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Objectives Antinuclear antibodies (ANA) are important for the diagnosis of various autoimmune diseases. ANA are usually detected by indirect immunofluorescence assay (IFA) using HEp-2 cells (HEp-2 IFA). There are many variables influencing HEp-2 IFA results, such as subjective visual reading, serum screening dilution, substrate manufacturing, microscope components and conjugate. Newer developments on ANA testing that offer novel features adopted by some clinical laboratories include automated computer-assisted diagnosis (CAD) systems and solid phase assays (SPA). Methods A group of experts reviewed current literature and established recommendations on methodological aspects of ANA testing. This process was supported by a two round Delphi exercise. International expert groups that participated in this initiative included (i) the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group “Autoimmunity Testing”; (ii) the European Autoimmune Standardization Initiative (EASI); and (iii) the International Consensus on ANA Patterns (ICAP). Results In total, 35 recommendations/statements related to (i) ANA testing and reporting by HEp-2 IFA; (ii) HEp-2 IFA methodological aspects including substrate/conjugate selection and the application of CAD systems; (iii) quality assurance; (iv) HEp-2 IFA validation/verification approaches and (v) SPA were formulated. Globally, 95% of all submitted scores in the final Delphi round were above 6 (moderately agree, agree or strongly agree) and 85% above 7 (agree and strongly agree), indicating strong international support for the proposed recommendations. Conclusions These recommendations are an important step to achieve high quality ANA testing.

Only case reports and small clinical series report the effects of booster vaccination with BNT162b2 in patients with rheumatoid arthritis (RA). We studied 200 patients with RA in clinical remission evaluated with the DAS28. All patients were vaccinated for SARS CoV-2 with the BNT162b2 mRNA vaccine. The value of anti-SARS-CoV 2 Spike RBD IgG antibodies was determined at T1 (3 weeks after first vaccination) and T2 (3 weeks after booster). In addition, patients underwent assessment of lymphocyte subpopulations by flow cytometry analysis before starting the vaccination cycle (T0). Furthermore, the serum antibody levels of 96 health care workers (HCWs) were analyzed for comparison. DAS28 values at T0, T1, and T2 indicated remission or low disease activity in all patients. Levels of anti-SARS CoV-2 IgG at T1 were higher in HCWs than in patients' groups: 1562.00 BAU WHO/mL [975.00-1632.00] vs 416.00 BAU WHO/mL [110.00, 1581.00], p0.001. Anti-SARS COV2 IgG levels at T1 and at T2 were slightly lower in patients taking b/tsDMARDs than in patients under csDMARDs. Regression analysis evidenced age, treatment with abatacept (ABA), JAK inhibitors, and rituximab (RTX) as negative predictors of higher anti-SARS CoV-2 IgG levels at T1. Moreover, treatment with anti-IL6, anti-JAK, and anti-tumor necrosis factor (TNF) emerged as positive predictors of higher levels of anti-SARS CoV-2 IgG at T2. Our data show that despite the booster vaccine with BNT162b2, seroconversion in patients with rheumatoid arthritis is influenced by the background therapy, particularly for patients being treated with ABA and RTX.

Objectives Evaluating anti-SARS-CoV-2 antibody levels is a current priority to drive immunization, as well as to predict when a vaccine booster dose may be required and for which priority groups. The aim of our study was to investigate the kinetics of anti-SARS-CoV-2 Spike S1 protein IgG (anti-S1 IgG) antibodies and neutralizing antibodies (NAbs) in an Italian cohort of healthcare workers (HCWs), following the Pfizer/BNT162b2 mRNA vaccine, over a period of up to six months after the second dose. Methods We enrolled 57 HCWs, without clinical history of COVID-19 infection. Fluoroenzyme-immunoassay was used for the quantitative anti-S1 IgG antibodies at different time points T1 (one month), T3 (three months) and T6 (six months) following the second vaccine shot. Simultaneously, a commercial surrogate virus neutralization test (sVNT) was used for the determination of NAbs, expressed as inhibition percentage (% IH). Results Median values of anti-S1 IgG antibodies decreased from T1 (1,452 BAU/mL) to T6 (104 BAU/mL) with a percent variation of 92.8% while the sVNT showed a percent variation of 34.3% for the same time frame. The decline in anti-S1 IgG antibodies from T1 to T6 was not accompanied by a loss of the neutralizing capacity of antibodies. In fact at T6 a neutralization percentage Conclusions Our findings reveal that the decrease of anti-S1 IgG levels do not correspond in parallel to a decrease of NAbs over time, which highlights the necessity of using both assays to assess vaccination effectiveness.

Objectives Rheumatoid arthritis (RA) is a systemic autoimmune disease characterised by the presence of autoantibodies that are used for classification of the disease. Though routine diagnostics is commonly restricted to measuring rheumatoid factor (RF) and anti-citrullinated protein antibodies, detection of RF IgM, IgG and IgA isotypes, may increase the power of RA serodiagnosis by reducing the number of seronegative patients as well as provide prognostic information. The agglutination-based RF assays, such as nephelometry or turbidimetry, are unable to differentiate isotypes. We compared three different immunoassays used in current laboratory practice to detect RF isotypes. Methods We tested 117 consecutive serum samples that were positive for total RF at nephelometry, from 55 RA and 62 non-RA subjects. IgA, IgG, and IgM isotypes of RF were tested by immunoenzymatic (ELISA, Technogenetics), fluoroenzymatic (FEIA, ThermoFisher) and chemiluminescence (CLIA, YHLO Biotech Co.) immunoassays. Results Diagnostic performance differed considerably between the assays, especially with regard to RF IgG isotype. Agreement among methods by Cohen’s kappa ranged from 0.05 (RF IgG CLIA vs. FEIA) to 0.846 (RF IgM CLIA vs. FEIA). Conclusions The poor agreement observed in this study indicates substantial lack of comparability among assays for RF isotypes. Harmonization of these tests requires further efforts before their measurement can be used in clinical practice.

Background The introduction of multiparametric autoantibody tests has been proposed to improve the accuracy of the immunological diagnosis of autoimmune diseases (AID) and to accelerate time for completing the diagnostic process. Multiplex tests are capable of detecting many autoantibodies in a single run whereas a traditional immunoassay uses a single antigen to detect only a single specificity of autoantibodies. The reasons why multiplex tests could replace conventional immunoassays lie in the evidence that they allow for more efficient handling of large numbers of samples by the laboratory, while ensuring greater diagnostic sensitivity in AID screening. Content This review aims to highlight the important role that multiparametric tests could assume when designed for defined profiles they are used not only for diagnostic purposes but also to predict the onset of AID to identify clinical phenotypes and to define prognosis. Furthermore, differences in the antibody profile could identify which subjects will be responsive or not to a specific pharmacological treatment. Summary The use of autoantibody profiles, when specifically requested and performed with clinically validated technologies, can represent a significant step toward personalized medicine in autoimmunology.

Background: In recent years, the involvement of the soluble urokinase Plasminogen Activator Receptor (suPAR) in the pathophysiological modulation of Rheumatoid Arthritis (RA) has been documented, resulting in the activation of several intracellular inflammatory pathways. Methods: We investigated the correlation of urokinase Plasminogen Activator (uPA)/urokinase Plasminogen Activator Receptor (uPAR) expression and suPAR with inflammation and joint damage in RA, evaluating their potential role in a precision medicine context. Results: Currently, suPAR has been shown to be a potential biomarker for the monitoring of Systemic Chronic Inflammation (SCI) and COVID-19. However, the effects due to suPAR interaction in immune cells are also involved in both RA onset and progression. To date, the literature data on suPAR in RA endorse its potential application as a biomarker of inflammation and subsequent joint damage. Conclusion: Available evidence about suPAR utility in the RA field is promising, and future research should further investigate its use in clinical practice, resulting in a big step forward for precision medicine. As it is elevated in different types of inflammation, suPAR could potentially work as an adjunctive tool for the screening of RA patients. In addition, a suPAR system has been shown to be involved in RA pathogenesis, so new data about the therapeutic response to Jak inhibitors can represent a possible way to develop further studies.

Anti-double-stranded DNA antibodies (anti-dsDNA) are considered a specific marker for systemic lupus erythematosus (SLE). Though the Farr technique was once the reference method for their detection, it has been almost entirely replaced by more recently developed assays. However, there is still no solid evidence of the commutability of these methods in terms of diagnostic accuracy and their correlation with the Crithidia luciliae immunofluorescence test (CLIFT). Anti-dsDNA antibody levels were measured in 80 subjects: 24 patients with SLE, 36 disease controls drawn from different autoimmune rheumatic diseases (14 systemic sclerosis, 10 Sjögren's syndrome, nine autoimmune myositis, three mixed connective tissue disease), 10 inflammatory arthritis and 10 apparently healthy blood donors by eight different methods: fluorescence enzyme immunoassay, microdot array, chemiluminescent immunoassay (two assays), multiplex flow immunoassay, particle multi-analyte technology immunoassay and two CLIFT. At the recommended manufacturer cut-off, the sensitivity varied from 67% to 92%, while the specificity ranged from 84% to 98%. Positive agreement among CLIFT and the other assays was higher than negative agreement. Mean agreement among methods assessed by the Cohen's kappa was 0.715, ranging from moderate (0.588) to almost perfect (0.888). Evaluation of the concordance among quantitative values by regression analysis showed a poor correlation index (mean r2, 0.66). The present study shows that current technologies for anti-dsDNA antibody detection are not fully comparable. In particular, their different correlation with CLIFT influences their positioning in the diagnostic algorithm for SLE (either in association or sequentially). Considering the high intermethod variability, harmonization and commutability of anti-dsDNA antibody testing remains an unachieved goal.

Calprotectin (S100A8/A9) has been identified as a biomarker that can aid in predicting the severity of disease in COVID-19 patients. This study aims to evaluate the correlation between levels of circulating calprotectin (cCP) and the severity of COVID-19.Sera from 245 COVID-19 patients and 110 apparently healthy individuals were tested for calprotectin levels using a chemiluminescent immunoassay (Inova Diagnostics). Intensive care unit (ICU) admission and type of respiratory support administered were used as indicators of disease severity, and their correlation with calprotectin levels was assessed.Samples from patients in the ICU had a median calprotectin concentration of 11.6 µg/ml as compared to 3.5 µg/ml from COVID-19 patients who were not in the ICU. The median calprotectin concentration in a cohort of healthy individuals collected before the COVID-19 pandemic was 3.0 µg/ml (95% CI: 2.820-2.969 µg/ml). Patients requiring a Venturi mask, continuous positive airway pressure, or orotracheal intubation all had significantly higher values of calprotectin than controls, with the increase of cCP levels proportional to the increasing need of respiratory support.Calprotectin levels in serum correlate well with disease severity and represent a promising serological biomarker for the risk assessment of COVID-19 patients.

Background: We investigated how the non-medical switching (NMS) between Etanercept (ETN)/originator and SB4/biosimilar affects treatment efficacy in a rheumatic disease (RD) cohort, evaluating some laboratory parameters as loss of efficacy predictors after NMS. Methods: We enrolled 124 patients with RD (rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis): 79 switchers from ETN/originator to SB4 and 45 naïve patients receiving SB4 (first biological treatment). At baseline, 6 (T1), and 12 months (T2), clinical and laboratory parameters were evaluated. Results: In naïve patients, TNF-α significantly increased at T1 in responders (NR) and non-responders (NNR). TNF-α was lower in NNR than in NR at T1 and T2. In NR and NNR, drug levels (DL) increased between T1 and T2. However, DLs were lower in NNR than in NR at T1 and T2. TNF-α was higher in switcher responders (SR) than in non-responders (SNR) at T1 and T2. In SNR, DLs were higher at baseline than in SR, but they decreased significantly at T1 and T2. Conclusions: We observed a decrease in DL and TNF-α levels after NMS in SNR. Moreover, in naïve patients, DL and TNF-α levels were higher in NR than in NNR. Monitoring DL and TNF-α levels may represent a future precision medicine approach to predict loss of efficacy after NMS.

Objectives Currently available computer-aided diagnosis (CAD) systems for the detection of anti-nuclear antibodies (ANA) by indirect immunofluorescence (IIF) assay enable a standardized measurement of system-specific fluorescent intensity (FI) measures. We aimed to evaluate an internal quality control (iQC) program that controls the total ANA IIF process in routine practice. Methods In addition to the kit iQC materials, supplemental quality indicators were integrated in a total quality assurance (QA) program: patient-derived iQC’s samples (negative, 1/160 fine speckled and 1/160 homogeneous), median sample FI per run and percentage of ANA IIF positive samples per run. Analytical rejection criteria were based on the imprecision of the positivity index (PI) measure of the Zenit PRO system (Menarini). Clinical rejection criteria were based on changes in FI that correspond to a change in ANA IIF titer of ≥2. To evaluate the QA program, different artificial errors were introduced during the ANA IIF process. After every run, quality indicators were evaluated and compared to the pre-set target values. Results Rescanning the ANA IIF slides five times, using an old conjugate and a needle obstruction resulted in analytically and even clinically relevant errors in ANA IIF results. All errors were correctly detected by the different defined quality indicators. Traditional Westgard rules, including analytically (and clinically) defined rejection limits were useful in monitoring quality indicators. Conclusions The integration of a total process iQC program in CAD systems, based on the specific FI measurands and performance criteria of the system, adds value to QA.

Since January 2020, the whole world has been facing the worst epidemic for a century. SARS-CoV- 2 infection has so far caused more than one million deaths, with the only measures capable of containing the spread of the virus being social distancing, frequent hand washing, and the wearing of masks. Vaccine development was urgently needed and there are now more than 90 candidate vaccines being developed using different technologies. The European Medicines Agency has recently approved a second mRNA-based vaccine, but the introduction of vaccines has raised some doubts about patients with rheumatic disease, who are at high risk of infection because of disease activity and the therapies used to treat it. The aim of this study was to investigate how vaccines may interact with the immune system and treatment of such patients, and how to monitor the post-vaccine antibody titres and T cell responses in order to assess their efficacy and safety.

In early undifferentiated arthritis (EUA), the relationship between inflammatory biomarkers and disability is still unclear. The aim of this study was to correlate inflammatory biomarkers with the Arthritis Impact Measurement Scales (AIMS) in EUA.Seventy patients with EUA were compared with 20 patients with established rheumatoid arthritis (RA). The association of AIMS [mobility, physical impairment (PI), dexterity, household activities, activities of daily living (ADL), social activity, pain, anxiety, depression] with serum laboratory [phase acute reactants, calprotectin, interleukin-6, tumour necrosis factor (TNF)-α, rheumatoid factor, anti-nuclear and anti-citrullinated peptide antibodies, HLA-DRB], clinical [Clinical Disease Activity Index (CDAI), fatigue, pain and stiffness NRS], x-ray and ultrasound biomarkers was analysed with non-parametric Spearman's rank correlation and Mann-Whitney U tests.No differences in AIMS were found between EUA and established RA patients, or between EUA patients that evolved into early RA (n=17) and those that remained EUA (n=53) at six months of follow-up. In EUA, erythrocyte sedimentation rate correlated with mobility impairment, PI and depression (p=0.04, p=0.03 and p=0.022, respectively), TNF-α correlated with PI (p=0.01) and calprotectin with anxiety (p=0.02). HLA-DRB1*11-positive EUA patients had lower ADL deficiency (p=0.006), depression (p=0.0004) and anxiety (p=0.01). CDAI correlated with PI (p=0.01) and pain (p=0.01), fatigue with PI (p=0.0001) and ADL (p=0.009), stiffness with PI (p=0.01), and Power Doppler ultrasound synovitis with PI (p=0.02) and pain (p=0.007).In EUA, physical and mood disorders are associated with new and old inflammatory serological, clinical and imaging biomarkers. HLA-DRB1*11-positivity may be protective against these disease-related features.

The soluble urokinase plasminogen activator receptor (suPAR) is the bioactive form of uPAR, a membrane-bound glycoprotein, and it is primarily expressed on the surface of immunologically active cells. Mirroring local inflammation and immune activation, suPAR has gained interest as a potential prognostic biomarker in several inflammatory diseases. Indeed, in many diseases, including cancer, diabetes, cardiovascular diseases, kidney diseases, and inflammatory disorders, higher suPAR concentrations have been associated with disease severity, disease relapse, and mortality. Our review describes and discusses the supporting literature concerning the promising role of suPAR as a biomarker in different autoimmune rheumatic and non-rheumatic diseases.

The establishment of the International Consensus on ANA Patterns (ICAP) in 2014–2015 was welcomed by members of the medical community as a significant improvement in guiding harmonization of ANA test interpretation and reporting. In the subsequent years, several itinerant meetings and continuous interaction with the community contributed to disseminate the ICAP harmonization on the immunofluorescence patterns observed in the indirect immunofluorescence assay on HEp-2 cells (HEp-2 IFA) and to promote progressive improvement in the classification of HEp-2 IFA patterns. The 6th ICAP Workshop was held in person on September 6, 2021 as a satellite meeting of the 15th Dresden Symposium on Autoantibodies. This article summarizes the major discussions at the meeting as well as outlining the current plans for the ICAP committee.

The identification of anti-NXP2 antibodies is considered a serological marker of dermatomyositis (DM), with calcinosis, severe myositis and, in some reports, with cancer. Historically, these associations with anti-NXP2 antibodies have been detected by immunoprecipitation (IP), but in the last few years commercial immunoblotting assays have been released. The aim of this collaborative project was to analyse the clinical features associated to anti-NXP2 antibodies, both with commercial line blot (LB) and IP. Myositis-specific and myositis-associated autoantibodies were detected in single centres by commercial line blot (LB); available sera were evaluated in a single centre by protein and RNA immunoprecipitation (IP), and IP-Western blot. Sixty patients anti-NXP2+ (NXP2+) positive by LB were compared with 211 patients anti-NXP2 negative with idiopathic inflammatory myositis (IIM). NXP2+ showed a younger age at IIM onset (p = 0.0014), more frequent diagnosis of dermatomyositis (p = 0.026) and inclusion-body myositis (p = 0.009), and lower rate of anti-synthetase syndrome (p

Results of the anti-nuclear antibodies-indirect immunofluorescence assay (anti-cell antibodies test) on HEp-2 cell substrates should be communicated to clinicians in a standardized way, adding value to laboratory findings and helping with critical clinical decisions. This paper proposes a test report based on the practices informed by 118 laboratories in 68 countries, with recommendations from the International Consensus on ANA Patterns (ICAP) group. Major focus is placed on the report format containing endpoint titers, immunofluorescence patterns together with anti-cell (AC) nomenclature, remarks on follow-up or reflex testing, and possible other autoantibody associations. ISO 15,189 directives were integrated into the test report. Special situations addressed include serum screening dilutions and endpoint titers, relevance of immunofluorescence patterns with special attention to cytoplasmic patterns, mixed and compound patterns, and how to report different titers corresponding to multiple patterns or autoantibodies in the same sample. This paper suggests a subtitle for the HEp-2-IIFA, namely anti-cell antibodies test, which could gradually substitute the original outdated ANA nomenclature. This ICAP pro forma report represents a further step in harmonizing the way relevant clinical information could be provided by laboratories.

The authors wish to make the following correction to this paper [...]

Several commercial surrogate Virus Neutralization Tests (sVNTs) have been developed in the last year. Neutralizing anti-SARS-CoV-2 antibodies through interaction with Spike protein Receptor Binding Domain (S-RBD) can block the virus from entering and infecting host cells. However, there is a lack of information about the functional activity of SARS-CoV-2 antibodies that may be associated with protective responses. For these reasons, to counteract viral infection, the conventional virus neutralization test (VNT) is still considered the gold standard. The aim of this study was to contribute more and detailed information about sVNTs' performance, by determiningEighty-eight samples were tested using two chemiluminescence assays (Snibe and Mindray) and two ELISA assays (Euroimmun and Diesse). The antibody titers were subsequently detected and quantified by VNT.The overall agreement between each sVNT and VNT was 95.45% for Euroimmun and 98.86% for Diesse, Mindray and Snibe. Additionally, we investigated whether the sVNTs were closer to the gold standard than traditional anti-SARS-CoV-2 antibody assays S-RBD or S1 based, finding a higher agreement mean value for sVNTs (98.01 ± 1.705% vs 95.45 ± 1.921%;Our data revealed a good agreement between VNT and sVNTs. Despite the VNT still remains the gold standard, the sVNT might be a valuable tool for screening wider populations.

Along years, the advent of biological therapy widely modified treatment of rheumatic diseases and other disorders. However, many agents may elicit in anti-drug antibodies (ADAbs) upon consecutive infusions, with a loss of response. For the right strategy of a personalized medicine, the therapeutic monitoring of TNF-α inhibitors and ADAbs represents an important effort in diagnostic-therapeutic pathway, to improve overall patient management and favoring an appropriate clinical approach. A raising number of diagnostic tests have been designed to elucidate the efficacy and/or safety of a specific drug or class of drugs for a targeted patient's group. Our paper reviewed the current understanding of the immunogenicity of biological drugs employed in the treatment of inflammatory diseases underlying the laboratory role.

Rheumatoid arthritis (RA) is a chronic inflammatory disorder that affects joints with swelling and progressive joint destruction. The pathology leads to a progressive disability with an impact on the quality of life of the patients. Strategies to reduce in-patient care costs could have a considerable impact on lowering the direct medical costs of RA in Italy. Abatacept, a selective T-cell costimulation modulator, is a valuable treatment option for patients with moderate-to-severe RA. A search using the keywords "cost-effectiveness analysis", "budget impact model", "abatacept", and "rheumatoid arthritis" was carried out on PubMed. Abatacept in the first- and second-treatment lines has been evaluated in our research. We evaluated patients with inadequate MTX response, inadequate anti-TNF agents response, switch studies and real-world data. Furthermore, in our research, we evaluated the main head-to-head studies published.

The vulnerable population of kidney transplant recipients (KTRs) are low responders to COVID-19 vaccines, so specific immune surveillance is needed. The interferon-gamma (IFN-γ) release assay (IGRA) is effective in assessing T cell-mediated immunity. We assessed SARS-CoV-2-directed T cell responses in KTRs with absent antibody production after a third dose of the mRNA-1273 vaccine, using two different IGRAs. A cohort of 57 KTRs, who were actively followed up, received a third dose of the mRNA-1273 vaccine. After the evaluation of humoral immunity to SARS-CoV-2, 14 seronegative patients were tested with two commercial IGRAs (SD Biosensor and Euroimmun). Out of 14 patients, one and three samples were positive by IGRAs with Euroimmun and SD Biosensor, respectively. The overall agreement between the two assays was 85.7% (κ = 0.444). In addition, multivariate linear regression analysis showed no statistically significant association between the IFN-γ concentration, and the independent variables analyzed (age, gender, years since transplant, total lymphocytes cells/mcl, CD3+ cells/mcl, CD3+ CD4+ cells/mcl, CD3+ CD8+ cells/mcl, CD19+ cells/mcl, CD3-CD16+CD56+ cells/mcl) (p > 0.01). In a vulnerable setting, assessing cellular immune response to complement the humoral response may be advantageous. Since the two commercial IGRAs showed a good agreement on negative samples, the three discordant samples highlight the need for further investigations.