IntroductionAnalysis of autoantibodies (AAB) by indirect immunofluorescence (IIF) is a basic tool for the serological diagnosis of systemic rheumatic disorders. Automation of autoantibody IIF reading including pattern recognition may improve intra- and inter-laboratory variability and meet the demand for cost-effective assessment of large numbers of samples. Comparing automated and visual interpretation, the usefulness for routine laboratory diagnostics was investigated.MethodsAutoantibody detection by IIF on human epithelial-2 (HEp-2) cells was conducted in a total of 1222 consecutive sera of patients with suspected systemic rheumatic diseases from a university routine laboratory (n = 924) and a private referral laboratory (n = 298). IIF results from routine diagnostics were compared with a novel automated interpretation system.ResultsBoth diagnostic procedures showed a very good agreement in detecting AAB (kappa = 0.828) and differentiating respective immunofluorescence patterns. Only 98 (8.0%) of 1222 sera demonstrated discrepant results in the differentiation of positive from negative samples. The contingency coefficients of chi-square statistics were 0.646 for the university laboratory cohort with an agreement of 93.0% and 0.695 for the private laboratory cohort with an agreement of 90.6%, P < 0.0001, respectively. Comparing immunofluorescence patterns, 111 (15.3%) sera yielded differing results.ConclusionsAutomated assessment of AAB by IIF on HEp-2 cells using an automated interpretation system is a reliable and robust method for positive/negative differentiation. Employing novel mathematical algorithms, automated interpretation provides reproducible detection of specific immunofluorescence patterns on HEp-2 cells. Automated interpretation can reduce drawbacks of IIF for AAB detection in routine diagnostics providing more reliable data for clinicians.
BackgroundRecurrent airway obstruction (RAO), an asthma‐like disease, is 1 of the most common allergic diseases in horses in the northern hemisphere. Hypersensitivity reactions to environmental antigens cause an allergic inflammatory response in the equine airways. Cytosine‐phosphate‐guanosine‐oligodeoxynucleotides (CpG‐ODN) are known to direct the immune system toward a Th1‐pathway, and away from the pro‐allergic Th2‐line (Th2/Th1‐shift). Gelatin nanoparticles (GNPs) are biocompatible and biodegradable immunological inert drug delivery systems that protect CpG‐ODN against nuclease degeneration. Preliminary studies on the inhalation of GNP‐bound CpG‐ODN in RAO‐affected horses have shown promising results.ObjectivesThe aim of this study was to evaluate the clinical and immunological effects of GNP‐bound CpG‐ODN in a double‐blinded, placebo‐controlled, prospective, randomized clinical trial and to verify a sustained effect post‐treatment.Animals and MethodsTwenty‐four RAO‐affected horses received 1 inhalation every 2 days for 5 consecutive administrations. Horses were examined for clinical, endoscopic, cytological, and blood biochemical variables before the inhalation regimen (I), immediately afterwards (II), and 4 weeks post‐treatment (III).ResultsAt time points I and II, administration of treatment rather than placebo corresponded to a statistically significant decrease in respiratory effort, nasal discharge, tracheal secretion, and viscosity, AaDO 2 and neutrophil percentage, and an increase in arterial oxygen pressure.Conclusion and Clinical ImportanceAdministration of a GNP‐bound CpG‐ODN formulation caused a potent and persistent effect on allergic and inflammatory‐induced clinical variables in RAO‐affected horses. This treatment, therefore, provides an innovative, promising, and well‐tolerated strategy beyond conventional symptomatic long‐term therapy and could serve as a model for asthma treatment in humans.
IntroductionDiagnosis of antiphospholipid syndrome (APS) still remains a laboratory challenge due to the great diversity of antiphospholipid antibodies (aPL) and their significance regarding APS-diagnostic criteria.MethodsA multi-line dot assay (MLDA) employing phosphatidylserine (PS), phosphatidylinositol (PI), cardiolipin (CL), and beta2-glycoprotein I (β2 GPI) was used to detect aPL, immunoglobulin G (IgG) and immunoglobulin M (IgM) in 85 APS patients, 65 disease controls, and 79 blood donors. For comparison, anti-CL and anti-β2 GPI IgG and IgM were detected by enzyme-linked immunosorbent assay (ELISA).ResultsThe level of agreement of both methods was good for anti-CL IgG, moderate for anti-CL IgM, very good for anti-β2 GPI IgG, and moderate for anti-β2 GPI IgM (kappa = 0.641, 0.507, 0.803 and 0.506, respectively). The frequency of observed discrepancies for anti-CL IgG (1.75%), anti-CL IgM (3.93%), anti-β2 GPI IgG (1.75%), and anti-β2 GPI IgM (0.87%) was low (McNemar test, P < 0.05, not-significant, respectively). Sensitivity, specificity, positive (+LR) and negative (-LR) likelihood ratios for at least one positive aPL antibody assessed by ELISA were 58.8%, 95.8%, 14.1, and 0.4, respectively, and for at least three positive aPl IgM and/or one positive aPL IgG by MLDA were 67.1%, 96.5%, 19.3, and 0.3, respectively. The frequency of IgM to PI, PS and CL, and combination of three or more aPL IgM detected by MLDA was significantly higher in APS patients with cerebral transient ischemia (P < 0.05, respectively).ConclusionsThe novel MLDA is a readily available, single-step, sensitive diagnostic tool for the multiplex detection of aPL antibodies in APS and a potential alternative for single aPL antibody testing by ELISA.
Background In patients with rheumatoid arthritis (RA), seropositivity for either rheumatoid factor or anti-CCP antibodies has been identified as a predictive marker for treatment response to rituximab (RTX) in different studies. However, it is unclear, whether analysis of fine-reactivity against certain epitopes of citrullinated antigens or immunoglobulin subclass reactivity can contribute to the prediction of treatment response to RTX. Objectives To investigate whether reactivity patterns against epitopes of the mutated citrullinated vimentin (MCV) antigen and/or anti-MCV subclass reactivities can contribute to differentiation of responders to RTX. Methods Fine-reactivity against MCV was investigated in anti-MCV antibody positive patients with RA (n=34) under treatment with RTX. According to EULAR response criteria, 23 patients were classified as responders by reaching remission as defined by DAS28 <2.6 or low disease activity as defined by DAS28 <3.2. Follow-up analyses of anti-MCV response were performed using 88 synthetic overlapping MCV peptides in ELISA. The peptides used contained all known modifications of vimentin by mutation and citrullination1. In addition, influence of RTX treatment on anti-MCV IgG, IgM and IgA antibody reactivities was investigated by ELISA. Results At baseline or during follow-up, none of the responders showed an anti-MCV IgA antibody response. In contrast, non-responders were positive for anti-MCV IgA in 50% of the cases at baseline and showed no reduction of antibody subclass reactivities in the follow-up. Furthermore, anti-MCV antibody subclass reactivities showed that RTX-responders were characterized by a reduction of anti-MCV IgG antibody titers, and by a normalization of anti-MCV IgM titers in all initially positive cases. At baseline, subsequent responders to RTX treatment were characterized by only a minor different epitope recognition pattern compared to non-responders. Remarkably upon treatment, a reduction of the number of recognized MCV epitopes was observed in 16 out of 23 responders (69.6%) in contrast to 3 out of 11 (27.3%) non-responders in follow-up analyses. Conclusions Responders to RTX were characterized by a restricted immunoglobulin subclass reactivity against MCV lacking IgA anti-MCV antibodies at baseline, and a reduction of anti-MCV antibody titers as well as the epitope recognition pattern under treatment. Fine-reactivity against the MCV antigen can potentially contribute as a useful marker to predict response to RTX in anti-MCV positive RA patients. References Bang H, Egerer K, Gauliard A, Luthke K, Rudolph PE, Fredenhagen G, Berg W, Feist E, Burmester GR. Mutation and citrullination modifies vimentin to a novel autoantigen for rheumatoid arthritis. Arthritis Rheum. 2007 Jul 30;56(8):2503-2511 Disclosure of Interest L. Naumann Grant/Research support from: Roche Pharma AG, H. Bang Employee of: Orgentec, K. Egerer: None Declared, D. Meyer zum Büschenfelde: None Declared, B. Lehmann: None Declared, G. Fredenhagen Employee of: Orgentec, H. Bastian: Non...
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