Abstract Crohn's disease (CrD) is a chronic relapsing inflammatory bowel disease (IBD) potentially affecting any portion of the gastrointestinal tract from the mouth to the anus. CrD usually ...manifests between 15 and 30 years of age and presents typically with abdominal pain, fever, bloody or non-bloody diarrhoea, and weight loss. Paediatric patients may show failure to thrive, growth impairment, and delayed puberty additionally. Extraintestinal manifestations like arthritis, uveitis, and erythema nodosum are diagnosed in almost half of the patients. CrD is characterized by a discontinuous and ulcerous transmural inflammation often involving the ileocaecal region and leading to a stricturing or even fistulising phenotype in up to 50% of patients finally. Incidence and prevalence of CrD have been rising worldwide over the past decades. Although many details of the pathophysiology of CrD have been elucidated, no common aetiopathogenic model exists for all forms of CrD, presenting more an umbrella term for a phenotypically and genotypically heterogeneous clinical condition. In CrD, we see an inappropriate response of the innate and/or adaptive immune system to the intestinal microbiota in genetically predisposed individuals. The diagnosis of CrD is based mainly on patient's history and clinical examination and supported by serologic, radiologic, endoscopic, and histologic findings. Antibodies to Saccharomyces cerevisiae and autoantigenic targets such as glycoprotein 2 may aid in differentiating CrD from UC. Their single use, however, is limited by low sensitivity requiring antibody profiling for an appropriate serologic diagnosis. This review focuses on diagnostic and classification criteria of CrD.
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.
During the 12th International Workshop on Autoantibodies and Autoimmunity held in Sao Paulo, Brazil, on August 28, 2014, a full day session was devoted to establishing a consensus on the nomenclature ...of staining patterns observed in the antinuclear antibody (ANA) indirect immunofluorescence test on HEp-2 cells. The current report summarizes the collective agreements with input from the host Brazilian and international communities that represented research, clinical, and diagnostic service laboratories. Patterns are categorized in three major groups (nuclear, cytoplasmic, and mitotic patterns) and each pattern has been defined and described in detail. The consensus nomenclature and representative patterns are made available online at the international consensus on antinuclear antibody pattern (ICAP) website (www.ANApatterns.org). To facilitate continuous improvement and input, specific comments on ICAP are encouraged and these will be discussed in subsequent ICAP meetings. The ultimate goal with the establishment of the ICAP is to promote harmonization and understanding of autoantibody test nomenclature, as well as interpretation guidelines for ANA testing, thereby optimizing usage in patient care.
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.
Abstract Analysis of autoantibodies (AAB) by indirect immunofluorescence (IIF) remains the hallmark of diagnosing autoimmune diseases despite the introduction of multiplex techniques. Non-organ ...specific AAB are screened in routine diagnostics by IIF on HEp-2 cells. However, IIF results vary due to objective (e.g., cell fixation) and subjective factors (e.g., expert knowledge). Therefore, inter- and intralaboratory variance is relatively high. Standardisation of AAB testing by IIF remains a critical issue in and between routine laboratories and may be improved by automated interpretation systems. An overview of existing interpretation techniques will be given taking into account own data of the first fully automated reading system AKLIDES. The novel system provides fully automated reading of IIF images and software algorithms for the mathematical description of IIF AAB patterns. It can be used for screening and preclassification of non-organ specific AAB in routine diagnostics regarding systemic autoimmune and autoimmune liver diseases. Furthermore, this system paves the way for economic data processing of cell-based IIF assays and can contribute to the reduction of interlaboratory variance of AAB testing. More sophisticated pattern recognition algorithms and novel calibration systems will improve standardised quantifications of IIF image interpretation.
Abstract An increasing number of rheumatoid arthritis (RA)-associated autoantibodies (AAB) are available for AAB profiling that may improve the early diagnosis of RA and provide prognostic and, ...probably theranostic information. To select AAB specificities for optimal AAB combinations, known AAB should be evaluated with standardized methods by means of standardized study designs and subjected to statistical analysis. Profiling of anti-citrullinated peptide/protein antibodies (ACPA), anti-A2/RA-33 antibodies, and rheumatoid factors (RF) IgM and IgA improves the serologic diagnosis of RA using cut-offs corresponding to 98% specificity for each parameter included. Currently, the combination of anti-CCP antibodies with RF IgM and RF IgA is recommended either in parallel or by stepwise determination. Because anti-CCP antibody demonstrated the best diagnostic performance for profiling, this AAB should be used for first-line screening. The main advantage of AAB profiling is the increased sensitivity for RA diagnosis. Additional benefits of AAB profiles comprise the increased diagnostic specificity of particular AAB combinations (e.g., ACPA plus RF or RF IgM plus RF IgA) and their possible association with disease development and/or therapy response. The inclusion of novel RA-associated AAB (RAAB) and use of clinically evaluated multiplex assays may facilitate the use of profiling in diagnostic routine laboratories.
Neutrophil extracellular traps (NETs) are chromatin filaments decorated with enzymes from neutrophil cytoplasmic granules. Anti-neutrophil cytoplasmic antibodies (ANCAs) bind to enzymes from ...neutrophil cytoplasmic granules and are biomarkers for the diagnosis of systemic vasculitides. ANCA diagnostics are based on indirect immunofluorescence (IIF) of ethanol-fixed neutrophils. IIF shows a cytoplasmic staining pattern (C-ANCA) due to autoantibodies against proteinase 3 (PR3) or a perinuclear staining pattern (P-ANCA) due to autoantibodies against myeloperoxidase (MPO). The distinct ANCA-staining patterns are an artifact of ethanol fixation. Here, we tested NETs as a substrate for the detection of ANCAs in human sera. We observed that P-ANCAs specifically stained NETs, while C-ANCAs targeted the cell bodies of netting neutrophils. The distinct ANCA-staining patterns were caused by the presence of MPO, but not PR3, in NETs. Using NETs as a substrate for IIF, we characterized ANCAs in sera of patients with ANCA-associated vasculitis (AAV). Furthermore, we inhibited serine proteases by diisopropylfluorophosphate to prevent chromatin unfolding and the release of NETs and thus generated neutrophils with MPO-positive nuclei and PR3-positive cytoplasm, which resembled the appearance of ethanol-fixed neutrophils. In conclusion, our data suggest that NETs are selectively loaded with antigens recognized by P-ANCAs, and netting neutrophils provide a physiological substrate for ANCA detection in patients with AAV.
We determined the prevalence of anti-nuclear autoantibodies (ANAs) in the German adult population and examined the association between ANAs and cardiovascular and metabolic disorders.
We used data ...and blood samples from the pretest phases of the German National Cohort, obtained from six of the 18 study centers (n = 1199). All centers applied standardized instruments including face-to-face interviews, anthropometric measurements and collection of blood samples. Self-reported histories of diabetes mellitus, heart attack and elevated blood cholesterol and/or lipids were recorded. Height, weight and blood pressure were measured. ANAs were detected using a semi-automated system (AKLIDES®; Medipan GmbH, Dahlewitz, Germany). A positive ANA was defined as a titer ≥ 1:80. ANA were classified as weakly (1:80 or 1:160), moderately (1:320 or 1:640) or strongly (≥1:1280) positive. Specific autoantibodies against nuclear antigens were detected with second-step assays according to the ANA staining pattern. Associations between the assessed disorders and ANA positivity and pattern were examined using sex and age-adjusted mixed-effects logistic regression models.
Thirty-three percent (95% confidence interval; 31-36%) of the 1196 participants (measurements could not be obtained from three samples) were ANA positive (titer ≥ 1:80). The proportions of weakly, moderately and strongly positive ANA were 29%, 3.3% and 1.3%, respectively. ANA positivity was more common among women than men across all titers (χ
, p = 0.03). ANA positivity, even when stratified according to height of titer or immunofluorescent pattern, was not associated with diabetes, elevated blood cholesterol and/or lipids, obesity or hypertension. Second-step autoantibody assays were positive in 41 of the 83 samples (49%) tested, with anti-DFS70 (n = 13) and anti-dsDNA (n = 7) being most frequent. These subgroups were too small to test for associations with the disorders assessed.
The prevalence of ANA positivity in the German general population was similar to values reported from other countries. Contrary to other studies, there was no association with selected self-reported and objectively measured cardiovascular and metabolic variables.
Abstract Asialoglycoprotein receptor (ASGPR) has attracted the attention of liver immunologists for many years. This liver-specific lectin was found to be a major B and T cell autoantigenic target in ...patients with autoimmune liver diseases, and in particular in autoimmune hepatitis (AIH). This review discusses the biological significance of ASGPR and its relevance to the pathogenesis of autoimmune and virus-triggered liver diseases. We also discuss emerging data on the diagnostic and clinical relevance of anti-ASGPR antibodies in light of recent reports based on commercially available anti-ASGPR enzyme-linked immunosorbent assays. Finally, we critically revisit the data reporting on disease-specific cellular immune responses against ASGPR and their relevance in relation to the pathogenesis of AIH.
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