Adopting the International Consensus on ANA Patterns (ICAP) classification for reporting: the experience of Italian clinical laboratories.

The indirect immunofluorescence assay on HEp-2 cells (HEp-2 IFA) is still considered the reference method to detect anti-nuclear antibodies (ANA) because of its high sensitivity and represents a relevant tool for the diagnosis of autoimmune rheumatic diseases. During the last decade, the International Consensus on ANA Patterns (ICAP) initiative promoted harmonization and understanding of HEp-2 IFA staining pattern nomenclature, as well as promoting their use in patient care by providing interpretation for HEp-2 IFA test results. In conjunction with a nationwide survey on the evolution of autoantibody diagnostics in autoimmune rheumatic diseases, we focused on the adherence of the Italian laboratories to the ICAP nomenclature analyzing its lights and shadows. The recent ICAP-oriented report, largely used today among Italian laboratories, also represents a further step in harmonizing and improving communication with the clinicians, adding value to laboratory findings and helping with critical clinical decisions.

An Italian nationwide survey on the evolution of autoantibody diagnostics in autoimmune rheumatic diseases.

OBJECTIVES: The advent of new technologies and the discovery of new antigenic targets of autoantibodies has led to significant changes in autoimmune diagnostics worldwide. To address the extent to which autoimmunology laboratories adhere to such innovation in testing and reporting practices, the Italian Society of Clinical Pathology and Laboratory Medicine launched a national survey to assess the current status of autoimmune diagnostics and to provide direction for further harmonisation. METHODS: A questionnaire covering topics related to the diagnosis of autoimmune systemic rheumatic diseases was distributed to 152 Italian autoimmunology laboratories. The 59 closed-answer questions were subdivided into four main sections: 1. the setting (university, hospital or private laboratory) and the number of tests carried out; 2. the technologies used and their level of automation; 3. the analytical phase (antibody tests and methods), including awareness of the International Consensus on ANA Patterns (ICAP) initiative; 4. reporting of results and clinician relations. RESULTS: A total of 121 laboratories (79.6%) responded to the survey (15% universities, 70 hospitals, and 15% private laboratories). Indirect immunofluorescence is used by 94.8% of respondents, chemiluminescence by 78.4%, fluoro-immuno-enzymatic assays by 67.5%, immunodot by 52.6%, line-immunoassay by 47.4%, addressable laser bead immunoassay by 10.3% and radioimmunological methods by 10.2%. The great majority of respondents implemented complete automation of the listed methodologies. 65% of participants state that they add an interpretative comment in the report. 45% of participants enjoy a collaborative relationship with clinicians; counselling activities are provided by almost half of participants. CONCLUSIONS: Survey results indicated that almost all respondent laboratories have broadened their antibody panel and that high-throughput technologies have been widely introduced. Gaps identified by the survey include a still incomplete compliance with guidelines in antibody profiles (e.g. in antiphospholipid syndrome and rheumatoid arthritis and reporting of test results. Awareness of these differences provides insights that may further contribute to achieving harmonisation in autoimmune diagnostics.

Lack of comparability of immunoassays for rheumatoid factor isotypes.

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.

Detection of antinuclear antibodies: recommendations from EFLM, EASI and ICAP.

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.

Soluble urokinase Plasminogen Activator Receptor (suPAR) levels are predictive of COVID-19 severity: an Italian experience.

BACKGROUND: The soluble urokinase Plasminogen Activator Receptor (suPAR) has been identified as a reliable marker of COVID-19 severity, helping in personalizing COVID-19 therapy. This study aims to evaluate the correlation between suPAR levels and COVID-19 severity, in relation to the traditional inflammatory markers. METHODS: Sera from 71 COVID-19 patients were tested for suPAR levels using Chorus suPAR assay (Diesse Diagnostica Senese SpA, Italy). suPAR levels were compared with other inflammatory markers: IL-1ß, IL-6, TNF-α, circulating calprotectin, neutrophil and lymphocyte counts, and Neutrophil/Lymphocytes Ratio (NLR). Respiratory failure, expressed as P/F ratio, and mortality rate were used as indicators of disease severity. RESULTS: A positive correlation of suPAR levels with IL-6 (r = 0.479, p = 0.000), TNF-α (r = 0.348, p = 0.003), circulating calprotectin (r = 0.369, p = 0.002), neutrophil counts (r = 0.447, p = 0.001), NLR (r = 0.492, p = 0.001) has been shown. Stratifying COVID-19 population by suPAR concentration above and below 6 ng/mL, we observed higher levels of circulating calprotectin (10.1 µg/mL, SD 7.9 versus 6.4 µg/mL, SD 7.5, p < 0.001), higher levels of P/F ratio (207.5 IQR 188.3 vs 312.0 IQR 127.8, p = 0.013) and higher mortality rate. Median levels of suPAR were increased in all COVID-19 patients requiring additional respiratory support (Nasal Cannula, Venturi Mask, BPAP and CPAP) (6.5 IQR = 4.9) compared to the group at room air (4.6 IQR = 4.2). CONCLUSION: suPAR levels correlate with disease severity and survival rate of COVID-19 patients, representing a promising prognostic biomarker for the risk assessment of the disease.

Clinical utility of circulating calprotectin to assist prediction and monitoring of COVID-19 severity: An Italian study.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: Calprotectin levels in serum correlate well with disease severity and represent a promising serological biomarker for the risk assessment of COVID-19 patients.

Comparison of current methods for anti-dsDNA antibody detection and reshaping diagnostic strategies.

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.

Cardiovascular safety, cancer and Jak-inhibitors: Differences to be highlighted.

Rheumatoid arthritis (RA) is a chronic inflammatory disease whose natural history leads to articular and extra-articular damage. Both cardiovascular risk and malignancy risk results higher in RA patients, compared to general population. Janus kinase inhibitors (JAKis) are oral targeted synthetic disease modifying antirheumatic drugs (tsDMARDs) that disrupt cytokine cascade and exert anti-inflammatory effects by interfering with signaling through the JAK-STAT intracellular pathways. A recent RCT comparing tofacitinib 5 mg twice daily, tofacitinib 10 mg twice daily and anti-TNF in rheumatoid arthritis demonstrated an increased risk of MACE HR 1.33 and cancer HR 1.49 at a follow-up of 4 years. This has led the FDA to class warnings for tofacitinib, baricitinib and upadacitinib. Cumulative RCT data, RCT extension data demonstrated a safety profile for Jak inhibitors. Conflicting data results from real life registries; the different selectivity for JAKs (JAK1, JAK2, JAK3 and Tyk2) probably determines differences in efficacy and safety profiles among the members of this group which should actually be evaluated. In order to better understand the cardiovascular and neoplastic risk linked to these class of drugs, we aim to provide a literature review on existing evidence of the safety of Jak-Inhibitors in rheumatoid arthritis.

The role of neutralizing antibodies by sVNT after two doses of BNT162b2 mRNA vaccine in a cohort of Italian healthcare workers.

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 <20% IH was observed only in 3.51% of HCWs. 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.

Assessing T-Cell Immunity in Kidney Transplant Recipients with Absent Antibody Production after a 3rd Dose of the mRNA-1273 Vaccine.

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.

Closing the serological gap in the diagnostic testing for COVID-19: The value of anti-SARS-CoV-2 IgA antibodies.

During coronavirus disease 2019 (COVID-19) pandemic, the early diagnosis of patients is a priority. Serological assays, in particular immunoglobulin (Ig)M and IgG anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have today several applications but the interpretation of their results remains an open challenge. Given the emerging role of the IgA isotype in the COVID-19 diagnostics, we aimed to identify the SARS-CoV-2 IgA antibodies in a COVID-19 population seronegative for IgM. A total of 30 patients hospitalized in San Giovanni di Dio Hospital (Florence, Italy) for COVID-19, seronegative for IgM antibodies, have been studied for anti-SARS-CoV-2 antibodies. They all had a positive oro/nasopharyngeal swab reverse transcription-polymerase chain reaction result. Assays used were a chemiluminescent assay measuring SARS-CoV-2 specific IgM and IgG (S + N) and an ELISA, measuring specific IgG (S1) and IgA antibodies against SARS-CoV-2. Among the 30 patients, eight were positive for IgA, seven were positive for IgG (N + S), and two for IgG (S1), at the first point (5-7 days from the onset of symptoms). The IgA antibodies mean values at the second (9-13 days) and third (21-25 days) time points were even more than twice as high as IgG assays. The agreement between the two IgG assays was moderate (Cohen's K = 0.59; SE = 0.13). The inclusion of the IgA antibodies determination among serological tests of the COVID-19 diagnostic is recommended. IgA antibodies may help to close the serological gap of the COVID-19. Variations among anti-SARS-CoV-2 IgG assays should be considered in the interpretation of results.

Vaccination against SARS-CoV-2 in patients with rheumatic diseases: doubts and perspectives.

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.

Do new and old biomarkers of early undifferentiated arthritis correlate with Arthritis Impact Measurement Scales?

OBJECTIVES: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Integrating quality assurance in autoimmunity: the changing face of the automated ANA IIF test.

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.

Diagnosing and Monitoring Celiac Patients with Selective IgA Deficiency: Still an Open Issue.

Although, the association between celiac disease (CD) and selective immunoglobulin A deficiency (SIgAD) has been known for more than fifty years, the procedures for diagnosing and monitoring patients with both conditions are still far from definitive. When serological markers were introduced as pre-bioptic investigations, it was immediately clear that searching for specific IgA antibodies without checking total serum IgA could lead to a failure in diagnosing IgA-deficient CD patients, while specific IgG antibodies could be useful as additional tests, because they are frequently found in the serum of affected patients. Nonetheless, until recently the diagnosis of CD in IgA-deficient patients was based on the few, fragmentary and often contradictory data available in literature. The introduction of the European Society for Pediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) guidelines in 2012 provided the current criteria for diagnosing CD in IgA-deficient patients, although some issues remained open, such as the selection of patients who should undergo specific IgG antibody testing and the choice of the most reliable IgG-based test for both diagnosis and follow-up. A real-life study recently assessed the impact of the 2012 ESPGHAN guidelines in diagnosing and monitoring CD in SIgAD patients, highlighting several pitfalls that can lead to operational uncertainties and difficulties in patient management. In the present report, the evolution of diagnostic tools and criteria for CD in SIgAD patients has been critically assessed, both strengths and open issues have been highlighted, and future perspectives for improving the current diagnostic protocols have been suggested.

Diagnostic accuracy of an automated chemiluminescent immunoassay for anti-SARS-CoV-2 IgM and IgG antibodies: an Italian experience.

A pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been spreading throughout the world. Though molecular diagnostic tests are the gold standard for COVID-19, serological testing is emerging as a potential surveillance tool, in addition to its complementary role in COVID-19 diagnostics. Indubitably quantitative serological testing provides greater advantages than qualitative tests but today there is still little known about serological diagnostics and what the most appropriate role quantitative tests might play. Sixty-one COVID-19 patients and 64 patients from a control group were tested by iFlash1800 CLIA analyzer for anti-SARS CoV-2 antibodies IgM and IgG. All COVID-19 patients were hospitalized in San Giovanni di Dio Hospital (Florence, Italy) and had a positive oro/nasopharyngeal swab reverse-transcription polymerase chain reaction result. The highest sensitivity with a very good specificity performance was reached at a cutoff value of 10.0 AU/mL for IgM and of 7.1 for IgG antibodies, hence near to the manufacturer's cutoff values of 10 AU/mL for both isotypes. The receiver operating characteristic curves showed area under the curve values of 0.918 and 0.980 for anti-SARS CoV-2 antibodies IgM and IgG, respectively. iFlash1800 CLIA analyzer has shown highly accurate results for the anti-SARS-CoV-2 antibodies profile and can be considered an excellent tool for COVID-19 diagnostics.

Algorithm for antinuclear antibodies in subjects with clinical suspicion of autoimmune diseases.

OBJECTIVES: Antinuclear antibodies (ANA) are fundamental in the diagnosis of systemic autoimmune rheumatic diseases (SARDs). Different assays for ANA screening are available, such as indirect immunofluorescence (IIF) on HEp-2 cells and Multiplex fluorescent immunoassay (MFI). This study aimed to clarify the importance of ANA detected only by IIF in the future development of SARDs and to recommend a laboratory algorithm that integrates the available diagnostic approaches to optimise the diagnosis of ANA IIF+MFI- subjects. METHODS: A total of 9,291 subjects with clinical suspicion of SARDs were evaluated for ANA by IIF and MFI. One hundred and ninety-eight subjects (2.1%) were ANA IIF+MFI-, who were followed up for 2 years. ANA were evaluated using IIF on HEp-2 cells and MFI on the BioPlex 2200. RESULTS: The ANA IIF+MFI- cohort included 106 subjects with SARDs, 26 subject with other autoimmune diseases (not-SARDs) and 66 subjects with minor symptoms or ANA requested in check-ups. Only 94 subjects underwent re-evaluation. After a 2-year follow-up, most re-evaluated subjects (51 patients) became ANA negative for both assays (mainly rheumatoid arthritis, polymyalgia and inflammatory bowel disease patients) and 35 subjects remained ANA IIF+MFI- (principally systemic sclerosis and systemic lupus erythematosus patients). A new algorithm for ANA evaluation was suggested. CONCLUSIONS: According to the proposed algorithm, ANA IIF+MFI- subjects should be screened by an alternative solid-phase assay such as line-immunoassay or ELISA.

Two Novel Technologies for the Detection of Anti-cardiolipin and Anti ß2-Glycoprotein Antibodies in the Real Life: Chemiluminescent in Comparison to the Addressable Laser Bead Immunoassays.

In the present study, we evaluated two novel technologies, the chemiluminescent immunoassay (CIA) QUANTA Flash on BIO-FLASH (Inova Diagnostics, San Diego, CA, USA) and the addressable laser bead immunoassay (ALBIA) on BioPlex™ 2200 (Bio-Rad, Hercules, CA, USA) for the detection of anti-cardiolipin IgG/IgM (aCL) and anti-ß2-glycoprotein IgG/IgM (aß2GPI) antibodies. The study was performed on 134 samples from consecutive patients (59 males and 75 females, mean age 54 ± 10 years) who consulted a rheumatologist because thrombosis and/or pregnancy complications were present or another immunological disease (Sjogren's syndrome, inflammatory arthritis). Fourteen patients of the total fulfilled 25the Sydney criteria for APS and for these patients previous results of aPLs were available. Sera were tested for aCL and aß2GPI of IgG and IgM isotypes using CIA (BIO-FLASH) and ALBIA (BioPlex™ 2200). Overall agreement between CIA and ALBIA ranged from 88.1% (aCL IgG) to 97.8% (aß2GPI IgG). Cohen's kappa coefficient ranged from 0.53 to 0.91, implying moderate to almost perfect agreement. Almost perfect agreement was found between BioPlex™ 2200 and BIO-FLASH aß2GPI IgG and aCL IgM with Cohen's kappa of 0.91 and 0.88, respectively. On the other hand, moderate agreement was found between BioPlex™ 2200 and BIO-FLASH aCL IgG and ß2GPI IgM assays with Cohen's kappa of 0.57 and 0.53, respectively. The two novel technologies look promising and comparable but further studies with larger cohorts are needed to contribute to the better understanding of the new aPLs antibodies assays performance.

The Laboratory Role in anti-TNF Biological Therapy Era.

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.

Only monospecific anti-DFS70 antibodies aid in the exclusion of antinuclear antibody associated rheumatic diseases: an Italian experience.

Background The dense fine speckled (DFS) is one of the most common patterns that can be observed as a result of the anti-nuclear antibodies (ANA) test on HEp-2 cells and is mostly caused by antibodies to DFS70 as the main antigenic target. As was recently demonstrated, isolated anti-DFS70 positivity can be used as an aid in the exclusion of ANA associated rheumatic diseases (AARD) due to the opportunity to better interpret unexplained positive IIF ANA results. Methods Our study included 333 subjects with AARD, 51 undifferentiated connective tissue disease (UCTD) patients, 235 disease controls and 149 healthy blood donors from an Italian cohort. All samples were tested for anti-DFS70 and anti-ENA antibodies using QUANTA Flash assays (Inova Diagnostics, San Diego, CA, USA). Results No differences in the prevalence of anti-DFS70 antibodies were seen among AARD, non-AARD and UCTD (2.1% [7/333] vs. 2.3% [9/384] vs. 5.9% [3/51], respectively; p-value = 0.188). AARD patients positive for anti-DFS70 antibodies showed in all cases an accompanying anti-ENA specificity. In contrast, monospecific anti-DFS70 antibodies showed a significantly different distribution with a clear trend across the main groups (AARD vs. non-AARD vs. UCTD: 0% [0/7] vs. 22% [2/9] vs. 100% [3/3], p = 0.007). Anti-DFS70 antibody levels among AARD, non-AARD and UCTD patients were not significantly different (p = 0.094). Within the anti-DFS70 antibody positive cases, AARD cohort showed a higher variability (median [min-max]: 3.2 [3.2-450.8] CU) compared to non-AARD (median [min-max]: 3.2 [3.2-75.7] CU) and UCTD patients (median [min-max]: 3.2 [3.2-59.0] CU). Conclusions Our preliminary data showed a similar frequency of anti-DFS70 antibodies in AARD, UCTD and non-AARD cohorts. Monospecificity of anti-DFS70 antibodies but not their mere presence is the key element in the diagnostic algorithm. Mono-specific anti-DFS70 antibodies might be a helpful biomarker to discriminate individuals with AARD from non-AARD presenting with a positive ANA.