenous groups of patients that can be compared across studies for clinical trials and research purposes. The measurement of autoantibodies is a relevant aspect in the definition of classification criteria, with a significant weight in the scores necessary to classify patients with autoimmune rheumatic diseases. The impact of autoantibodies has gradually increased over the years, contributing to the evolution and improvement of the classification criteria. However, these criteria often do not take into consideration how autoantibodies are measured, i.e. differences in diagnostic accuracy of the methods. This is a critical point especially when obsolete analytical methods that are no longer used in many clinical laboratories are taken into consideration. In this review we have critically examined assays and methods for the determination of autoantibodies that are (or could be) included among the classification criteria of autoimmune rheumatic diseases in light of more recent evidence and technology evolution., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Background and Aims: Diagnosis of fish may represent an important challenge for the allergists. This study aimed to evaluate the diagnostic sensitivity of an in vitro multiplex assay using a comprehensive panel of fish allergens and the cross-reactivity patterns between different molecular components., Methods: 56 subjects with fish allergy were enrolled. All patients underwent specific IgE measurement using the Allergy Explorer-Alex 2™ multiplex assay (Macroarray Diagnostics, Vienna, Austria) RESULTS: The single ß-parvalbumins Clu h 1, Cyp c 1, Gad m 1, Sal s 1, Sco s 1, Thu a 1 and Xyp g 1 scored positive in 75.0%, 67.8%, 62.5%, 80.3%,80.3%, 78.8% and 73,2% patients, respectively. 14.3% scored positive for the α-parvalbumin (Raj c-parvalbumin), and 16.1% for the aldolase + enolase (Gad m 2 + 3) components. 92.8% reacted to at least one ß-parvalbumin and 96.4% to at least one of the allergens tested. Overall sensitivity was higher than that obtained using commercial extracts of cod, salmon and tuna for skin prick test (75.8%) and IgE detection (92.3%)., Conclusions: The Alex 2 showed high diagnostic sensitivity and it might be used as an additional assay to investigate the cross-reactivity patterns between different molecular components, looking for potentially safe fish species., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Autoantibodies against extractable nuclear antigens (ENA) play a pivotal role in the diagnosis and classification of systemic autoimmune rheumatic diseases (SARD). In recent years, newly developed methods have enabled the simultaneous and quantitative detection of multiple anti-ENA reactivities. However, data regarding the comparability of results obtained using different technologies across different platforms are scarce. In this study we compared eight different immunoassays, commonly used in current laboratory practice for detection of anti-ENA antibodies., Methods: Sixty patients suffering from different SARD, 10 inflammatory arthritis patients (disease controls) and 10 healthy blood donors were included in this comparative study. Sera were collected in 15 centers belonging to the Study Group on Autoimmune Diseases of the Italian Society of Clinical Pathology and Laboratory Medicine. We evaluated the analytical sensitivity, specificity and diagnostic accuracy of each method for antibodies to Sm, RNP, Ro60, Ro52, Scl70, CENP-B and Jo1. Cohen's kappa was used to analyze the agreement among methods., Results: Average agreement among methods was 0.82, ranging from substantial (k = 0.72) to almost perfect (k = 0.92). However, while the specificity was very good for all methods, some differences emerged regarding the analytical sensitivity., Conclusions: Diagnostic performance of current technologies for anti-ENA antibody detection showed good comparability. However, as some differences exist among methods, laboratory scientists and clinicians must be aware of the diagnostic accuracy of the testing method in use., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Food insecurity affects one in seven households with children in the United States, disproportionately impacts households headed by women and minorities, and is associated with childhood comorbidities, including obesity. While food insecurity likely contributes to poor health through its effect on diet, such a simplistic understanding likely obscures the effects of poverty-related stress and other Adverse Childhood Experiences, on metabolic health., Methods: Over two summers, 100 children, ages 8-12 years, will be recruited from low-income households in an urban, Rhode Island community, to participate in an 8-week trial designed to isolate the experience of food insecurity. Summer represents a natural risk period of food insecurity in children, such that children will be randomized to receive weekly shipments of five breakfast and lunch meals that mimic school meals or to experience the likely onset of summertime food insecurity and receive a weekly newsletter on community resources that is not expected to affect food insecurity. Through assessment visits at baseline, mid-summer and end of summer, we will examine group differences in change in diet quality, biomarkers of Metabolic Syndrome, inflammation, and stress, BMI z-scores, and child measures of behavior and anxiety and depression symptoms. We will also explore the impact of caregiver mood and stress on the health effects of food insecurity., Conclusions: Findings stand to clarify the mechanisms by which food insecurity affects child health outcomes and to inform how to best address food insecurity in the context of poverty-related stress., Trial Registration: This trial has been registered at ClinicalTrials.gov (NCT04968496)., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background and Aims: Long-term kinetics of anti-RBD IgG and neutralizing antibodies were analyzed in a cohort of COVID-19 naïve health care workers (HCW) undergoing SARS-CoV-2 vaccination., Methods: An anti-RBD IgG immunoassay and a surrogate virus neutralization test (sVNT) were performed at different time points up to 6 months after vaccination in 57 HCWs. Values of anti-RBD IgG predicting an high neutralizing bioactivity (>60%) were also calculated., Results: Mean (range) values of anti-RBD IgG were 294.7 (11.6-1554), 2583 (398-8391), 320.4 (42.3-1134) BAU/mL at T1 (21 days after the 1st dose [T0]), T2 (30 days after the 2nd dose) and T3 (+180 days after T0), respectively. Mean (range) percentages of neutralization (NS%) were 24 (0-76), 86 (59-96) and 82 (52-99) at T1, T2 and T3, respectively. Anti-RBD IgG values and NS% were positively correlated at T2 and T3 while anti-RBD IgG value predicting a NS% > 60 markedly differed at T2 and T3 (594 vs. 108 BAU/mL, respectively)., Conclusion: While a high neutralizing bioactivity was maintained at least 6 months after vaccination in almost all individuals, the mean values of anti-RBD-IgG showed a marked decline at 6 months. The absolute value of anti-RBD IgG is a poor marker of neutralizing bioactivity., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: The serological screening for celiac disease (CD) is currently based on the detection of anti-transglutaminase (tTG) IgA antibodies, subsequently confirmed by positive endomysial antibodies (EMA). When an anti-tTG IgA positive/EMA IgA negative result occurs, it can be due either to the lower sensitivity of the EMA test or to the lower specificity of the anti-tTG test. This study aimed at verifying how variation in analytical specificity among different anti-tTG methods could account for this discrepancy., Methods: A total of 130 consecutive anti-tTG IgA positive/EMA negative samples were collected from the local screening routine and tested using five anti-tTG IgA commercial assays: two chemiluminescence methods, one fluoroimmunoenzymatic method, one immunoenzymatic method and one multiplex flow immunoassay method., Results: Twenty three/130 (17.7%) patients were diagnosed with CD. In the other 107 cases a diagnosis of CD was not confirmed. The overall agreement among the five anti-tTG methods ranged from 28.5% to 77.7%. CD condition was more likely linked to the positivity of more than one anti-tTG IgA assay (monopositive = 2.5%, positive with ≥ three methods = 29.5%; p = 0.0004), but it was not related to anti-tTG IgA antibody levels (either positive or borderline; p = 0.5)., Conclusions: Patients with positive anti-tTG/negative EMA have a low probability of being affected by CD. Given the high variability among methods to measure anti-tTG IgA antibodies, anti-tTG-positive/EMA-negative result must be considered with extreme caution. It is advisable that the laboratory report comments on any discordant results, suggesting to consider the data in the proper clinical context and to refer the patient to a CD reference center for prolonged follow up., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The commercial tests currently available as second-level tests to detect ANA sub-specificities are generally used independently from the ANA immunofluorescence (IIF) pattern. The aim of this study was to evaluate the efficacy of the use of a customizable pattern-oriented antigenic panel by immunoblot (IB) using the International Consensus on ANA Patterns (ICAP) classification scheme, in order to introduce a novel and updated autoimmune diagnostic flowchart. 710 sera referred for routine ANA testing were selected on the basis of the ANA pattern according to the ICAP nomenclature (nuclear speckled AC-2,4,5; nucleolar AC-8,9,10,29; cytoplasmic speckled AC-18,19,20) and on an IIF titer ≥1:320. They were then assayed by three experimental IB assays using a panel of selected antigens. ICAP-oriented IB detected 515 antibody reactivities vs. 457 of traditional anti-ENA in the nuclear speckled pattern group, 108 vs. 28 in the nucleolar pattern group, and 43 vs. 34 in the cytoplasmic speckled pattern. This pilot study may lead the way for a new approach introducing an ICAP pattern-oriented follow up testing as a valid alternative to the existing standard panels, thus enabling more patients with autoimmune rheumatic disease to be accurately diagnosed., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Molecular allergy has significantly improved the quality of allergy diagnosis; however, the positioning of singleplex and multiplex assays in the diagnostic algorithm is still a matter of debate., Methods: Aim of the study was to test the analytical performance of the recently commercialized Allergy Explorer-ALEX® in a selected population (105 allergic patients and 15 negative controls), comparing it with the reference ImmunoCAP® method and with skin prick test (SPT)., Results: Inter-assay qualitative comparison showed a substantial agreement between ALEX® and SPT (k = 0.64). A substantial agreement between ALEX® and ImmunoCAP® was shown on the detection of IgE to extracts (k = 0.64 for inhalants and k = 0.51 for food allergens), whereas a higher agreement was shown on detection of molecular components (k = 0.92 for inhalants and k = 0.72 for food allergens). Quantitative comparison showed a poor correlation between ALEX® and ImmunoCAP®., Conclusion: The simultaneous detection of both extracts and molecular components with ALEX® assay can potentially overcome some of the major limitations of the multiplex assay currently in use. However, before using ALEX® as routine method, the analytical performance (in particular for extracts) needs to be further investigated on a larger scale., (Copyright © 2019. Published by Elsevier B.V.)

Background: "Gluten-related disorders" is a term that encompasses different diseases induced by the ingestion of gluten-containing food. Because of their incidence the scientific community has been intensively studying them., Aim: To support gastroenterologists with a correct nomenclature and diagnostic approach to gluten-related disorders in adulthood., Methods: The Italian Association of Hospital Gastroenterologists and Endoscopists (AIGO) commissioned a panel of experts to prepare a position statement clarifying the nomenclature and diagnosis of gluten-related disorders, focusing on those of gastroenterological interest. Each member was assigned a task and levels of evidence/recommendation have been proposed., Results: The panel identified celiac disease, wheat allergy and non-celiac gluten sensitivity as the gluten-related disorders of gastroenterological interest. Celiac disease has an autoimmune nature, wheat allergy is IgE-mediated while the pathogenesis of non-celiac gluten sensitivity is still unknown as is the case of non-IgE mediated allergy. Diagnosis should start with the serological screening for celiac disease and wheat allergy. In case of normal values, the response to a gluten-free diet should be evaluated and a confirmatory blind food challenge carried out., Conclusions: Gluten-related disorders are clinically heterogeneous. Patients should be carefully managed and specific protocols applied for a correct differential diagnosis in gastroenterological setting., (Copyright © 2016 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Background: Autoantibodies to the DFS70 (dense fine speckles 70) protein have been identified among the antinuclear antibodies (ANA) in patients with various disorders. However, the ANA test in indirect immunofluorescence (IIF) is not a reliable method to identify anti-DFS70 antibodies. We undertook this study to evaluate the diagnostic performance of two new immunoblot methods for the detection of anti-DFS70 antibodies and to investigate whether their different DFS70 antigen composition could affect diagnostic accuracy in detecting anti-DFS70 antibodies., Methods: 62 samples showing a DFS70 staining pattern by IIF were tested by dot blot (Alphadia) and line blot (Euroimmun) methods. The dot blot method employs a truncated sequence of the DFS70 antigen (residues 349-435), while the line blot uses the full-length protein (aa 1-530). The 62 samples were previously assayed by a chemoluminescent (CLIA) method also using a truncated antigen (aa 349-435): 27 were CLIA positive and 35 were CLIA negative. 120 sera from subjects with infectious diseases were used as controls., Result: Both immunoblot methods were positive in the 27 IIF/CLIA positive samples; in addition, the Alphadia dot blot identified another seven DFS70 samples and the Euroimmun line blot was positive in five samples that were negative by CLIA. Among the 120 control samples, two false positives were recorded for the CLIA method, six for the Alphadia method and four for the Euroimmun method. Therefore, in this selected series of samples, sensitivity and specificity were 43.5% and 98.3% for the CLIA method, 54.8% and 95% for the dot blot and 51.6% and 96.6% for the line blot, respectively., Conclusions: Because of great inconsistency in assessing the DFS70 pattern using the ANA-IIF test, specific assays should be used to confirm anti-DFS70 antibodies. The results of this study show that there is no difference in the overall diagnostic accuracy among methods that use the truncated or the full-length DFS70 antigenic sequence and that it is likely that antibodies directed against antigens other than DFS70 may be responsible for producing a DFS70-like ANA-IIF pattern., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: The determination of the upper reference limit (URL) for thyroid peroxidase autoantibodies (TPOAbs) is a contentious issue, because of the difficulty in defining the reference population. The aim of this study was to establish the URL (eURL) for TPOAbs, according to the National Academy of Clinical Biochemistry (NACB) guidelines and to compare them with those obtained in a female counterpart, by the use of six commercial automated platforms., Methods: 120 healthy males and 120 healthy females with NACB-required characteristics (<30years, TSH between 0.5 and 2.0mIU/L, normal thyroid ultrasound, without personal/family history of thyroid and non-thyroid autoimmune diseases) were studied. Sera were analyzed for TPOAbs concentration using six immunoassay methods applied in automated analyzers: Advia Centaur XP (CEN), Siemens Healthcare Diagnostics; Maglumi 2000 Plus, Shenzen New Industries Biomedical Engineering; Architect ci4100, Abbott; Cobas e411 (COB) Roche Diagnostics; Unicel DxI (UNI) and Lumipulse G1200, Fujirebio., Results: Within each method, TPOAbs values had a high degree of dispersion and the eURLs were lower than those stated by the manufacturer. A statistically significant difference (p<0.05) between medians of males and females was observed only for COB and for UNI. However, the comparison of the male and female proportions positive for TPOAbs using the eURL of the counterpart, showed the lack of clinical significance of the above differences (Chi-square test, p>0.05)., Conclusions: Despite the analytical harmonization, the wide dispersion of the results and the differences of the eURLs between methods suggest the need of further studies focusing on TPO antigen preparations as the possible source of variability between different assays. In addition, the lack of clinical significant difference between males and females, in terms of TPOAb eURLs, confirms the suitability of the NACB recommendations., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: The liver asialoglycoprotein receptor (ASGPR) is the only organ-specific autoantigenic target in autoimmune hepatitis (AIH) patients and corresponding autoantibodies (Abs) have been suggested aiding in the serology of autoimmune liver diseases (ALD)., Methods: A novel enzyme-linked immunosorbent assay (ELISA) employing purified rabbit ASGPR was used to detect ASGPR Abs in patients with ALD and controls. ASGPR Ab was determined in sera from 172 patients with AIH type 1, AIH type 2 (n=42), primary biliary cirrhosis (PBC) (n=113), cryptogenic liver disease (n=30), toxic liver disease (n=11), primary sclerosing cholangitis (PSC) (n=27), HCV infection (n=25), non-alcoholic steatohepatitis (n=43) and 100 blood donors. ASGPR Ab positivity was compared with AIH-related Abs (ANA, ASMA, Abs to LKM-1, LC-1, and SLA/LP) in patients with AIH., Results: Patients with AIH-1 and AIH-2 demonstrated an ASGPR Ab prevalence of 29.1% and 16.7%, respectively. ASGPR Ab positivity in patients with AIH-1 and AIH-2 was not significantly different to those in patients with PSC and HCV (p>0.05, respectively). ASGPR Ab levels in all study cohorts were significantly different with the highest medians in patients with AIH, PSC, and HCV infection (p<0.0001). ASGPR Ab can be found as only AIH-specific Ab determined by LIA and ELISA in 24.4% of AIH patients (48/197)., Conclusions: The novel ASGPR Ab ELISA is a specific diagnostic tool for ASGPR Ab detection in AIH. In addition to AIH, patients with PSC can demonstrate elevated ASGPR Ab amongst those with ALD suggesting a tolerance break to ASGPR in PSC., (Copyright © 2015 Elsevier B.V. All rights reserved.)

1,25-Dihydroxyvitamin D is a steroid hormone derived from vitamin D, playing an important role in maintaining an adequate serum level of calcium and phosphorus. It is now clear that vitamin D exerts an endocrine action on the cells of the immune system, generating anti-inflammatory and immunoregulatory effects. The mechanisms underlying the role of vitamin D in autoimmunity are not completely understood. Lower vitamin D levels have been found in several autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, type 1 diabetes mellitus, multiple sclerosis, inflammatory bowel diseases, autoimmune thyroid diseases (i.e. Hashimoto's thyroiditis and Graves' disease) and autoimmune gastritis. Several genetic studies have demonstrated an association between thyroid autoimmunity susceptibility and gene polymorphisms of vitamin D receptor, vitamin D binding protein, 1-alpha-hydroxylase and 25-hydroxylase. Of note, some papers do not confirm this connection. With regard to the role of vitamin D in autoimmune thyroid diseases, available data remain controversial. Only few reports have analyzed the supposed association between autoimmune thyroid diseases and vitamin D concentration with inconclusive results. In our experience, low serum levels of vitamin D do not correlate either with Hashimoto's thyroiditis or with Graves' disease. The inability to achieve an unambiguous conclusion is in part due to the limitations in study design. In fact, most of the studies are cross-sectional surveys with a small number of subjects. In addition, the heterogeneity of the study population, seasonal variation of blood sampling, inter-method analytical variability of vitamin D assays and different definitions of vitamin D deficiency/insufficiency contribute to contradicting results. Therefore, further randomized, controlled, prospective trials are needed in order to demonstrate the causality of vitD in AITD and consequently the role of vitamin D supplementation in prevention or improvement of AITD, providing also information on the best formulation, dose and timing of supplementation., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Objective: To evaluate the autoantibody profile in patients with primary biliary cirrhosis (PBC) using a new multiplexed line-blot assay specifically designed for the diagnosis of autoimmune liver diseases., Methods: Sera of 58 consecutive PBC patients and 191 disease controls (144 with autoimmune liver diseases other than PBC, and 67 with non-autoimmune chronic liver diseases) were tested by both the multiplexed line-blot Autoimmune Liver Disease Profile 2 (ALD2) and by IIF on HEp-2 cells and on rat kidney/liver/stomach tissues. ALD2 contains the following PBC-associated antigens: AMA-M2, natively purified from bovine heart; M2-E3, a recombinant fusion protein including the E2 subunits of PDC, BCOADC and OGDC; sp100, PML and gp210 recombinant proteins., Results: With the ALD2 assay, a positive reaction to AMA-M2, M2-E3, sp100, PML and gp210 in PBC patients was observed in 77.6%, 84.5%, 34.5%, 15.1% and 18.9%, respectively, of the PBC sera. The overall sensitivity and specificity for PBC were 98.3% and 93.7%. Using IIF, positivity rates to AMA, and to antinuclear autoantibodies with membranous/rim-like and multiple nuclear dot patterns were 86.2%, 8.6% and 22.4%, respectively. The overall sensitivity and specificity for PBC of the IIF method were 86.2% and 97.9%, respectively., Conclusions: The ALD2 line-blot showed a good diagnostic accuracy for PBC and a higher sensitivity than the IIF method to detect sp100 and gp210 autoantibodies., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Indirect immunofluorescence (IIF) plays an important role in immunological assays for detecting and measuring autoantibodies. However, the method is burdened by some unfavorable features: the need for expert morphologists, the subjectivity of interpretation, and a low degree of standardization and automation. Following the recent statement by the American College of Rheumatology that the IIF technique should be considered as the standard screening method for the detection of anti-nuclear antibodies (ANA), the biomedical industry has developed technological solutions which might significantly improve automation of the procedure, not only in the preparation of substrates and slides, but also in microscope reading., Methods: We collected 104 ANA-positive sera from patients with a confirmed clinical diagnosis of autoimmune disease and 40 ANA-negative sera from healthy blood donors. One aliquot of each serum, without information about pattern and titer, was sent to six laboratories of our group, where the sera were tested with the IIF manual method provided by each of the six manufacturers of automatic systems. Assignment of result (pos/neg), of pattern and titer was made by consensus at a meeting attended by all members of the research team. Result was assigned if consensus for pos/neg was reached by at least four of six certifiers, while for the pattern and for the titer, the value observed with higher frequency (mode) was adopted. Seventeen ANA-positive sera and six ANA-negative sera were excluded. Therefore, the study with the following automatic instrumentation was conducted on 92 ANA-positive sera and on 34 ANA-negative sera: Aklides, EUROPattern, G-Sight (I-Sight-IFA), Helios, Image Navigator, and Nova View. Analytical imprecision was measured in five aliquots of the same serum, randomly added to the sample series., Results: Overall sensitivity of the six automated systems was 96.7% and overall specificity was 89.2%. Most false negatives were recorded for cytoplasmic patterns, whereas among nuclear patterns those with a low level of fluorescence (i.e., multiple nuclear dots, midbody, nuclear rim) were sometimes missed. The intensity values of the light signal of various instruments showed a good correlation with the titer obtained by manual reading (Spearman's rho between 0.672 and 0.839; P<0.0001 for all the systems). Imprecision ranged from 1.99% to 25.2% and, for all the systems, it was lower than that obtained by the manual IIF test (39.1%). The accuracy of pattern recognition, which is for now restricted to the most typical patterns (homogeneous, speckled, nucleolar, centromere, multiple nuclear dots and cytoplasmic) was limited, ranging from 52% to 79%., Conclusions: This study, which is the first to compare the diagnostic accuracy of six systems for automated ANA-IIF reading on the same series of sera, showed that all systems are able to perform very well the task for which they were created. Indeed, cumulative automatic discrimination between positive and negative samples had 95% accuracy. All the manufacturers are actively continuing the development of new and more sophisticated software for a better definition in automatic recognition of patterns and light signal conversion in end-point titer. In the future, this may avert the need for serum dilution for titration, which will be a great advantage in economic terms and time-saving., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The antiphospholipid syndrome (APS) is an autoimmune disease defined by the co-occurrence of clinical and serological symptoms [presence of at least one of the antiphospholipid autoantibodies (aPL), such as anti-cardiolipin (aCL) IgG/IgM and anti-β2glycoprotein I (aβ2GPI) IgG/IgM]. The measurement of these autoantibodies constitutes the first-line approach for the diagnosis of APS. Recently the advent of multiplex proteomic technologies seems to be an optimal solution for the parallel detection of autoantibodies (IgG, IgA, IgM) related to APS. The BioPlex 2200 is an automated commercial platform based on the multi-analyte profiling technology that allows the detection of different types of autoantibodies, particularly ANA, ENA, dsDNA, PR3, MPO, GBM. We performed firstly a study to evaluate the diagnostic accuracy of this analytical system in a group of APS patients. The BioPlex system showed a good diagnostic accuracy for all test evaluated, very similar to that of the other established commercial singleplex immunoassays. In our study, the simultaneous detection of aCL and aβ2GPI of IgA isotype in addition to IgG and IgM isotypes did not increase the diagnostic sensitivity for APS. The good diagnostic accuracy, the high level of automation, and the high throughput make this multiplex platform a very useful and practical tool for the laboratory diagnosis of aPL in daily practice., (© 2013 Elsevier B.V. All rights reserved.)

Background: Methods to detect anti-nucleosome antibodies (ANuA) have been available for more than 10 years and the test has demonstrated its good sensitivity and high specificity in diagnosing systemic lupus erythematosus (SLE). Despite these data produced through clinical and laboratory research, the test is little used., Objective: To verify the diagnostic performance of methods for measuring ANuA and to compare them with those for anti-dsDNA antibodies., Data Sources: A systematic review of English and non-English articles using MEDLINE and EMBASE with the search terms "nucleosome", "chromatin", "anti-nucleosome antibodies" and "anti-chromatin antibodies". Additional studies were identified checking reference lists in the selected articles., Study Selection: We selected studies reporting on anti-nucleosome tests performed by quantitative immunoassays, on patients with SLE as the index disease (sensitivity) and a control group (specificity). A total of 610 titles were initially identified with the search strategy described. 548 publications were subsequently excluded based on abstract and title. Full-text review was undertaken as the next step on 62 publications providing data on anti-nucleosome testing; 25 articles were then excluded because they did not include either SLE patients or a control group, and 37 articles were selected for the metanalysis. Finally, a sub-metanalysis study was conducted on the 26 articles providing data on both ANuA and anti-dsDNA antibody assays in the same series of patients., Data Extraction: Extraction of data from selected articles was performed by two authors independently, using predefined criteria: the number of patients with SLE as the index case, and the number of healthy or diseased controls; specification of the analytical method used to detect anti-nucleosome and anti-dsDNA antibodies; the cut-off used in the study; and the sensitivity and specificity of the assay. Demographic and clinical data on the population investigated (adults or children; lupus patients with or without nephritis; patients with active or inactive disease) were also recorded and analyzed in a separate evaluation., Results: The systematic review and metanalysis showed that the overall sensitivity of the ANuA assay is 61% (confidence interval-CI, 60-62) and the specificity 94% (CI, 94-95). The overall positive likelihood ratio is 13.81 (CI, 9.05-21.09) and the negative likelihood ratio 0.38 (CI, 0.33-0.44). The odds ratio for having SLE in ANuA-positive patients is 40.7. The comparative analysis on anti-dsDNA antibodies conducted on the 26 studies which provided data for both antibodies showed that ANuA have greater diagnostic sensitivity (59.9% vs 52.4%) and a specificity rating only slightly higher (94.9% vs 94.2%). The probability that a subject with positive ANuA have SLE is 41 times greater than a subject with negative ANuA, while for anti-dsDNA the probability is 28 times greater. These figures are even more impressive in children, in whom ANuA have an odds ratio for the diagnosis of SLE of 146, compared to 51 for anti-dsDNA antibodies. In selected studies, ANuA (p<0.0001) but not anti-dsDNA antibodies (p=0.256) were significantly associated with disease activity measured by the international score systems. However, neither antibody appears to correlate with kidney involvement., Conclusions: Data from the metanalysis have shown that ANuA have equal specificity but higher sensitivity and prognostic value than anti-dsDNA antibodies in the diagnosis of SLE. Despite a certain heterogeneity among the various studies, the use of ANuA appears more efficacious than anti-dsDNA., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Systemic sclerosis (SSc) is a heterogeneous autoimmune disorder characterized by microvascular injury, fibrosis of the skin and other organs, and presence of antinuclear autoantibodies (ANA) with a prevalence varying from 80 to 98%. The ANA classically detected in SSc include anti-centromere (ACA) and anti-topoisomerase I (ATA), which are positive in 50-60% of the patients. Even if other autoantibodies, such as anti-fibrillarin (AFA), anti-RNA polymerase III (RNAP III), anti-PMScl, anti-Th/To, and anti-hUF/NOR-90, are almost specific for SSc, until recently they were not routinely looked for, since the techniques for their identification were not suitable for routine use. In recent years, the advances in the knowledge of the biochemistry and of the immunoreactive sites of the autoantigens led to the development of new immunoassays using recombinant proteins as autoantigens. We evaluated a new multiplex line immunoblot assay (LIA) for the simultaneous detection of 13 different SSc-associated autoantibodies, in a cohort of 210 SSc Italian patients. The sensitivity and the specificity of this assay were as follows: 30.5% and 97.3% for ACA (anti-CENP-B), 29.5% and 96% for ACA (anti-CENP-A), 20% and 99.3% for ATA, 5.7% and 99.3% for anti-RNAP III (RP-155), 5.2% and 100% for anti-RNP III (RP-11), 6.7% and 98% for anti-PMScl (PMScl-100), 10.9% and 93.3% for anti-PMScl (PMscl-75), 3.3% and 98.7% for anti-Th/To, 0.48% and 100% for AFA, 4.8% and 96.7% for anti-hUF/NOR-90, 4.7% and 96% for anti-Ku, 0.95% and 100% for anti-Platelet-Derived Growth Factor Receptor, and 18.1% and 50% for anti-Ro-52, respectively. These results, which are similar to those obtained in other studies using traditional techniques, show that the LIA assay can be considered a more rapid and a more practical method than immunoprecipitation assays for studying SSc-related antibodies in the diagnostic work-up of SSc patients., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Anti-fibrillarin (AFA), anti-RNA polymerase (anti-RNAP), and anti-PM-Scl autoantibodies are useful markers for the diagnosis of systemic sclerosis (SSc) in patients who are anti-centromere- (ACA) or anti-topoisomerase I (anti-topo I)-negative, but, until recently, the only specific method for their identification was the radio-immunoprecipitation assay. The aim of this study was to evaluate the clinical accuracy of the new enzyme-linked immunosorbent assays (ELISA) developed by Phadia for their detection., Methods: Sera of 50 ACA and anti-topo I-negative SSc patients, and, as control group, sera of 122 patients (42 with SSc, ACA or anti-topo I-positive, 40 with systemic lupus erythematosus and 40 with rheumatoid arthritis) were studied., Result: Using the cutoff proposed by the manufacturer (10 AU/mL), sensitivity and specificity were: for AFA, 22% and 92.6%; for anti-RNAP, 16% and 97.5%; and for anti-PM-Scl, 8% and 98.8%, respectively. Using a cutoff corresponding to 98.8% specificity for all three antibodies, sensitivity was 10%, 14% and 8%, respectively. The combined use of these three antibody assays enabled identification of 32% of ACA- and anti-topo I-negative SSc patients., Conclusions: These new ELISA methods for AFA, anti-RNAP III and anti-PM-Scl detection have good diagnostic specificity, and may help identify a subset of SSc patients ACA and anti-topo I-negative., (Copyright 2010 Elsevier B.V. All rights reserved.)

Elevated B-Lymphocyte Stimulator (BLyS) and April (a proliferation-inducing ligand) expressions characterize several autoimmune diseases. We here analysed the possible role of BLyS and April in autoimmune thyroid diseases (AITD), comprising Hashimoto's thyroiditis (HT) and Graves' disease (GD). Seventy-seven patients with AITD and 77 blood donors (HBD) were enrolled in the study. Serum BLyS and April levels were assessed by ELISA. Results indicated a significant upregulation of BLyS in AITD patients (1.12+/-0.39 ng/ml versus 0.666+/-0.240 ng/ml in HBD; p<0.0001), with GD patients presenting higher BLyS levels than HT patients (1.22+/-0.42 ng/ml versus 1.07+/-0.38 ng/ml; p=0.0393). In contrast, April levels were downregulated, but only in HT patients [9.9+/-36.6 (median 0) ng/ml versus 7.4+/-22.1 (median 1.16) ng/ml in HBD; p=0.003; and versus 4.2+/-5.9 ng/ml (median 0.9) ng/ml in GD; p=0.0353]. In HT patients, Levo-thyroxine supplementation further increased BLyS and tended to normalize April levels. Neither BLyS nor April did correlate with the levels of the pathognomonic autoantibodies (TPOAb, TgAb, TRAb). Data are preliminary, but, for the first time, we provide the analyses of BLyS and April levels in AITD patients, suggesting new tools for the diagnosis, prognosis and possible therapeutic management of AITD., (Copyright 2009 Elsevier B.V. All rights reserved.)

Different types of infection are implicated in the pathogenesis of autoimmune thyroid diseases (AITD) through molecular mimicry or other mechanisms, but their role is disputed. Human studies support direct or indirect evidence of involvement of some viral and bacterial agents, but reports have provided conflicting and inconclusive results. Using a new automated multiplex array platform for the detection of antibodies, we determined seroreactivity against Toxoplasma gondii, Treponema pallidum, rubella virus, cytomegalovirus, and Epstein-Barr virus in a large group of Italian AITD patients and healthy controls. Only IgG concentrations against T. gondii were significantly higher in AITD patients than in controls, suggesting that these protozoa may be involved in the initiation of both Hashimoto's thyroiditis and Graves' disease.

Background: To evaluate the diagnostic characteristics of commercially available IgG anti-tTG assays in selective IgA deficiency (SIgAD), we tested different IgG anti-tTG methods and compared the results with those obtained from two other tests: one for IgG anti-gliadin (AGA) and one for IgG to deaminated gliadin peptides (DGP)., Methods: 20 CD patients with SIgAD and 113 controls (9 patients with SIgAD without CD; 54 patients with chronic liver disease; 50 healthy subjects) were tested with 9 IgG anti-tTG assays (2 of which are enriched with gliadin peptides), one IgG AGA assay and one IgG anti-DGP assay., Results: Using optimal cutoffs as determined by ROC curves, the sensitivity of IgG anti-tTG methods ranged from 75% (1 kit) to 95% (7 kits) and the specificity from 94% (1 kit) to 100% (5 kits). Sensitivity and specificity were 40% and 87% for IgG AGA, and 80% and 98% for IgG anti-DGP, respectively., Conclusions: All IgG anti-tTG methods evaluated are reliable serologic assays for the diagnosis of CD in patients with SIgAD and perform better than the gliadin-based assays used in this study. The tests containing both tTG and gliadinic peptides are burdened by a lower specificity than the anti-tTG assays.

Objective: To evaluate the role of anti-prothrombin (anti-PT) antibodies in predicting thrombosis in patients with systemic lupus erythematosus (SLE)., Methods: An inception cohort of 101 SLE patients (12 males, 89 females; mean age 30 +/- 8 years), was considered. Clinical and laboratory evaluations were regularly performed during a 15-year follow-up (median 108 months) with a special focus on thromboembolic events. Serum samples were collected at time of diagnosis and at least once a year thereafter. IgG and IgM anti-PT, anti-cardiolipin (aCL) and anti-beta(2)glycoprotein I (beta(2)GPI) antibodies were measured by enzyme-linked immunosorbent assay (ELISA); lupus anticoagulant (LAC) was assayed by the dilute Russell's viper venom time and activated partial thromboplastin time tests. The analytical specificity of anti-PT ELISA was investigated. The timing of thrombosis occurrence was calculated using the Kaplan-Meier method., Results: In the 15-year follow-up, thrombosis occurred in 14 out of the 101 patients: venous thrombosis in nine cases and arterial thrombosis in five. IgG and/or IgM anti-PT, anti-beta(2)GPI and aCL antibodies, and LAC activity were detected in ten, nine, seven, and nine cases, with sensitivity for thrombosis of 71.4%, 64.3%, 50% and 64.3%, respectively. Thrombosis-free survival was 90% at 5 years and 85.8% at 10 and 15 years, respectively. Thrombosis was predicted by anti-PT (P = 0.001), anti-beta(2)GPI antibodies (P = 0.002) and LAC activity (P = 0.001). Moreover, the risk of thrombosis progressively increased with the number of positive antiphospholipid antibody tests. The presence of four positive antibody tests was associated with a risk of thrombosis thirtyfold higher than in their absence., Conclusions: This longitudinal study shows that IgG anti-PT antibodies are predictors of thrombosis in SLE patients.

Arrays are one of the technologies able to detect autoantibodies by measuring simultaneously many thousands of markers from a unique biological sample. The main purpose of a diagnostic test is making an early and accurate diagnosis. From a statistical point of view, multiple testing increases the probability of false positive and false negative results. Some correction methods are available to account for this problem for instance family-wise error rate or false discovery rate. From an ethical point of view, the decision to accept or decline a test not requested has to be made autonomously. Some people may seek clarification about tests and implications of their choices. A scarcity of proven measures to reduce mortality has to be considered too. Reasons may also include avoidance of psychological harm or anxiety. Moreover, protection of confidentiality and privacy has to be respected. In conclusion, the fact that testing is optional and that surveillance advice can be offered on the basis of risk alone without a test should be discussed in the consultation. The implication of a positive test result should be discussed to make a decision about the degree to which early treatment of the condition is better than late (or no) treatment.

Array technology and proteomics are about to launch the era of multiplexed analysis, which allows simultaneous detection of numerous autoantibody specificities and the possibility of defining broad autoantibody profiles. This will probably improve disease staging, risk stratification, prognosis and treatment. However, although these technologies are very promising, they are still in their infancy, and therefore need to undergo strict analytical and clinical validation processes. The latter should involve clinicians and pathologists in prospective, multicentric studies conducted on large numbers of patients to define the specific significance of the various autoantibody profiles. Establishing common standards for the publication and sharing of microarray-generated data will be important for this purpose. Only when these studies have been completed will these new technologies find a place in clinical laboratories. Although we are entering a decade which will probably see a radical change in the diagnostic approach to autoimmune diseases, we do not yet have sufficient knowledge to apply proteomic technologies on a large scale. For the time being, therefore, it is advisable to continue using well-established approaches and diagnostic algorithms such as those reported in the international guidelines, which have been prepared in accordance with the principles of appropriateness and evidence-based medicine.

Background: Conflicting results were obtained in the assay of anti-transglutaminase (anti-tTG) autoantibodies in patients with chronic liver disease. In order to establish whether this was attributable to methodological differences, anti-tTG antibodies were assayed in a large number of patients suffering from liver cirrhosis (LC)., Methods: 54 patients with LC and 29 patients suffering from celiac disease (CD), used as controls, were tested for IgA and IgG anti-tTG with 11 different commercial methods., Results: In the patients with LC, positivity ranged from 0% to 33.3% for IgA anti-tTG and from 0% to 11.1% for anti-tTG of the IgG class. The largest number of false positives was found with methods that used tTG in association with gliadin peptides as antigen substrate. A significant association was found between IgA anti-tTG antibodies and serum immunoglobulin concentration., Conclusions: The results of the various methods of assaying anti-tTG antibodies in patients with LC are highly variable, and the positives found are generally false positives, partly due to the high immunoglobulin concentration.

Objectives: We tested a simple noninvasive method for cardiac resynchronization therapy (CRT) optimization using standard finger photoplethysmography (FPPG)., Background: CRT can increase left ventricular cardiac output in patients with heart failure and ventricular conduction delay. Optimal therapy delivery depends on an appropriate AV delay. Multiple invasive and noninvasive methods have been attempted to identify patients and the best AV delay for CRT, but all suffer from a combination of high patient risk, cost, complexity, and low reproducibility., Methods: FPPG and invasive aortic pressure data were simultaneously collected from 57 heart failure patients during intrinsic rhythm alternating with very brief periods of pacing at 4 to 5 AV delays. After correcting data for artifacts, the median percentage responses for each AV delay were classified as positive, negative, or neutral compared to baseline (Wilcoxon rank test)., Results: FPPG correctly identified positive aortic pulse pressure responses with 71% sensitivity (95% CI: 60-80%) and 90% specificity (95% CI: 84-94%) and negative aortic pulse pressure responses with 57% sensitivity (95% CI: 44-69%) and 96% specificity (95% CI: 91-98%). The magnitude of FPPG changes were strongly correlated with positive aortic pulse pressure changes (R(2) = 0.73, P < .0001) but less well correlated with negative aortic pulse pressure changes (R(2) = 0.43, P < .0001). FPPG selected 78% of the patients having positive aortic pulse pressure changes to CRT and identified the AV delay giving maximum aortic pulse pressure change in all selected patients., Conclusions: FPPG can provide a simple noninvasive method for identifying significant changes in aortic pulse pressure with high specificity, including identifying patients in whom aortic pulse pressure increases with CRT and the AV delay giving the maximum aortic pulse pressure.

Celiac disease (CD) is an autoimmune enteropathy triggered by the ingestion of gluten in susceptible individuals, and is one of the most frequent genetically based diseases, with a prevalence of 1:200 in the general population. The association between CD and connective tissue diseases (CTD) and autoimmune diseases of the digestive tract (DT) has been described in several case reports but in few extensive studies, with varying prevalence. A high rate of false positive results were observed when low specific tests, such as the anti-gliadin and the guinea pig tissue transglutaminase (tTG) antibody assays were used. In a study of 400 patients with CTD and 218 with autoimmune DT disease, tested for IgA and IgG anti-tTG using the more specific human recombinant antigen, 12 cases (1.9%) of anti-tTG antibody positivity were found, but only 2 (0.3%) were confirmed as affected by CD following small bowel biopsy. Most of the patients testing false positive had primary biliary cirrhosis. In this short review we describe the association between CD and CTD, inflammatory bowel disease and primary biliary cirrhosis, with special emphasis on the diagnostic accuracy of CD antibody assays.

This study was performed by the Italian Society of Laboratory Medicine (SIMeL) in order to establish the variability between the different analytical systems currently used in clinical laboratories for the detection of autoantibodies diagnostic of systemic autoimmune disease. Sixteen industrial, and two university laboratories participated in this study which entailed the determination of anti-nuclear (ANA), anti-dsDNA and anti-ENA antibodies in 11 sera from patients with clinically diagnosed systemic rheumatic disease, using reagents produced by these companies and different methodologies (indirect immunofluorescence, immunoenzymatic assay, counterimmunolectrophoresis, immuno and western blotting). We found 93.5% agreement between the methods used for the detection of ANA, 85.2% for anti-dsDNA antibodies, and 86.9% for anti-ENA antibodies. Among the anti-ENA antibodies, regardless of the method used, detection percentages were excellent for anti-RNP and anti-SSB/La (100%), good for anti-SSA/Ro (93%), but unacceptable for the anti-Jo-1 (67%), anti-Scl70 and anti-Sm (47%) antibodies. This further stresses the need for rigorous standardisation of commercial reagents and analytical procedures, as well as the introduction of external quality assessment (EQA) programs, and a complete definition of operative protocols adjusted to the sensitivity and specificity of the various methods.

We report a new partially automated method for the measurement of the adherence of PMN in vitro. Adherence to a plastic surface was detected by measuring leukocyte alkaline phosphatase activity of the adherent cells, with a Titertek Multiscan system. Using three different cellular concentrations (1 X 10(6), 5 X 10(5), 2.5 X 10(5) PMN/ml) the response curve was linear to 45 min and adhesion was maximal by 30 min. The specificity of the reaction was acceptable as was the assay reproducibility (intra-assay coefficient of variation less than 8%; inter-assay coefficient of variation less than 11%).