Abstract Fully automated multiplex immunoassays are increasingly used as first line screening for antinuclear antibodies. The diagnostic performance of such multiplex assays in untreated patients at ...the time of diagnosis has not been reported. Antinuclear antibodies were measured by indirect immunofluorescence (IIF) (dilution 1:160) and by BioPlex 2200 ANA screen (antibodies to dsDNA, chromatin, ribosomal protein, SSA-52, SSA-60, SSB, Sm, SmRNP, RNP-A, RNP-68, Scl-70, Jo-1, and centromere B) in 236 patients with a systemic rheumatic disease at the time of diagnosis, 149 blood donors, 139 patients with chronic fatigue syndrome (CFS), and 134 diseased controls. BioPlex ANA screen and IIF were positive in, respectively, 79% and 90% of patients with systemic lupus erythematosus (SLE), 60% and 60% with cutaneous lupus, 72% and 93% with systemic sclerosis (SSc), 100% and 100% with mixed connective tissue disease (MCTD), 89% and 56% with primary Sjögren's (SS) syndrome, 36% and 36% with polymyositis/dermatomyositis, 5.4% and 6% of blood donors, 7.2% and 3.6% of patients with CFS, and 11% and 18% of diseased controls. BioPlex test result interval specific likelihood ratios increased with increasing antibody concentration. The simultaneous presence of at least three antibodies by BioPlex was found in 35% of patients with SLE, 4% with SSc, 100% with MCTD, 64% with SS, 7% with inflammatory myopathy, 0.7% of CFS and diseased controls, and none of the blood donors. In conclusion, test result specific likelihood ratios and the presence of multiple autoantibodies help with the interpretation of data generated by multiplex immunoassays.
Immunofluorescence (IF) is an important immunochemical technique that allows for detection and localization of a wide variety of antigens in different types of tissues of various cell preparations. ...IF allows for excellent sensitivity and amplification of signal in comparison to immunohistochemistry, employing various microscopy techniques. There are two methods available, depending on the scope of the experiment or the specific antibodies in use: direct (primary) or indirect (secondary). Here, we describe preparation of specimens preserved in different types of media and step-by-step methods for both direct and indirect immunofluorescence staining.
The indirect immunofluorescence assay (IIFA) on HEp-2 cells is widely used for detection of antinuclear antibodies (ANA). The dichotomous outcome, negative or positive, is integrated in diagnostic ...and classification criteria for several systemic autoimmune diseases. However, the HEp-2 IIFA test has much more to offer: besides the titre or fluorescence intensity, it also provides fluorescence pattern(s). The latter include the nucleus and the cytoplasm of interphase cells as well as patterns associated with mitotic cells. The International Consensus on ANA Patterns (ICAP) initiative has previously reached consensus on the nomenclature and definitions of HEp-2 IIFA patterns. In the current paper, the ICAP consensus is presented on the clinical relevance of the 29 distinct HEp-2 IIFA patterns. This clinical relevance is primarily defined within the context of the suspected disease and includes recommendations for follow-up testing. The discussion includes how this information may benefit the clinicians in daily practice and how the knowledge can be used to further improve diagnostic and classification criteria.
The vascular endothelium responds to the shear stress generated by blood flow and changes function to maintain tissue homeostasis and adapt to injury in pathological conditions. Shear stress in the ...retinal circulation is altered in patients with retinal vascular diseases, such as diabetic retinopathy. Therefore, we aimed to study the effect of laminar shear stress on barrier properties and on the release of proinflammatory cytokines in human retinal microvascular endothelial cells (HRMEC). HRMEC were cultured in Ibidi flow chambers and exposed to laminar shear stress (0–50 dyn/cm2) for 24–48 h. Tight junction distribution (ZO-1 and claudin-5) and cytokine production were determined by immunofluorescence and ELISA, respectively. The chemotactic effect of conditioned media exposed to shear stress was determined by measuring lymphocyte transmigration in Transwells. We found that cells exposed to moderately low shear stress (1.5 and 5 dyn/cm2) showed enhanced distribution of membrane ZO-1 and claudin-5 and decreased production of the proinflammatory cytokines IL-8, CCL2, and IL-6 compared to static conditions and high shear stress values. Moreover, conditioned media from cells exposed to low shear stress, had the lowest chemotactic effect to recruit lymphocytes compared to conditioned media from cells exposed to static and high shear stress conditions. In conclusion, high shear stress and static flow, associated to impaired retinal circulation, may compromise the inner blood retinal barrier phenotype and barrier function in HRMEC.
•CandModerately low shear stress enhanced membrane ZO-1 and claudin-5 and decreased production of IL-8, CCL2, and IL-6 compared to static and high shear stress conditions.•Shear stress modulates iBRB function, highlighting that static culture does not allow proper iBRB function in vitro.
Abstract Background This retrospective study investigated the clinical utility of a positive antinuclear antibody (ANA) test performed outside of the rheumatology setting. Prior studies have ...investigated the frequency of ANA positivity within the general population. The purpose of this investigation was to evaluate the clinical utility of a positive ANA test result in a real-world setting by reviewing the final diagnoses of patients who were referred to a tertiary rheumatology clinic for evaluation of a positive ANA test result. Methods We reviewed the records of patients presenting to the authors between July 2007 and July 2009. Patients were included in the evaluation if they were referred for a positive ANA test result. All relevant descriptive and laboratory data were collated, as were the initial reasons for ordering ANA testing and the ultimate diagnoses reached. Positive predictive values for a “positive ANA test result” were calculated for all antinuclear antibody-associated rheumatic diseases and for lupus specifically. Results A total of 232 patients were referred for a positive ANA test result. The positive predictive value of a positive ANA test result in this cohort was 2.1% for lupus and 9.1% for any antinuclear antibody-associated rheumatic disease. No antinuclear antibody-associated rheumatic disease was identified in patients with an ANA < 1:160. The most common reason for ordering ANA testing was widespread pain (54/232, 23.2%). Conclusions In this retrospective study, more than 90% of patients who were referred to a tertiary rheumatology clinic for a positive ANA test result had no evidence for an ANA-associated rheumatic disease. The poor predictive value of a positive ANA in this cohort was largely attributable to unnecessary testing in patients with low pretest probabilities for ANA-associated rheumatic disease.
Objective
Growing evidence suggests increasing frequencies of autoimmunity and certain autoimmune diseases, but findings are limited by the lack of systematic data and evolving approaches and ...definitions. This study was undertaken to investigate whether the prevalence of antinuclear antibodies (ANA), the most common biomarker of autoimmunity, changed over a recent 25‐year span in the US.
Methods
Serum ANA were measured by standard indirect immunofluorescence assays on HEp‐2 cells in 14,211 participants age ≥12 years from the National Health and Nutrition Examination Survey, with approximately one‐third from each of 3 time periods: 1988–1991, 1999–2004, and 2011–2012. We used logistic regression adjusted for sex, age, race/ethnicity, and survey design variables to estimate changes in ANA prevalence across the time periods.
Results
The prevalence of ANA was 11.0% (95% confidence interval 95% CI 9.7–12.6%) in 1988–1991, 11.5% (95% CI 10.3–12.8%) in 1999–2004, and 15.9% (95% CI 14.3–17.6%) in 2011–2012 (P for trend < 0.0001), which corresponds to ~22 million, ~27 million, and ~41 million affected individuals, respectively. Among adolescents age 12–19 years, ANA prevalence increased substantially, with odds ratios (ORs) of 2.02 (95% CI 1.16–3.53) and 2.88 (95% CI 1.64–5.04) in the second and third time periods relative to the first (P for trend < 0.0001). ANA prevalence increased in both sexes (especially in men), older adults (age ≥50 years), and non‐Hispanic whites. These increases in ANA prevalence were not explained by concurrent trends in weight (obesity/overweight), smoking exposure, or alcohol consumption.
Conclusion
The prevalence of ANA in the US has increased considerably in recent years. Additional studies to determine factors underlying these increases in ANA prevalence could elucidate causes of autoimmunity and enable the development of preventative measures.
Thrombospondin type 1 domain-containing 7A (THSD7A) is a target antigen identified in adult membranous nephropathy (MN) along with the major antigen phospholipase A
receptor 1 (PLA
R1). The ...prevalence of THSD7A-Ab-positive patients is unknown, and it is unclear whether the clinical presentation differs between patients positive for PLA
R1-Ab or THSD7A-Ab. We screened serum samples of 1276 patients with MN from three different cohorts for the presence of THSD7A-Ab by Western blot analysis and a newly developed indirect immunofluorescence test (IFT). Compared with Western blot analysis, the IFT had a 92% sensitivity and a 100% specificity. The prevalence of THSD7A-associated MN in a prospective cohort of 345 patients with MN was 2.6%, and most were women. In this cohort, the percentage of patients with THSD7A-associated MN and malignant disease significantly exceeded that of patients with PLA
R1-associated MN and malignant disease. In all cohorts, we identified 40 patients with THSD7A-associated MN, eight of whom developed a malignancy within a median time of 3 months from diagnosis of MN. In one patient with THSD7A-associated MN and metastases of an endometrial carcinoma, immunohistochemistry showed THSD7A expression on the metastatic cells and within follicular dendritic cells of the metastasis-infiltrated lymph node. We conclude that the IFT allows sensitive and specific measurement of circulating THSD7A-Ab in patients with MN. Patients with THSD7A-associated MN differ in their clinical characteristics from patients with PLA
R1-associated MN, and more intensive screening for the presence of malignancies may be warranted in those with THSD7A-associated MN.
The detection of autoantibodies that target intracellular antigens, commonly termed anti-nuclear antibodies (ANA), is a serological hallmark in the diagnosis of systemic autoimmune rheumatic diseases ...(SARD). Different methods are available for detection of ANA and all bearing their own advantages and limitations. Most laboratories use the indirect immunofluorescence (IIF) assay based on HEp-2 cell substrates. Due to the subjectivity of this diagnostic platform, automated digital reading systems have been developed during the last decade. In addition, solid phase immunoassays using well characterized antigens have gained widespread adoption in high throughput laboratories due to their ease of use and open automation. Despite all the advances in the field of ANA detection and its contribution to the diagnosis of SARD, significant challenges persist. This review provides a comprehensive overview of the current status on ANA testing including automated IIF reading systems and solid phase assays and suggests an approach to interpretation of results and discusses meeting the problems of assay standardization and other persistent challenges.
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