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.

SARS-CoV-2 antibody responses before and after a third dose of the BNT162b2 vaccine in Italian healthcare workers aged ≤60 years: One year of surveillance.

This study monitored the anti-spike-receptor-binding domain (RBD) and neutralizing antibodies induced by the Pfizer/BioNTech mRNA BNT162b2 vaccine in a cohort of 163 healthcare workers aged ≤60 years. We have taken advantage of two study groups, both of whom received the first two doses in the same time window, but Group 1 (54 HCWs) received the third dose 2 months before Group 2 (68 HCWs) did. The cohorts were monitored from the 12th day after the first vaccine dose up to 1 month after the third vaccine dose for a total of eight time points and about 1 year of surveillance (T1 = 12 days after the first dose; T2 = 10 days after the second dose; T3 = 1 month after the second dose; T4 = 3 months after the second dose; T5 = 4 months after the second dose; T6 = 5 months after the second dose; T7 = 7 months after the second dose; T8 = 1 month after the third dose for Group 1; T8* = 9 months after the second dose for Group 2; T9 = 1 month after the third dose for Group 2). The mean value of anti-spike antibodies decreased faster over time, but at T7, its decline was significantly slowed (T7 vs. T8*). After the third dose, the anti-spike titer rose about 34-fold (T7 vs. T8 and T8* vs. T9) and the booster improved the anti-spike titer by about three times compared with that of the second dose (T3 vs. T8 and T3 vs. T9), and no difference was noted between the two groups. The neutralizing titer was evaluated at T3, T7, T8, and T9. Anti-spike and neutralizing antibodies were found to be strongly correlated (r2 = 0.980; p < 0.001). At T3, 70% of the participants had a neutralizing antibody titer >91% of total anti-spike antibodies that increased to 90% after the third dose (T8 and T9). However, when the anti-spike titer reached its lowest value (T7), the neutralizing antibody levels decreased even further, representing only 44% of total anti-spike antibodies (p < 0.0001). Our findings show that the third vaccine dose improves the humoral response, but the wane of the anti-spike and neutralizing antibody titers over time is more marked in the neutralizing antibodies.

Current technologies for anti-ENA antibody detection: State-of-the-art of diagnostic immunoassays.

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.

Detecting Autoantibodies by Multiparametric Assays: Impact on Prevention, Diagnosis, Monitoring, and Personalized Therapy in Autoimmune Diseases.

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.

Standardization and Quality Assessment Under the Perspective of Automated Computer-Assisted HEp-2 Immunofluorescence Assay Systems.

The recent availability of automated computer-assisted diagnosis (CAD) systems for the reading and interpretation of the anti-nuclear antibody (ANA) test performed with the indirect immunofluorescence (IIF) method on HEp-2 cells, has improved the reproducibility of the results and initiated a process of harmonization of this test. Furthermore, CAD systems provide quantitative expression of fluorescence intensity, allowing the introduction of objective quality control procedures to the monitoring of the entire process. The calibration of the reading systems and the automated image interpretation are essential prerequisites for obtaining reproducible and harmonized IIF test results and form the basis for standardization, regardless of the computer algorithms used in the different systems. The use of automated CAD systems, facilitating control procedures, represents a step forward for the quality certification of the laboratory.

Positive tissue transglutaminase antibodies with negative endomysial antibodies: Unresolved issues in diagnosing celiac disease.

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.

A new diagnostic algorithm for pattern-oriented autoantibody testing according to the ICAP nomenclature: A pilot study.

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.

Anti-DFS70 antibodies detected by specific methods in patients with thrombosis or recurrent pregnancy loss: no evidence of an association.

A dense fine speckled pattern (DFS) caused by antibodies to the DFS70 kDa nuclear protein is a relatively common finding while testing for anti-nuclear antibodies (ANA) by indirect immunofluorescence (IIF) on HEp-2 cells. However, despite many efforts and numerous studies, the clinical significance of anti-DFS70 antibodies is still unknown as they can be found in patients with various disorders and even in healthy subjects. In this study we aimed at verifying whether these antibodies are associated with thrombotic events or with unexplained recurrent pregnancy loss (RPL). We studied 443 patients with venous or arterial thrombosis or RPL and 244 controls by IIF on HEp-2 cells and by a DFS70-specific chemiluminescent immunoassay (CIA). The DFS pattern was observed in IIF in 31/443 (7.0%) patients and in 6/244 (2.5%) controls (p = 0.01) while anti-DFS70 specific antibodies were detected by CIA in 11 (2.5%) patients and in one (0.4%) control (p = 0.06). Positive samples, either by IIF or by CIA, were then assayed by a second DFS70-specific line-immunoassay (LIA) method: 83.3% of the CIA positive samples were confirmed DFS70 positive versus only 29.7% of the IIF positive samples. These findings show that IIF overestimates anti-DFS70 antibody frequency and that results obtained by specific CIA and LIA assays do not indicate that venous or arterial thrombosis or RPL are linked to a higher prevalence of anti-DFS70 antibodies.

Laboratory Monitoring of Biological Therapies in Rheumatology: The Role of Immunogenicity.

Biological drugs, such as proteins and immunogens, are increasingly used to treat various diseases, including tumors and autoimmune diseases, and biological molecules have almost completely replaced synthetic drugs in rheumatology. Although biological treatments such as anti-tumor necrosis factor (TNF) drugs seem to be quite safe, they cause some undesirable effects, such as the onset of infections due to weakening of the immune system. Given the biological nature of these drugs, they might be recognized as extraneous; this would induce an immune reaction that neutralizes their effectiveness or lead to more serious consequences. Laboratories play a pivotal role in appropriate therapeutic management. The aim of this review was to underline the production of anti-drug antibodies during treatment with biological drugs and highlight the role of laboratories in ensuring appropriate use of these drugs.

With antitransglutaminase antibodies in the breast milk, is breastfeeding beneficial or harmful?

Breast milk is fundamental to induce an appropriate immunotolerance in the newborn. Here, we present the case of a young mother who was diagnosed with celiac disease while breastfeeding. She is dealing with the problem of knowing that celiac disease-specific autoantibodies are present in her breast milk, but not knowing if they are potentially harmful to her baby.

Chemiluminescent immunoassay technology: what does it change in autoantibody detection?

Diagnostic technology is rapidly evolving, and over the last decade, substantial progress has been made even for the identification of antibodies, increasingly approaching this type of diagnostic to that of automated clinical chemistry laboratory. In this review, we describe the analytical and diagnostic characteristics of chemiluminescence technology in its strength and in its applicability for a more rapid and accurate diagnosis of autoimmune diseases. The wide dynamic range, greater than that of immunoenzymatic methods, the high sensitivity and specificity of the results expressed in quantitative form, the high degree of automation and the clinical implications related to the reduction in the turnaround time, and the ability to run a large number of antibody tests (even of different isotypes), directed towards large antigenic panels in random access mode, make this technology the most advanced in the clinical laboratory, with enormous repercussions on the workflow and on the autoimmunology laboratory organisation. Further improvements are expected in the coming years with the development of new analytical platforms such as the flow-injection chemiluminescent immunoassay, the two-dimensional resolution for chemiluminescence multiplex immunoassay and the magnetic nanoparticles chemiluminescence immunoassay, which will likely result in additional increases in the clinical efficacy of antibody tests.

Lack of Clinical Relevance of ANA and ASMA Positivity in Patients with Liver Transplantation without a History of Autoimmune Diseases.

The relevance of isolated autoimmunity elevation in orthotopic liver transplantation (OLT) patients is unknown. Our aim was to analyse how serum autoantibodies change in time and to evaluate their clinical relevance in OLT patients. Patients were invited to provide samples to evaluate ANA, AMA, ASMA, and LKM at the time of enrolment (T0), after 6 months (T6), and after 12 months (T12). We included 114 patients in the study (76% males, median age 62.5 years), finding isolated elevation of at least one serum antibody in up to 80% of them. We described fluctuating positive autoantibodies in the one year of observation, with only 45.6% of patients positive for ANA and less than 2% positive for ASMA, at all three times. Isolated elevation of tissue antibodies was not related to gender, age, HCC at transplant, early rejection, cause of transplantation, immunotherapy taken, and age at the time of the study. We did not detect a higher prevalence of positive autoimmunity in patients with signs of liver injury. ANA and ASMA evaluation in patients with liver transplantation and no history of autoimmune disease has no clinical relevance, since it varies in time and is not related to any risk factors or liver injury. Routine autoimmunity evaluation should be avoided.

The ANA-reflex test as a model for improving clinical appropriateness in autoimmune diagnostics.

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