Recent advances in genomic methodologies have significantly enhanced our understanding of immune-mediated rheumatic diseases. Specific structural variants (SVs), such as substantial DNA deletions or insertions, including chromosomal aberrations, have been implicated in diseases of immune dysregulation. Regrettably, SVs are frequently overlooked in next-generation sequencing (NGS) targeted-gene panels, whole exome sequencing (WES) and whole genome sequencing (WGS). In view of a case of chromosome 18p deletion syndrome, characterized by hypogammaglobulinemia and an autoinflammatory phenotype, we provide a comprehensive review on chromosome aberrations associated with multiple immune-mediated conditions, highlighting the clinical aspects of the various chromosome aberrations associated with immune-mediated diseases. Further investigations and development of functional tests should contribute to elucidate the mechanistic connection between chromosome aberrations and Primary Immune Regulatory Disorders (PIRD), bringing novel perspectives in the field of autoinflammatory and autoimmune diseases.

OBJECTIVE:This study was undertaken to describe a case series of Brazilian new onset refractory status epilepticus (NORSE) patients and evaluate the sensitivity and specificity of a clinical score to predict cryptogenic etiology (c-NORSE score) after autoimmune encephalitis (AE) testing. METHODS:Thirty-seven patients with NORSE from the Brazilian Autoimmune Encephalitis Network were investigated with brain magnetic resonance imaging (MRI), cerebrospinal fluid (CSF) analysis, and complementary testing with tissue-based assays and cell-based assays for antineuronal and antiglial antibodies (abs) in serum/CSF. Final diagnoses were compiled after chart review. Patients were allocated into two groups according to c-NORSE score: (≥5) high score (HS) or (<5) low score (LS), and clinical variables were compared. c-NORSE score sensitivity and specificity were calculated. RESULTS:We found that 23 (62%) of the NORSE patients were children, 49% were female, 22% had AE, and 51% were classified as c-NORSE. Eleven patients (30%) were allocated to the HS group and 26 (70%) to LS. Bilateral and symmetric MRI findings were more frequent in the HS group (HS 100% vs. LS 43%, p = .018), whereas memory or behavioral symptoms were less common (HS 27% vs. LS 89%, p = .001). All HS patients had c-NORSE, whereas 69% of the LS patients had other diagnoses, such as AE (n = 8), herpes simplex virus encephalitis (n = 4), paraneoplastic encephalomyelitis (n = 1), acute disseminated encephalomyelitis (n = 1), and other causes (n = 4). The sensitivity of the c-NORSE score for predicting cryptogenic cases was 57.9% (95% confidence interval [CI] = 36%-80%), and the specificity was 100% (95% CI = 84%-100%). SIGNIFICANCE:In this cohort, AE was the most identified cause of NORSE, and 51% were cryptogenic. c-NORSE score ≥ 5 had a specificity of 100% (95% CI = 84%-100%) for identifying cryptogenic cases, whereas a score < 5 indicates additional investigation should be ordered. Although the sensitivity of the c-NORSE score was lower than previously reported, suggesting it may vary depending on complementary investigation, it is a useful tool for bedside NORSE evaluation in low-income countries, where access to antineuronal abs is limited.

Background The VII Brazilian Consensus on Autoantibodies against HEp-2 Cells (BCA/HEp-2), dedicated to the identification of autoantibodies targeting cellular constituents in HEp-2 cells, was held on October 5–6, 2023, in Goiânia (GO, Brazil), as part of the 40th Brazilian Congress of Rheumatology. Main body Nineteen researchers and experts from universities and clinical laboratories participated in this meeting, which resulted in important advancements. Among the key updates, two new nuclear patterns were incorporated into the Brazilian classification tree: the nuclear fine speckled with mitotic plate (AC-30) and the myriad discrete speckled pattern strongly associated with antibodies to SS-A/Ro60, previously categorized as AC-4a and now recognized as AC-31. Additionally, the mitotic pattern AC-28 (mitotic chromosomal), characterized by punctate staining of chromosomes in pro- and metaphase stages without interphase cell staining, was included. Adjustments to the classification tree were made to redistribute patterns based on morphological characteristics, harmonize composite pattern designations with ICAP nomenclature, and incorporate the AC-XX group into the classification structure. Guidance was also provided to laboratory analysts on the limitations of identifying patterns with weak reactivity, recommending that reports in such cases be issued according to the appropriate level of analytical competency. Conclusion The recommendations aimed to establish clear guidelines for laboratories and clinicians, building upon those from the V and VI Consensuses while further aligning with ICAP recommendations. This consensus marks a significant step forward in adapting ICAP guidelines to the Brazilian context, refining the classification tree and addressing practical challenges faced by the scientific and clinical community.

The indirect immunofluorescence assay (IFA) on HEp-2 cells is the prevailing method used to screen for autoantibodies in the investigation of systemic autoimmune diseases (SAID). When positive, the titer provides a semi-quantitative assessment of the autoantibody serum concentration whereas the immunofluorescence pattern indicates the possible autoantibody specificities. The Brazilian Consensus on ANA Patterns (BCA) and the International Consensus on ANA Patterns (ICAP) provide recommendations for the harmonization on the pattern nomenclature and test reporting. Nuclear patterns are among the most frequent in the clinical laboratory and some of them are highly relevant in the diagnosis of SAID. Nuclear patterns with stained metaphase plate (MP) indicate autoantibodies against chromatin components or against chromatin-bound antigens. These include the nuclear homogeneous (AC-1), nuclear dense fine speckled (AC-2), Topo 1-like (AC-29), and nuclear fine speckled with stained MP (AC-30) patterns. The Brazilian consensus has also classified the quasi-homogeneous nuclear pattern (QH). The correct identification of these patterns is important because each one is associated with different autoantibody specificities and clinical scenarios. However, the recognition of the nuances in texture of the staining pattern and other specific features that characterize each of them may be challenging for the analyst at the microscope. This review focuses on the morphological characteristics, immunological identities, and clinical relevance of nuclear patterns with stained MP. The aim is to assist laboratory analysts and clinicians in identifying and interpreting these patterns, thus optimizing the use of the HEp-2 IFA test in the investigation of patients under suspicion of SAID.

External quality assurance (EQA) programs play a pivotal role in monitoring laboratory practices, allowing each laboratory to evaluate the consistency of results across different methods as well the ability of individual laboratories to compare and improve over time their own performance.The objective of our study was to analyze the UK NEQAS EQA reports for the "Antibodies to Nuclear and Related Antigens" program from 2013 to 2023, to assess the overall level of harmonization of the responses for anti-nuclear antibody (ANA) testing by indirect immunofluorescence (IIF), in terms of both pattern and titer consensus. As a second aim, we analyzed the impact of the introduction in UK NEQAS EQA reports of the International Consensus on ANA Patterns (ICAP) nomenclature and of digital image recognition on the harmonization of the ANA HEp-2 IIF test.The percentage of consensus for positive/negative results was significantly higher (90.9 +/- 1.4) in 2023 than in 2013 (64.0 +/- 7.8, p <0.001). Common to all years in the investigated period, consensus on pattern recognition was significantly lower for the homogenous pattern (70.5 +/- 16.0) compared to the centromere (84.9 +/- 14.9), the speckled (90.3 +/- 12.3), and the negative (84.5 +/- 18.6, p <0.001) samples, while it was significantly higher for titers 1:80-1:320 than for titers >1:320 (p <0.001).The difference between manual reading and digital reading was not significant (93.8 % vs. 92.4 %; p =0.078), but it was significant between the pre- and post-use of the ICAP nomenclature (82.6 % vs. 93.8 %; p <0.001).This study shows that the variability in ANA recognition and reporting is pattern (homogeneous >speckled >centromere) and titer (high titer >low titer) dependent. While we did not find any difference between the use of manual reading compared to digital reading, the adoption of the ICAP nomenclature greatly improved the harmonization of ANA reporting.

Introduction MOGAD encephalitis and ADEM share several clinical features with autoimmune encephalitis (AE) associated with antineuronal antibodies (ANeA); nonetheless, treatment and prognosis differ. Anti-MOG antibodies (abs) are not routinely tested in possible AE, and epidemiological studies on MOGAD encephalitis are scarce. Objectives To determine the frequency of anti-MOG abs in the serum and CSF in a cohort of possible AE and to compare the clinical characteristics of MOGAD patients and those with seropositive AE. Methods 481 patients with possible AE from the Brazilian Autoimmune Encephalitis Network underwent tissue-based assay and cell-based assay (CBA) for ANeA. Anti-MOG abs were assessed in serum and CSF with in-house CBA. Clinical and laboratory characteristics of MOGAD and seropositive AE patients were compared. Results Of the 481 patients, 87 (18%) had ANeA, and 17 (3.5%) had anti-MOG abs. Three AE patients with anti-MOG abs and ANeA were excluded from further analysis. Anti-MOG abs were detected in 4 (1.2%) of the 328 adults and 10 (6.5%) of the 153 children. Of the 14 patients with MOGAD, nine had ADEM (mostly children), and five had encephalitis (including three adults). Only one patient with ADEM had anti-MOG abs exclusively in CSF. All patients with MOGAD encephalitis were seropositive for anti-MOG abs, and three had normal brain MRI. Patients with MOGAD had fewer behavioral changes (MOGAD 21% x AE 96%, p ≤ 0.0001) and movement disorders (MOGAD 42% x AE 81%, p=0.0017) and more demyelinating symptoms, such as myelitis and optic neuritis (MOGAD 14% x AE 0%, p=0.013). Conclusion Approximately 3.5% of patients with possible AE harbor anti-MOG abs, and 0.9% of the adults had MOGAD encephalitis. Anti-MOG abs were more frequent than other ANeAs regularly tested in AE. We provide evidence that MOGAD is a differential diagnosis in possible AE, even in adult patients with normal brain MRI, and that serum anti-MOG should be considered as an add-on diagnostic tool in AE among adults and pediatric patients.

Background/Objective: Myasthenia Gravis (MG) is an autoimmune disorder characterized by pathogenic autoantibodies (AAbs) targeting nicotinic acetylcholine receptors (AChR), disrupting neuromuscular communication. RadioImmunoPrecipitation Assay (RIPA) is recommended to detect AChR AAbs, but its complexity and radioactive requirements limit widespread use. We compare non-RIPA anti-AChR immunoassays, including Cell-Based Assay (CBA) and two ELISA kits, against the gold standard RIPA. Methods/Results: 145 samples were included with medical indication for anti-AChR testing. By the RIPA method, 63 were negative (RIPA-Neg < 0.02 nmol/L), 18 were classified as Borderline (≥0.02 –1 nmol/L), and 64 were positive (RIPA-Pos > 1 nmol/L). The competitive ELISA showed poor agreement with RIPA (Kappa = 0.216). The indirect ELISA demonstrated substantial agreement with RIPA (Kappa = 0.652), with ∼76% sensitivity and ∼94% specificity for MG diagnostic. The CBA, where fixed cells expressing clustered AChR were used as substrate, exhibited almost perfect agreement with RIPA (Kappa = 0.984), yielding ∼98% sensitivity and 96% specificity for MG. In addition, a semiquantitative analysis showed a strong correlation between CBA titration, indirect ELISA, and RIPA levels (r = 0.793 and r = 0.789, respectively). Conclusions: The CBA displayed excellent analytical performance for MG diagnostic when compared to RIPA, making it a potential replacement for RIPA in clinical laboratories. Some solid-phase assays (such as the indirect ELISA applied here), as well as CBA titration, offer reliable options to estimate anti-AChR AAb levels after confirming positivity by the CBA.∥

AbstractBackground/ObjectivesAnti-centromere antibodies are associated with limited cutaneous Systemic Sclerosis (lcSSc) and in general, with a more favorable prognosis for SSc. Centromere pattern (AC-3) observed in the immunofluorescence assay in HEp-2 cells (HEp-2-IFA) suggests the presence of antibodies against CENP antigens, mainly CENP-B/A. In this study, we evaluated the frequency of reactivity to CENP-B and CENP-A in samples with an AC-3 pattern in the HEp-2-IFA. We also correlated the demographic and clinical characteristics of SSc patients according to the presence of the AC-3 pattern.MethodsSamples from 39 SSc patients with AC-3 pattern were included and evaluated for CENP-B/A reactivity by line-blot and indirect ELISA. As controls, 48 lcSSc without the AC-3 pattern (Non-AC-3 group) were included. Clinical characteristics were recovered for 69 patients, 21 with AC-3 and all those in the Non-AC-3 group.ResultsOut of 39 samples with AC-3, 33 (84.6%) were reactive against CENP-B and 32 (82.1%) were reactive against CENP-A using the line-blot assay. Using anti-CENP-B ELISA, 36 (92.3%) of the samples with AC-3 were positive for anti-CENP-B. There was 79.5% concordance for CENP-B reactivity between ELISA and line-blot. Using the immunoblot assay, 27 (69.2%) of the samples with AC-3 were reactive against CENP-B and CENP-A, and one sample was positive only for CENP-A. Altogether, 38 samples (97.4%) were reactive against CENP-B by at least one method and all 39 samples (100%) were positive for either CENP-B or CENP-A in at least one of the tests. Regarding the clinical features, interstitial lung disease was less frequent in patients with AC-3 pattern compared to Non-AC-3 (10%versus54.2%; p=0.001). Other organ involvement parameters had similar frequencies between the groups.ConclusionsIn conclusion, the HEp-2-IFA method was 100% sensitive in detecting antibodies to CENP-A and CENP-B. Anti-CENP-B is the predominant autoantibody in samples yielding the AC-3 pattern, but exclusive anti-CENP-A reactivity was observed less frequently. Among lcSSc patients, anti-centromere reactivity was associated with less frequent lung involvement.

The International Consensus on ANA Patterns (ICAP) is an ongoing international initiative dedicated to harmonizing technical and interpretation aspects of the HEp-2 IFA test. Comprised of internationally recognized experts in autoimmunity and HEp-2 IFA testing, ICAP has operated for the last 10 years by promoting accurate reading, interpretation, and reporting of HEp-2 IFA images by professionals involved in various areas related to autoimmune diseases, such as clinical diagnostic laboratories, academic research, IVD industry, and patient care. ICAP operates through continuous information exchange with the international community and encourages the participation of younger experts from all over the world. The 7th ICAP workshop has addressed several aspects that originated from this interaction with the international community and has effectively established objective goals and tasks to be delivered over the next two years. Some of these are outlined in this article, including the planning of three audio-visual educational modules to be posted at the www.anapattern.org website, the classification of two novel HEp-2 IFA patterns, the implementation of a project dedicated to continuously updating the information on the clinical and immunologic relevance of the HEp-2 IFA patterns, and the launch of two additional branches of the HEp-2 Clinical and Immunological (HEp-2 CIC) project.

Idiopathic inflammatory myopathies, especially antisynthetase syndrome, often appear outside of the muscles as interstitial lung disease (ILD). Another typical finding is the presence of mechanic’s hands. The aim of the present study was to describe the clinical, functional, tomographic, and serological data of patients with ILD and mechanic’s hands and their response to treatment and survival rates. This is a retrospective study of ILD with concurrent myopathy. Among the 119 patients initially selected, 51 had mechanic’s hands. All the patients were screened for anti-Jo-1 antibodies. An expanded panel of myopathy autoantibodies was also performed in 27 individuals. Of the 51 patients, 35 had 1 or more antibodies. The most common were anti-Jo-1, anti-PL-7, and anti-PL-12, while of the associated antibodies, anti-Ro52 was present in 70% of the 27 tested individuals. A significant response to treatment was characterized by an increase in predicted forced vital capacity (FVC) of at least 5% in the last evaluation done after 6 to 24 months of treatment. A decrease in predicted FVC of at least 5%, the need for oxygen therapy, or death were all considered treatment failures. All patients were treated with corticosteroids, and 71% with mycophenolate. After 24 months, 18 patients had an increase in FVC, 11 had a decrease, and 22 remained stable. After a median follow-up of 58 months, 48 patients remained alive and three died. Patients with honeycombing on high-resolution chest tomography (log-rank = 34.65; P < .001) and a decrease in FVC ≥5% (log-rank = 18.28, P < .001) had a poorer survival rate. Patients with ILD and mechanic’s hands respond well to immunosuppressive treatment.

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.

Objetivo: A urticária crônica espontânea é uma dermatose caracterizada pelo aparecimento de lesões na pele com duração superior a 6 semanas e com grande impacto na qualidade de vida do paciente. Até o momento, o seu diagnóstico é feito pela inoculação do soro autólogo na pele do paciente para visualização da pápula urticariforme, o que pode ser desagradável e perigoso em alguns casos. O teste de ativação de basófilos é um teste in vitro, caracterizado pela detecção da molécula CD63, presente nos grânulos de histamina de basófilos. Quando há ativação, ocorre a fusão dos grânulos com a membrana celular, aumentando a expressão de CD63 na sua superfície celular. Devido ao envolvimento de autoanticorpos IgE ou a seus receptores em basófilos, a avaliação do marcador CD63 pode ser uma alternativa segura para o diagnóstico da urticária crônica espontânea. Método: Basófilos de doadores hígidos são colocados em contato com o soro a ser testado e um tampão com IL-3. Controles que mimetizam reações IgE e não IgE mediadas, assim como apenas o tampão com IL-3, também são utilizados. Após incubação a 37ºC na presença do anticorpo CD63, é feita a aquisição por citometria de fluxo e avaliada a frequência de basófilos ativados após comparação com a expressão basal. Considera-se positivo quando a expressão for ≥5%. Conclusão: Até o momento foram analisados os soros de três pacientes com diagnóstico clínico: o primeiro com ausência de lesões apresentou 1,74% de ativação; o segundo com lesões recentes obteve 4,25% de ativação. Ambos estavam sob uso de anti-histaminicos. E no terceiro paciente, em início de tratamento, a expressão foi de 5,3%. Soros de indivíduos controles e com urticária física apresentaram expressão inferior a 5%. Apesar do baixo número de pacientes avaliados até o momento, o teste de ativação de basófilos tem se mostrado uma boa e segura alternativa para o diagnóstico de urticária crônica espontânea.

Objective: Published studies have shown associations between anti-ribosomal P (anti-P) antibody and systemic lupus erythematosus with hepatic manifestations. This has been reported also in autoimmune hepatitis. However, the consistency of the latter association remains controversial. This study aimed to evaluate the frequency of anti-P antibodies in autoimmune hepatitis using two different immunoassays. Methods: One-hundred and seventy-seven patients with autoimmune hepatitis were screened, and 142 were analyzed for anti-P antibody positivity. The samples were first analyzed using two different immunoassays: enzyme-linked immunosorbent assay (ELISA) and chemiluminescence and then compared with a group of 60 patients with systemic lupus erythematous. The positive samples were subjected to western blot analysis. Results: Anti-P was found in 5/142 autoimmune hepatitis cases (3.5%) by chemiluminescence and in none by ELISA. Among the five chemiluminescence-positive autoimmune hepatitis samples, on anti-P western blot analysis one was negative, two were weakly positive, and two were positive. In contrast, anti-P was detected in 10/60 patients with systemic lupus erythematosus (16.7%) and presented higher chemiluminescence units than the autoimmune hepatitis samples. Conclusion: A low frequency of anti-P antibodies was observed in autoimmune hepatitis, suggesting that this test is not useful for the diagnosis or management of this disease.