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Background The objective of the study was to determine whether the staining pattern and titer of indirect immunofluorescence assay on HEp-2 cells (HEp-2 IFA) are associated with systemic lupus erythematosus (SLE) disease activity. Methods A total of 269 consecutive patients meeting the ACR and SLICC criteria for SLE were classified into three groups according to the SLE Disease Activity Index 2000 (SLEDAI2K): Remission (SLEDAI2K = 0; n = 47); Intermediate (SLEDAI2K = 1-5; n = 111); Active (SLEDAI2K ≥ 6; n = 111). All subjects were assessed for HEp-2 IFA titer and staining pattern and nine traditional parameters of SLE disease activity. After a 6 to 12-month interval, 101 of the 269 patients were reassessed. Results HEp-2 IFA homogeneous nuclear pattern (AC-1) occurred more frequently in the Active Group compared to the Remission Group (p < 0.001). Fine speckled nuclear pattern (AC-4) tended to occur more frequently in the Remission Group compared to the Active Group (p = 0.054). Subjects with AC-1 pattern had higher SLEDAI (8.8 ± 7.6) than those with AC-4 (4.8 ± 5.2) (p < 0.001). HEp-2 IFA titer and anti-nuclear antibody by enzyme-linked immunosorbent assay (ANA-ELISA) values were lower in the Remission Group compared to the other two groups (p < 0.001). Multivariate analyses identified only ELISA anti-dsDNA as an independent variable associated with disease activity. In follow-up analysis, HEp-2 IFA titer decreased significantly in the 33 subjects with decreased disease activity (p = 0.002). Receiver operator characteristic (ROC) curve analysis for determination of disease activity showed equivalent areas under the curve (AUC) for HEp-2 IFA titer and traditional disease activity parameters. Conclusions HEp-2 IFA pattern and titer can reflect SLE disease activity and may be considered in conjunction with other laboratory and clinical parameters in the assessment of SLE disease activity.
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Anticuerpos Antinucleares/sangre , Ensayo de Inmunoadsorción Enzimática/métodos , Lupus Eritematoso Sistémico/inmunología , Adulto , Línea Celular , ADN/inmunología , Femenino , Humanos , Lupus Eritematoso Sistémico/sangre , Masculino , Persona de Mediana Edad , Nucleosomas/inmunología , Estudios ProspectivosRESUMEN
Background International autoantibody standards, traditionally based on material obtained from plasmapheresis of single subjects, represent individual immune response and may not comprehend the heterogeneity of the general population. The anti-DFS70 autoantibody yields a characteristic dense fine speckled (DFS) nuclear pattern on indirect immunofluorescence assay on HEp-2 cells (HEp-2 IFA) and speaks against autoimmunity. We propose a novel strategy for developing autoantibody reference standards, based on stepwise pooling of serum samples from hundreds of individuals with anti-DFS70 antibodies. Methods Within a 2-year period, serum samples were selected from routine HEp-2 IFA according to the following criteria: DFS HEp-2 IFA pattern at titer ≥1:640; anti-DFS70 reactivity in three analyte-specific tests (Western blot [WB], enzyme-linked immunosorbent assay [ELISA] and chemiluminescent immunoassay [CLIA]). Aliquots of individual samples were combined into progressively larger pools with stepwise validation of intermediary pools as for individual samples. Validated intermediary pools were merged into a final pool for lyophilization. Results A total of 741 validated samples yielded a 750 mL final pool that was lyophilized into thousands of 200 µL-aliquots. Reconstituted aliquots yielded the expected anti-DFS70 reactivity in ELISA, CLIA and WB, as well as high-titer DFS HEp-2 IFA pattern. The appropriate anti-DFS70 reactivity of the lyophilized pool was confirmed by seven international expert centers, using HEp-2 IFA, ELISA, WB and immunoprecipitation. Conclusions This proof-of-concept study provides an innovative and efficient strategy to build serum reference standards for autoantibody testing. The anti-DFS70 standard will integrate the panel of standards of Autoantibody Standardization Committee (ASC, www.autoab.org), contributing to education for proper assay validation and interpretation of the DFS pattern and other HEp-2 IFA patterns.
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Proteínas Adaptadoras Transductoras de Señales/inmunología , Autoanticuerpos/metabolismo , Espectrometría de Masas/métodos , Prueba de Estudio Conceptual , Femenino , Humanos , MasculinoRESUMEN
BACKGROUND: Evaluation of fibrosis is crucial in the assessment of chronic hepatitis C (CHC). The enhanced liver fibrosis (ELF) is a serological panel including hyaluronic acid (HA), tissue inhibitor of matrix metalloproteinases-1 (TIMP-1), and amino-terminal propeptide of type III procollagen (PIIINP) that has shown good results in predicting liver fibrosis in distinct scenarios of chronic liver diseases. AIMS: We aimed to assess the performance of ELF on the detection of fibrosis and cirrhosis in a CHC patient cohort and to compare the results of ELF and transient elastography (TE-Fibroscan) using liver biopsy as reference. PATIENTS AND METHODS: One hundred twenty patients were prospectively evaluated by TE and ELF using an ADVIA Centaur automated system. The ELF score was calculated using the manufacturer's algorithm. Biopsies were classified according to the METAVIR score. Receiver operator characteristic curve analyses were performed to evaluate the accuracy of ELF and TE. RESULTS: The area under the receiver operator characteristic curve (AUROC) of ELF for the diagnosis of significant fibrosis was 0.81 [95% confidence interval (CI), 0.73-0.87], for advanced fibrosis was 0.82 (95% CI, 0.74-0.88), and for cirrhosis was 0.78 (95% CI, 0.70-0.85). Using the proposed cutoffs, ELF overestimated fibrosis in 66% (81/120) of cases and underestimated in 3% (3/120). We found no statistically significant difference when comparing the AUROC of ELF and TE for diagnosing fibrosis or cirrhosis. CONCLUSIONS: ELF panel is a good noninvasive fibrosis marker and showed similar results to TE in CHC patients. However, new cutoff points need to be established to improve its performance on patients with CHC.
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Ensayo de Inmunoadsorción Enzimática , Hepatitis C Crónica/sangre , Hepatitis C Crónica/diagnóstico , Ácido Hialurónico/sangre , Cirrosis Hepática/virología , Fragmentos de Péptidos/sangre , Procolágeno/sangre , Inhibidor Tisular de Metaloproteinasa-1/sangre , Adulto , Anciano , Algoritmos , Área Bajo la Curva , Biomarcadores/sangre , Biopsia , Diagnóstico por Imagen de Elasticidad , Femenino , Hepatitis C Crónica/complicaciones , Humanos , Cirrosis Hepática/diagnóstico , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Pronóstico , Estudios Prospectivos , Curva ROC , Reproducibilidad de los Resultados , Factores de RiesgoRESUMEN
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 <â0.02 nmol/L), 18 were classified as Borderline (≥0.02 -1 nmol/L), and 64 were positive (RIPA-Posâ>â1 nmol/L). The competitive ELISA showed poor agreement with RIPA (Kappaâ=â0.216). The indirect ELISA demonstrated substantial agreement with RIPA (Kappaâ=â0.652), with â¼76% sensitivity and â¼94% specificity for MG diagnostic. The CBA, where fixed cells expressing clustered AChR were used as substrate, exhibited almost perfect agreement with RIPA (Kappaâ=â0.984), yielding â¼98% sensitivity and 96% specificity for MG. In addition, a semiquantitative analysis showed a strong correlation between CBA titration, indirect ELISA, and RIPA levels (râ=â0.793 and râ=â0.789, 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.â¥.
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Autoanticuerpos , Ensayo de Inmunoadsorción Enzimática , Miastenia Gravis , Ensayo de Radioinmunoprecipitación , Humanos , Ensayo de Inmunoadsorción Enzimática/métodos , Miastenia Gravis/inmunología , Miastenia Gravis/diagnóstico , Sensibilidad y Especificidad , Receptores Colinérgicos/inmunología , Femenino , Masculino , Persona de Mediana Edad , Adulto , Anciano , Adulto JovenRESUMEN
OBJECTIVE: The aim of this study was to assess the laboratory performance of periostin associated with a panel of biomarkers to identify the inflammatory phenotype of Brazilian asthma patients. METHODS: We evaluated 103 Brazilian individuals, including 37 asthmatics and 66 nonasthmatic controls. Both groups underwent analyses for serum periostin, eosinophil levels in the peripheral blood, the fraction of exhaled nitric oxide (FeNO), total serum IgE, urinary leukotriene E4, and serum cytokines. RESULTS: Higher levels of periostin (p = 0.005), blood eosinophils (p = 0.012), FeNO (p = 0.001), total IgE (p < 0.001), and IL-6 (p ≤ 0.001) were found in the asthmatic patients than the controls. Biomarker analyses by the ROC curve showed an AUC greater than 65%. Periostin (OR: 12,550; 95% CI: 2,498-63,063) and IL-6 (OR: 7,249; 95% CI: 1,737-30,262) revealed to be suitable asthma inflammation biomarkers. Blood eosinophils, FeNO, total IgE, IL-6, TNF, and IFN-g showed correlations with clinical severity characteristics in asthmatic patients. Periostin showed higher values in T2 asthma (p = 0.006) and TNF in non-T2 asthma (p = 0.029). CONCLUSION: The panel of biomarkers proposed for the identification of the inflammatory phenotype of asthmatic patients demonstrated good performance. Periostin proved to be an important biomarker for the identification of T2 asthma.
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Asma , Inmunoglobulina E , Humanos , Brasil , Interleucina-6 , Asma/diagnóstico , Eosinófilos , Biomarcadores , Fenotipo , Óxido Nítrico/análisisRESUMEN
OBJECTIVE: Anti-P-ribosomal antibody is a biomarker of systemic lupus erythematosus mainly associated with renal, nervous, and hepatic involvement. Systemic lupus erythematosus may present with features similar to autoimmune hepatitis. This study aimed to investigate the association of Anti-P-ribosomal antibodies in systemic lupus erythematosus compared to autoimmune hepatitis in the general Brazilian population. Autoimmune hepatitis and systemic lupus erythematosus share several clinical features. ÛªAnti-P ribosomal antibody is a biomarker for systemic lupus erythematosus. The association between anti-P ribosomal antibody and autoimmune hepatitis has shown conflicting results. Our results showed no association between anti-P ribosomal antibody and autoimmune hepatitis. 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.
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Hepatitis Autoinmune , Lupus Eritematoso Sistémico , Humanos , Hepatitis Autoinmune/diagnóstico , Hepatitis Autoinmune/complicaciones , Autoanticuerpos , Lupus Eritematoso Sistémico/diagnóstico , Lupus Eritematoso Sistémico/complicaciones , Biomarcadores , Western BlottingRESUMEN
Background: The frequency of antibodies in autoimmune encephalitis (AIE) may vary in different populations, however, data from developing countries are lacking. To describe the clinical profile of AIE in Brazil, and to evaluate seasonality and predictors of AIE in adult and pediatric patients. Methods: We evaluated patients with possible AIE from 17 centers of the Brazilian Autoimmune Encephalitis Network (BrAIN) between 2018 and 2022. CSF and serum were tested with TBAs and CBAs. Data on clinical presentation, complementary investigation, and treatment were compiled. Seasonality and predictors of AIE in adult and pediatric populations were analyzed. Results: Of the 564 patients, 145 (25.7%) were confirmed as seropositive, 69 (12.23%) were seronegative according to Graus, and 58% received immunotherapy. The median delay to diagnosis confirmation was 5.97 ± 10.3 months. No seasonality variation was observed after 55 months of enrolment. The following antibodies were found: anti-NMDAR (n=79, 54%), anti-MOG (n=14, 9%), anti-LGI1(n=12, 8%), anti-GAD (n=11, 7%), anti-GlyR (n=7, 4%), anti-Caspr2 (n=6, 4%), anti-AMPAR (n=4, 2%), anti-GABA-BR (n=4, 2%), anti-GABA-AR (n=2, 1%), anti-IgLON5 (n=1, 1%), and others (n=5, 3%). Predictors of seropositive AIE in the pediatric population (n=42) were decreased level of consciousness (p=0.04), and chorea (p=0.002). Among adults (n=103), predictors of seropositive AIE were movement disorders (p=0.0001), seizures (p=0.0001), autonomic instability (p=0.026), and memory impairment (p=0.001). Conclusion: Most common antibodies in Brazilian patients are anti-NMDAR, followed by anti-MOG and anti-LGI1. Only 26% of the possible AIE patients harbor antibodies, and 12% were seronegative AIE. Patients had a 6-month delay in diagnosis and no seasonality was found. Findings highlight the barriers to treating AIE in developing countries and indicate an opportunity for cost-effect analysis. In this scenario, some clinical manifestations help predict seropositive AIE such as decreased level of consciousness, chorea, and dystonia among children, and movement disorders and memory impairment among adults.
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Enfermedades Autoinmunes del Sistema Nervioso , Corea , Adulto , Humanos , Niño , Brasil/epidemiología , Encéfalo , Anticuerpos , Receptores de N-Metil-D-AspartatoRESUMEN
OBJECTIVE: To identify features of antinuclear antibody (ANA)-HEp-2 test results that discriminate ANA-positive healthy individuals and patients with autoimmune rheumatic diseases (ARDs). METHODS: We sequentially retrieved data on 918 healthy individuals and 153 patients with ARDs after clinical assessment. ANA-positive healthy individuals for whom data were available were reevaluated after 3.6-5.0 years. An ANA-HEp-2 test result was considered positive when a clear ANA pattern was observed at 1:80 dilution in 2 distinct commercial HEp-2 slides by 2 blinded independent observers. RESULTS: ANAs were present in 118 healthy individuals (12.9%) and 138 patients with ARDs (90.2%). The ANA titer was higher in patients with ARDs than in healthy individuals (P<0.001). The ANA pattern profile was distinct in the 2 groups. Nuclear homogeneous, nuclear coarse speckled, and nuclear centromeric patterns appeared exclusively in patients with ARDs. The nuclear dense fine speckled pattern occurred only in healthy individuals. The most frequent ANA pattern in both groups was the nuclear fine speckled pattern, which occurred at lower titer in healthy individuals than in patients with ARDs (P<0.001). Anti-extractable nuclear antigen was present in 1 healthy individual (anti-SSA/Ro) and in 52 patients with ARDs (37.7%). None of the 40 reevaluated healthy individuals developed ARDs, and 29 (72.5%) remained ANA positive. All healthy individuals who became ANA negative had an ANA titer of 1:80 at baseline. CONCLUSION: Our findings suggest that the titer, and especially the pattern, on the ANA-HEp-2 test strongly enhances our ability to discriminate ANA-positive healthy individuals and patients with ARDs.
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Anticuerpos Antinucleares/inmunología , Enfermedades Autoinmunes/inmunología , Enfermedades Reumáticas/inmunología , Adolescente , Adulto , Factores de Edad , Anciano , Enfermedades Autoinmunes/diagnóstico , Western Blotting , Femenino , Humanos , Masculino , Persona de Mediana Edad , Curva ROC , Enfermedades Reumáticas/diagnóstico , Estadísticas no Paramétricas , Encuestas y CuestionariosRESUMEN
BACKGROUND: Increased transferrin saturation (TS) and ferritin are common in hereditary hemochromatosis (HH) but also in chronic liver diseases (CLD). Nontransferrin bound iron (NTBI) is believed to be associated with iron-induced cell damage. We aimed to evaluate NTBI in CLD and their relationship with liver damage. METHODS: Two groups of patients were studied. Group 1 (G1): 94 CLD patients from an Outpatient Hepatology Unit. Group 2 (G2): 36 healthy individuals form a Medical Checkup Clinic. Transferrin iron-binding capacity, TS, ferritin, AST, ALT, and red cell count were performed using standard tests. NTBI was assessed as enhanced labile plasma iron (eLPi). Levels of eLPi less than 0.4 µmol/l were considered within the normal range. RESULTS: Prevalence of increased iron tests (elevated TS and ferritin) was 14% in G1 and 5.5% in G2 ( P = 0.19). Positive NTBI was found in 12 patients (11 in G1 and 1 in G2). Positivity to NTBI was associated with increased iron tests ( P = 0.03), cirrhosis ( P = 0.03) and AST index (ASTI) ( P = 0.03). NTBI was associated with TS of more than 70% ( P = 0.002) but not to elevated ferritin ( P = 0.74). Variables strongly associated with a positive NTBI in univariate analysis (TS > 70%, cirrhosis and ASTI) were submitted to binary regression analysis. TS of more than 70% was the only independent predictive factor ( P = 0.049; odds ratio, 6.8). CONCLUSION: NTBI was associated with TS in CLD, but not with ferritin. NTBI testing could be useful for CLD patients with increased iron tests. Alternatively, a TS of more than 70% can be used as a surrogate marker.
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Hierro , Transferrina , Biomarcadores , Ferritinas , Humanos , Cirrosis Hepática/diagnóstico , Transferrina/análisis , Transferrina/metabolismoRESUMEN
BACKGROUND: The VI Brazilian Consensus on Autoantibodies against HEp-2 cells for determination of autoantibodies against cellular constituents on HEp-2 cells was held on September, 2019, in Fortaleza (CE, Brazil). The guidelines in this edition were formulated by the group of Brazilian experts discussing the classification of complex patterns, the classification of the nuclear discrete dots (few and multiple), the identification of the discrete fine speckled pattern (AC-4a) and improvements on the ANA report. MAINBODY: Sixteen Brazilian researchers and experts from universities and clinical laboratories representing the various geographical regions of Brazil participated in the meeting. Four main topics were discussed: (1) How to classify patterns with fluorescence in more than one cell compartment considering three relevant categoris: composite patterns, mixed patterns and multiple patterns; (2) The splitting of the discrete nuclear dots pattern into the multiple discrete nuclear dots (AC-6) and few discrete nuclear dots (AC-7) patterns, respectively; (3) Inclusion of a novel nuclear pattern characterized by discrete fine speckled pattern highly associated with antibodies to SS-A/Ro60, classified as AC-4a. In addition, adjustments on the Brazilian Consensus nomenclature were implemented aiming to harmonize the designation of some patterns with the International Consensus on ANA Patterns (ICAP). Furthermore, the designations of the PCNA-like pattern (AC-13), CENP-F-like pattern (AC-14) and Topo I-like pattern (AC-29) were adjusted in accordance to ICAP. Finally, there was a recommendation for adjustment in the test report in order to address the status of nuclear envelope staining. For all topics, the aim was to establish specific guidelines for laboratories and clinicians. All recommendations were based on consensus among participants. All recommendations from the V Consensus were maintained and there was relevant progress in the BCA/HEp-2 guidelines and further harmonization with ICAP. CONCLUSION: The VI BCA/HEp-2 edition was successful in establishing important recommendations regarding the classification of complex patterns, in supporting the identification of a novel pattern within the AC-4 group and in the harmonization process with the ICAP terminology.
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Anticuerpos Antinucleares , Autoanticuerpos , Brasil , Consenso , HumanosRESUMEN
Introduction: The indirect immunofluorescence assay on HEp-2 cells (HEp-2/IFA) is used worldwide for screening for autoantibodies to cellular antigens. Cell culture and fixation methods influence the cell distribution of autoantigens and the preservation of epitopes. Therefore, discrepancy of results obtained using different HEp-2/IFA kits (interkit nonreproducibility) is a common phenomenon in the clinical laboratory routine. Objective: This study evaluated the interkit nonreproducibility of HEp-2/IFA results using samples from patients with systemic autoimmune disease (SAD), nonautoimmune diseases (NAD), and healthy blood donors (HBD). Methods: Serum from 275 SAD patients, 293 NAD patients, and 300 HBD were processed at 1:80 dilution using four HEp-2 kits according to the manufacturers' instructions. Interkit reproducibility was determined for positive/negative results and patterns. The agreement of positive/negative results among kits for each sample was determined as the reactivity agreement score (RAS). The pattern reproducibility score (PRS) in each sample was calculated as a function of the number of kits showing equivalent patterns. Qualitative variables and ordinal variables were analyzed by the Chi-square and Mann-Whitney U tests, respectively. Results: A total of 402 samples were nonreactive in all kits and were considered devoid of autoantibodies. Further analysis included the 466 reactive samples (238 SAD, 119 NAD, 109 HBD). Reactivity to the nucleus had the highest interkit reproducibility (RAS = 83.6), followed by the metaphase plate (RAS = 78.9), cytoplasm (RAS = 77.4), and nucleolus (RAS = 72.4). Interkit reproducibility was higher in SAD (RAS = 78.0) than in NAD (RAS = 70.6) and HBD (RAS = 71.3) groups. Samples with strong reactivity (++++/4 and +++/4) had higher interkit reproducibility than those with weak reactivity (+/4). In the SAD group, RAS for nuclear reactivity was 87.5% for strongly reactive samples as opposed to 4.4% for weakly reactive samples, and the same was observed for NAD and HBD samples. The most robust patterns were the centromere AC-3 (PRS = 78.4), multiple nuclear dots AC-6 (PRS = 73.6), nuclear coarse speckled AC-5 (PRS = 71.3), nuclear homogeneous AC-1 (PRS = 67.9), and the reticular cytoplasmic AC-21 (PRS = 68.6). Conclusion: Interkit nonreproducibility in HEp-2/IFA is prevalent and occurs with the highest frequency with weakly reactive samples. International initiatives with the engagement of in vitro diagnostic industry are encouraged to promote the harmonization of the properties and performance of HEp-2/IFA commercial kits.
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Anticuerpos Antinucleares/inmunología , Autoanticuerpos/inmunología , Enfermedades Autoinmunes/inmunología , Técnica del Anticuerpo Fluorescente Indirecta/métodos , Juego de Reactivos para Diagnóstico/normas , Anticuerpos Antinucleares/sangre , Autoanticuerpos/sangre , Enfermedades Autoinmunes/sangre , Enfermedades Autoinmunes/diagnóstico , Línea Celular Tumoral , Humanos , Juego de Reactivos para Diagnóstico/clasificación , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Introduction: The morphological patterns in indirect immunofluorescence assay on HEp-2 cells (HEp-2 IFA) reflect the autoantibodies in the sample. The International Consensus on ANA Patterns (ICAP) classifies 30 relevant patterns (AC-0 to AC-29). AC-4 (fine speckled nuclear pattern) is associated to anti-SS-A/Ro, anti-SS-B/La, and several autoantibodies. Anti-SS-A/Ro samples may contain antibodies to Ro60 and Ro52. A variation of AC-4 (herein designated AC-4a), characterized by myriad discrete nuclear speckles, was reported to be associated with anti-SS-A/Ro. The plain fine speckled pattern (herein designated AC-4b) seldom was associated with anti-SS-A/Ro. This study reports the experience of four expert laboratories on AC-4a and AC-4b. Methods: Anti-Ro60 monoclonal antibody A7 was used to investigate the HEp-2 IFA pattern. Records containing concomitant HEp-2 IFA and SS-A/Ro tests from Durand Laboratory, Argentina (n = 383) and Fleury Laboratory, Brazil (n = 144,471) were analyzed for associations between HEp-2 IFA patterns and disease-associated autoantibodies (DAA): double-stranded DNA, Scl-70, nucleosome, SS-B/La, Sm, and U1-RNP. A total of 381 samples from Dresden Technical University (TU-Dresden), Germany, were assayed for HEp-2 IFA and DAA. Results: Monoclonal A7 recognized Ro60 in Western blot and immunoprecipitation, and yielded the AC-4a pattern on HEp-2 IFA. Analyses from Durand Laboratory and Fleury Laboratory yielded compatible results: AC-4a was less frequent (8.9% and 2.7%, respectively) than AC-4b (26.1% and 24.2%) in HEp-2 IFA-positive samples. Reactivity to SS-A/Ro occurred in 67.6% and 96.3% of AC-4a-pattern samples against 23% and 6.8% of AC-4b pattern samples. Reciprocally, AC-4a occurred in 24% and 47.1% of anti-SS-A/Ro-positive samples, and in 3.8% and 0.1% of anti-SS-A/Ro-negative samples. Data from TU-Dresden show that the AC-4a pattern occurred in 69% of 169 anti-SS-A/Ro-monospecific samples (62% of all anti-SS-A/Ro-positive samples) and in 4% of anti-SS-A/Ro-negative samples, whereas anti-SS-A/Ro occurred in 98.3% of AC-4a samples and in 47.9% of AC-4b samples. In all laboratories, coexistence of anti-SS-B/La, but not other DAA, in anti-SS-A/Ro-positive samples did not disturb the AC-4a pattern. AC-4a was predominantly associated with anti-Ro60 antibodies. Conclusions: This study confirms the association of AC-4a pattern and anti-SS-A/Ro in opposition to the AC-4b pattern. The results of four international expert laboratories support the worldwide applicability of these AC-4 pattern variants and their incorporation into ICAP classification under codes AC-4a and AC-4b, respectively. The AC-4 pattern should be maintained as an umbrella pattern for cases in which one cannot discriminate AC-4a and AC-4b patterns. The acknowledgment of the AC-4a pattern should add value to HEp-2 IFA interpretation.
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Anticuerpos Antinucleares/análisis , Autoantígenos/inmunología , Enfermedades Autoinmunes/diagnóstico , Núcleo Celular/inmunología , Técnica del Anticuerpo Fluorescente Indirecta , ARN Citoplasmático Pequeño/inmunología , Ribonucleoproteínas/inmunología , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Argentina , Enfermedades Autoinmunes/inmunología , Brasil , Línea Celular , Consenso , Florida , Alemania , Humanos , Variaciones Dependientes del Observador , Valor Predictivo de las Pruebas , Reproducibilidad de los ResultadosRESUMEN
OBJECTIVES: To determine the frequency of the antineutrophil cytoplasmic antibodies (ANCA), antiproteinase-3 and antimyeloperoxidase, in primary sclerosing cholangitis (PSC) with or without inflammatory bowel disease (IBD+ or IBD-) and in different types of autoimmune hepatitis (AIH). Additionally, to verify the agreement between ANCA patterns by indirect immunofluorescence and their antigenic specificities by ELISA. METHODS: For this study, 249 patients were enrolled (42 PSC/IBD+; 33 PSC/IBD-; 31 AIH type-1; 30 AIH type-2; 31 AIH type-3; 52 primary biliary cirrhosis; 30 healthy controls) whose serum samples were tested for ANCA autoantibodies. RESULTS: There were fewer female subjects in the PSC/IBD- group (p=0.034). Atypical perinuclear-ANCA was detected more frequently in PSC/IBD+ patients than in PSC/IBD- patients (p=0.005), and was significantly more frequent in type-1 (p<0.001) and type-3 AIH (p=0.012) than in type-2 AIH. Proteinase-3-ANCA was detected in 25 samples (only one with cytoplasmic-ANCA pattern), and more frequently in PSC/IBD+ than in PSC/IBD- patients (p=0.025). Myeloperoxidase-ANCA was identified in eight samples (none with the perinuclear-ANCA pattern). Among the 62 reactive samples for atypical perinuclear-ANCA, 13 had antigenic specific reactions for proteinase-3 and myeloperoxidase. CONCLUSIONS: PSC/IBD+ differed from PSC/IBD- in terms of sex and proteinase 3-ANCA and atypical perinuclear-ANCA reactivity, the latter of which was more frequently detected in type-1 and type-3 AIH than in type-2 AIH. There was no agreement between ANCA patterns and antigenic specificities in IBD and autoimmune liver diseases, which reinforces the need for proteinase-3 and myeloperoxidase antibody testing.
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Colangitis Esclerosante , Hepatitis Autoinmune , Anticuerpos Anticitoplasma de Neutrófilos , Autoanticuerpos , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , MasculinoRESUMEN
Background: Anti-NMDAR encephalitis is the most frequent cause of autoimmune encephalitis. Chikungunya (CHIK) is an arbovirus responsible for outbreaks of fever, cutaneous rash and arthritis in underdeveloped countries, and a trigger for autoimmunity. Case Presentation: We report a five-year-old male patient with fever, myalgia, headache and conjunctivitis for 5 days. After 1 week he developed tonic-clonic seizures and evolved with dystonia and oromandibular dyskinesia followed by onset of focal motor seizures, decreased level of consciousness, dysautonomia and central apnea. Brain MRI was normal, CSF analysis revealed 15 cells, protein 16.6 mg/dL and glucose 68 mg/dL. Anti-NMDAR antibodies were detected in serum and CSF after 3 weeks of symptom onset. CHIK serology was positive for both IgM and IgG, suggesting a recent infection. Dengue and Zika serologies were negative. CSF PCR for herpes viruses and arboviruses (CHIK, Dengue and Zika) were negative. Conclusion: We report the occurrence of anti-NMDAR encephalitis after acute CHIK infection. The biphasic course, positivity for both CHIK IgM and IgG and negative CHIK CSF PCR results, as well as a dramatic response to immunotherapy suggest an immune-mediated pathogenesis. Because of the global epidemic of CHIK infection and unknown mechanisms involving CHIK and autoimmunity, patients with acute CHIK infections and neurological manifestations should be considered for antineuronal antibody testing.
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After publication of the original article [1], we were notified that there is a mistake in Fig. 2.
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OBJECTIVES: To report on a novel IIF pattern specifically associated with antibodies to DNA topo I. METHODS: A novel compound IF pattern, designated Scl-70 pattern, was characterized in routine ANA-HEp-2 IIF screening. Within the last 3 years, all serum samples presenting the Scl-70 pattern at the ANA-HEp2 IIF screening were tested for anti-topo I reactivity. Conversely, 16 serum samples with known anti-topo I reactivity and affinity-purified anti-topo I antibody preparations were tested for the Scl-70 pattern. RESULTS: The Scl-70 pattern comprised the staining of five cellular regions: nucleus, nucleolus and cytoplasm in interphase cells; nucleolar organizing region (NOR) and chromosomes in mitotic cells. All 81 serum samples selected as Scl-70 pattern reacted with topo I. All 16 anti-topo I samples and antibody preparations reproduced the Scl-70 pattern. This compound IF pattern was consistently observed in different commercial HEp-2 cell slides and in home-made slides with HEp-2 cells and human fibroblasts fixed with alternative protocols. Double IIF experiments demonstrated the co-localization of topo I and human upstream binding factor at the NOR. CONCLUSIONS: The Scl-70 pattern belongs to the group of compound IF patterns that hold strong association with the respective autoantibody specificities, such as that observed with centromere protein F (CENP-F) and nuclear mitotic apparatus-1 (NuMA-1) protein. The identification of the Scl-70 pattern at routine ANA-HEp-2 IIF screening may lead to implementation of specific tests for the identification of anti-topo I antibodies. In addition, the Scl-70 pattern outlines cellular domains other than those previously reported for topo I, which is of interest for further understanding the roles of this enzyme in cell biology.
Asunto(s)
Autoanticuerpos/inmunología , Autoantígenos/sangre , ADN-Topoisomerasas de Tipo I/inmunología , Proteínas Nucleares/sangre , Línea Celular , Nucléolo Celular/química , Núcleo Celular/química , Cromosomas/química , Citoplasma/química , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Interfase , Mitosis , Región Organizadora del Nucléolo/química , Esclerodermia Sistémica/inmunologíaRESUMEN
Indirect immunofluorescence assay (IFA) has been used for detection of autoantibodies against cellular antigens for more than 50 years. Originally using rodent tissue as substrate, the method was optimized by using the human immortal HEp-2 cell line derived from a larynx epidermal carcinoma. The HEp-2/IFA platform allows for optimal visualization of several cellular domains recognized by autoantibodies in the samples being tested. Serial dilution allows for the estimation of the concentration (titer) of the autoantibodies in the sample. Judicious analysis of the topographic distribution of the immunofluorescence (pattern) provides useful hints on the most plausible autoantigens being recognized, vis-à-vis the cognate autoantibodies. The importance of the HEp-2/IFA pattern has been recently emphasized by the International Consensus on ANA Patterns (ICAP), an initiative that established a comprehensive classification of the most relevant and prevalent HEp-2/IFA patterns (designated anti-cell (AC) patterns) and harmonized its nomenclature. The former designation "antinuclear antibody test" has been progressively replaced by the term "anti-cell antibody test," due to the recognition that the HEp-2/IFA method in fact allows the detection of autoantibodies to several cellular domains, such as the cytoplasm and mitotic apparatus.The performance of the HEp-2/IFA test is strongly influenced by several technical details, including cell culture conditions, cell fixation and permeabilization methods, choice and titration of fluorochrome-conjugated secondary antibody, use and choice of blocking solutions, washing buffers, and antifading mounting medium. The several steps of the procedure must be carefully performed in order to avoid the formation of false positive fluorescent artifacts. The quality control of the assay involves the use of serum standards for negative, low positive and strongly positive reaction in each run of the assay. In addition, every new lot or new brand of HEp-2 slides should be evaluated by using a panel of standard sera yielding the most relevant AC patterns. Special attention should be dedicated to the training of personnel for the analysis of the slides at the microscope. These should be able to identify possible artifacts, recognize all relevant AC patterns, and formulate possible reflex tests according to the observed AC patterns.
Asunto(s)
Autoanticuerpos/análisis , Técnica del Anticuerpo Fluorescente Indirecta/métodos , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , HumanosRESUMEN
BACKGROUND: Anti-mitochondria autoantibodies (AMA) occur in > 95% primary biliary cholangitis (PBC) patients. Biochemically normal AMA-positive (BN/AMA+) individuals, occasionally noticed by indirect immunofluorescence (IIF) on HEp-2 cells and confirmed in AMA-specific assays, may represent early stages of PBC. The Enhanced Liver Fibrosis (ELF) score is a surrogate marker for liver fibrosis. This prospective study investigated the ELF score in BN/AMA+ individuals and PBC patients, considering autoantibody avidity and serum levels along the years. METHODS: 327 samples from 35 PBC and 59 BN/AMA+ were prospectively obtained in average 3.83 (range 0.50-7.40) years apart. Samples were tested by IIF on rat-kidney (IIF-AMA), western-blot for AMA (WB-AMA), and ELISA for antibodies against pyruvate-dehydrogenase (PDC-E2), gp210, sp100 and CENP-A/B. Anti-PDC-E2 avidity was determined by 6 M urea-elution ELISA. Alkaline phosphatase (ALP), gamma glutamyl transferase (ɣGT) and ELF score were measured by automated methods. RESULTS: Along the follow-up period BN/AMA+ subjects and PBC patients presented significant increase in serum anti-PDC-E2 (mean 10.45% and 8.86% per year; respectively), anti-PDC-E2 avidity (3.02% and 4.94%/year) and ELF score (3.24% and 2.71%/year). IIF-AMA and ɣGT increased in BN/AMA+ (6.59% and 2.36%) and decreased in PBC (- 4.89%/year and - 3.88%/year). In BN/AMA+ individuals there was positive correlation of ELF with IIF-AMA titer (r = 0.465; p < 0.001) and with anti-PDC-E2 levels (r = 0.239; p < 0.001). Expansion of autoantibody targets along time occurred in 39% BN/AMA+ and 49% PBC patients. The frequency of BN/AMA+ with high probability of having established PBC increased from 7 to 14%. CONCLUSIONS: BN/AMA+ individuals present an orchestrated increase in ELF score and humoral autoimmune response over time, indicating an opportunity for early therapeutic intervention and prevention in autoimmunity.
RESUMEN
BACKGROUND: The V Brazilian Consensus for determination of autoantibodies against cellular constituents on HEp-2 cells, held in Brasilia (DF, Brazil) on August 27, 2016, discussed the harmonization between the Brazilian Consensus on ANA (BCA) guidelines and the International Consensus on ANA Patterns (ICAP) recommendations ( www.anapatterns.org ). Initial guidelines were formulated by the group of Brazilian experts with the purpose of guiding and enabling Brazilian clinical laboratories to adopt recommendations and to provide a common standard for national and international consensuses. MAINBODY: Twenty Brazilian researchers and experts from universities and clinical laboratories representing the various geographical regions of the country participated in the meeting. Three main topics were discussed, namely the harmonization between the BCA guidelines and latest recommendations of the ICAP initiative, the adjustment of the terminology and report on HEp-2 patterns, and a reassessment of quality assurance parameters. For the three topics, our aim was to establish specific guidelines. All recommendations were based on consensus among participants. There was concrete progress in the adjustment of the BCA guidelines to match the ICAP guidelines. To a certain extent, this derives from the fact that ICAP recommendations were largely based on the algorithm and recommendations of the IV Brazilian ANA Consensus, as consistently recognized in the ICAP publications and presentations. However, although there is great overlap between the two Consensuses, there are some point divergences. These specific items were individually and extensively discussed, and it was acknowledged that in several points ICAP improved recommendations previously issued by the Brazilian ANA Consensus and these changes were readily implemented. Regarding some specific topics, the BCA panel of experts felt that the previously issued recommendations remained relevant and possibly will require further discussion with ICAP. The term anti-cell antibodies was adopted as the recommended designation, recognizing that the assay addresses antibodies against antigens in the nucleus and in other cell compartments. However, the acronym ANA HEp-2 was maintained due to historical and regulatory reasons. It was also signalized that the latest trend in ICAP is to adopt the term Indirect Immunofluorescent Assay on HEp-2 cell substrate (HEp-2 IIFA). In addition, the quality assurance strategies previously presented were ratified and emphasized. CONCLUSION: The V BCA edition was successful in establishing an overall harmonization with the ICAP recommendations for interpretation of the HEp-2 IIFA test, pinpointing the perspectives in filling the remaining gaps between both initiatives.