Antinuclear Antibody Staining Research Articles

Examination of antinuclear antibody staining patterns and titers in patients with childhood-onset systemic lupus erythematosus.

Antinuclear antibodies (ANA) staining patterns can provide useful information in systemic lupus erythematosus (SLE). In our study, we examined the frequency of ANA staining patterns in disease-related features in childhood-onset SLE patients. ANA and its staining patterns were assessed in childhood-onset SLE patients. Two hundred twenty-three patients were included (F/M = 3/1). Their median age at diagnosis was 14.3 (11.9-16.1) years. The anti-cell (AC)-4/5 (fine or large speckled) pattern was the most common nuclear ANA pattern (75.8%), while the AC-19 (dense fine speckled) pattern was the most frequently detected cytoplasmic ANA pattern (13.1%). The AC-4/5 (fine or large speckled) patterns were notably seen in fever, acute and chronic cutaneous lupus, arthritis, serositis, hematologic involvement, renal involvement, neuropsychiatric involvement, gastrointestinal involvement, and cardiopulmonary involvement (all p < .001). Conversely, the AC-1 (homogeneous) pattern was significantly detected in oral/nasal ulcers and non-scarring alopecia (both p < .001). Regarding the laboratory features, the AC-4/5 (fine or large speckled) patterns exhibited a predominant seen in autoimmune hemolytic anemia, leukopenia, thrombocytopenia, elevated ESR and CRP, hypocomplementemia, direct Coombs, anti-Smith (Sm), anti-SSA and SS-B, anti-ribonucleoprotein (RNP), anti-histone, anti-ribosomal P, lupus anticoagulant, anti-cardiolipin immunoglobulin (Ig)M/IgG, and anti-β2-glycoprotein IgM/IgG positivities (all p < .001). In contrast, the AC-1 (homogeneous) pattern was detected in anti-double-stranded (ds) DNA and anti-histone positivity (both p < .001). Our study showed that AC-4/5 and AC-1 patterns of ANA are frequently detected in many clinical and serological features of childhood-onset SLE patients. However, further studies are needed in larger populations to verify these results.

Analysis of antinuclear antibody in 9 528 pregnant women during early pregnancy in a hospital in Qingdao City

To analyze the positivity rate and titer of antinuclear antibody (ANA), as well as nuclear pattern and target antigen of ANA in healthy pregnant women during early pregnancy in Qingdao area. A prospective cohort study design was used to include a total of 9 528 healthy pregnant women registered at the Women and Children's Hospital Affiliated to Qingdao University from March 2023 to June 2024.Indirect immunofluorescence assay (IIF) was used to detect ANA, its titer and cell staining pattern. Fifteen specific antibodies were tested using the magnetic bar code immunofluorescent luminescence method. Logistic regression model was used to analyze the risk factors of pregnancy with autoimmune disease(AID). The results showed that among 9 528 pregnant women in early pregnancy, 1 346 cases (14.1%) were positive of ANA, including 1 011 cases with a titer of 1∶100 (10.6%), 236 cases (2.5%) with a titer of 1∶320, and 99 cases (1.0%) were detected at a titer >1∶320. Among the 1 346 ANA-positive pregnant women, nuclear granular type accounted for the highest proportion (483 cases, 35.9%), followed by speckled type (347 cases, 25.8%) and cytoplasmic type (176 cases, 13.1%).Then, pregnant women with ANA titers ≥1∶100 were detected 15 specific antibodies.Anti-SSA was tested in 121 cases accounted for the majority, followed by 110 cases with anti-Ro-52, 56 cases with anti-SSB, 51 cases with anti-mitochondrial M2 subtype antibodies and 37 cases with anti-centromere B. In conclusion,in healthy pregnant women in Qingdao area, ANA positivity rate was 14.1%, and the titer of ANA was mainly at 1∶100.The predominant nuclear patterns were nuclear granular and speckled types.The specific autoantibodies were mainly anti-SSA antibodies and anti-Ro-52 antibodies.The detection of ANA and specific autoantibodies is of great significance for early prediction, diagnosis, and intervention of autoimmune diseases during pregnancy.

Antinuclear antibody staining patterns by indirect immunofluorescence assay observed in patients from a tertiary health center in Latin America

Introduction/ObjectiveThis study aimed to describe the frequency of antinuclear antibody (ANA) staining patterns by indirect immunofluorescence assay observed in patients from a tertiary health center in Latin America. Materials and methodsThis retrospective, descriptive, and observational study evaluated data from all patients undergoing antinuclear antibody indirect immunofluorescence assay from a single-tertiary center (University Hospital Fundación Valle del Lili, Cali-Colombia) in 2020. ResultsOne thousand and eight patients met the inclusion criteria. The median patient age was 47 (34–59.2) years, and most were female (769, 75.3%). A positive ANA immunofluorescence assay was observed in approximately two-thirds of patients (664, 65.8%). ANA test results were primarily used to exclude a suspected diagnosis in approximately half of the patients (466, 46.2%). Thirty-seven percent (250/664) of the cohort with ANA-positive titers had a systemic autoimmune rheumatic disease (SARD). The most prevalent SARDs included rheumatoid arthritis (RA) (55, 8.2%) followed by systemic lupus erythematosus (SLE) (37, 5.5%). The vast majority of ANA-positive patients had a reported speckled pattern (anti-cell [AC]-2,4,5; 269; 40.5%) followed by homogenous (AC-1; 266; 40%), nucleolar (AC-8,9,10; 46; 6.9%), and centromere (AC-3; 16; 2.4%). The most frequent patterns observed among SLE patients included homogenous (AC-1) patterns in 17 (45.9%) patients, speckled (AC-2,4,5) nuclear patterns in 11 (29.7%) patients, mixed patterns in 7 (18.9%) patients, and reticular/anti-mitochondrial antibody (AMA, AC-21) cytoplasmic patterns in 2 (5.4%) patients. ConclusionThis study is the first to describe ANA patterns in a Colombian population. Speckled and homogenous patterns were predominant in patients with SARDs.

Macaque liver substrate for evaluating dense fine speckled-like patterns on HEp2010 cells

Antinuclear antibody (ANA) detection by indirect immunofluorescence (IIF) is instrumental in the evaluation of systemic autoimmune diseases (SADs). The dense fine speckled (DFS) ANA staining predominantly associates with anti-DFS70, an autoantibody that is thought to exclude the presence of SAD. However, the DFS pattern may mask the presence of other ANA patterns that may be clinically relevant. Our laboratory uses the HEp2010 substrate which contains both HEp2 and liver substates. The aim of this study was to determine whether negative liver nucleus immunofluorescence could exclude the presence of antibodies to extractable nuclear antigens (ENA) in sera with DFS-like patterns. One hundred consecutive sera samples suspicious for DFS pattern, along with 15 sera of control patterns (positive metaphase bars) were included in the study. Each sample was examined separately on HEp2010 (Euroimmun) and liver by two independent readers. Anti-DFS70 was assessed by line and chemiluminescent immunoassays. DFS-like sera were more likely to be liver nucleus-negative compared with control sera. Of the liver-negative sera, 61/64 (95.3%) were deemed anti-ENA negative. Using the liver substrate in the evaluation of anti-ENA had a sensitivity of 90.0% and a negative predictive value of 95.4%. In our cohort, concurrent evaluation of sera with the liver substrate helped rule out the presence of other anti-ENA. This technique may be a safeguard for DFS-like ANA patterns that may mask underlying anti-ENA on IIF.

Antinuclear antibody staining patterns by indirect immunofluorescence assay observed in patients from a tertiary health center in Latin America

Introduction/ObjectiveThis study aimed to describe the frequency of antinuclear antibody (ANA) staining patterns by indirect immunofluorescence assay observed in patients from a tertiary health center in Latin America. Materials and methodsThis retrospective, descriptive, and observational study evaluated data from all patients undergoing antinuclear antibody indirect immunofluorescence assay from a single-tertiary center (University Hospital Fundación Valle del Lili, Cali-Colombia) in 2020. ResultsOne thousand and eight patients met the inclusion criteria. The median patient age was 47 (34–59.2) years, and most were female (769, 75.3%). A positive ANA immunofluorescence assay was observed in approximately two-thirds of patients (664, 65.8%). ANA test results were primarily used to exclude a suspected diagnosis in approximately half of the patients (466, 46.2%). Thirty-seven percent (250/664) of the cohort with ANA-positive titers had a systemic autoimmune rheumatic disease (SARD). The most prevalent SARDs included rheumatoid arthritis (RA) (55, 8.2%) followed by systemic lupus erythematosus (SLE) (37, 5.5%). The vast majority of ANA-positive patients had a reported speckled pattern (anti-cell [AC]-2,4,5; 269; 40.5%) followed by homogenous (AC-1; 266; 40%), nucleolar (AC-8,9,10; 46; 6.9%), and centromere (AC-3; 16; 2.4%). The most frequent patterns observed among SLE patients included homogenous (AC-1) patterns in 17 (45.9%) patients, speckled (AC-2,4,5) nuclear patterns in 11 (29.7%) patients, mixed patterns in 7 (18.9%) patients, and reticular/anti-mitochondrial antibody (AMA, AC-21) cytoplasmic patterns in 2 (5.4%) patients. ConclusionThis study is the first to describe ANA patterns in a Colombian population. Speckled and homogenous patterns were predominant in patients with SARDs.

Negative ANA-IIF in SLE patients: what is beyond?

The antinuclear antibody (ANA) test has high sensitivity in diagnosing and classifying systemic lupus erythematosus (SLE).ObjectivesTo describe the immunological pattern of SLE patients through investigating specific antinuclear autoantibodies by enzyme dot immunoassay and studying their frequency in both positive and negative ANA indirect immunofluorescence assay (IIF) cases.MethodsIn a cross-sectional study, blood samples from 393 newly diagnosed SLE patients were analyzed using (IIF) on HEp-2 cells and ANA dot immunoassay by automated enzyme immunoassay (EIA) to detect 19 antibodies.ResultsNinety-one percent of the patients are females; their mean age was 37 ± 12.28. Antinuclear antibody (ANA) was detected by IIF in 82.4% of cases, with 181 (46.1%) speckled and 167 (42.4%) homogeneous ANA patterns. The majority of patients (96%) demonstrated autoantibodies via EIA. Among the ANA-IIF-negative patients, 97.2% demonstrated autoantibodies. There was a significant difference in the frequency of certain autoantibodies between SLE patients with negative and positive ANA-IIF (1.44 0.73, 3.12 2.09, p = 0.00) respectively.ConclusionThe results of analyzing 19 autoantibodies with the ANA staining pattern increased the significance of analyzing the immune profile even if IIF is negative when clinical symptoms strongly suggest SLE diagnosis. Certain autoantibodies may evade staining by the IFA approach while they are present in the patient’s serum, and they may not be detected by the ANA EIA profile if it does not contain that antigenic substrate.Key Points• Indirect immunofluorescence on Hep-2 is the conventional method for ANA detection and is regarded as the “gold standard” for testing in clinical practice for SLE.• In our study, ANA profile dot enzyme immunoassay (EIA)-based test was performed to evaluate 19 autoantibodies in SLE patients either positive or negative for ANA-IIF.• The presence of anti-dsDNA with ANA-IIF-negative serum in 32.4% of SLE patients provides evidence that not all anti-dsDNA antibodies are identified on standard HEp-2 substrates.• certain autoantibodies can evade staining by the ANA-IIF method despite being present in the SLE patient’s blood; this supports the ANA profile enzyme dot immunoassay as a more sensitive test.

Investigation of the Contribution of Autoantibodies to Clinical Diagnosis in Liver Pathologies and the Identification of Accompanying Autoimmune Diseases

Autoantibodies are frequently detected in the presence of autoimmune liver diseases (ALD) [autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC)] and are widely used to classify the disease clinically. The aim of this study was to investigate the contribution of autoantibodies for the diagnosis of ALD and the identification of other accompanying systemic autoimmune rheumatic diseases (SARD). In addition, it was aimed to compare the results of indirect immunofluorescence (IIF) antinuclear antibody (ANA) patterns and extractable nuclear antigen (ENA) antibodies. A total of 593 patients, including 544 patients with high liver function tests from general surgery/gastroenterology clinics and 49 patients referred from internal medicine/rheumatology clinics to investigate ALD, were included in the study. HBsAg and anti-HCV test results of the patients were found to be negative. ANA, anti-mitochondrial antibody (AMA)/anti-liver-kidney microsomal antibody (LKM), anti-smooth muscle antibody (ASMA), antineutrophil cytoplasmic antibody (ANCA) assays were performed by indirect immunofluorescence method (IIF) (Euroimmune AG, Luebeck, Germany). Extractable nuclear antigen (ENA) (nRNP/Sm, Sm, SS-A, SS-B, Scl-70, Jo-1, dsDNA, nucleosome, histone, ribosomal P-protein, AMA-M2, Ro-52, PM-Scl, CENP-B, PCNA, DFS70) and liver profiles [soluble liver antigen\liver pancreas antigen (SLA/LP), liver cytosolic antigen1(LC-1), LKM-1, anti-mitochondrial antibody M2(AMA-M2)] (Euroimmune AG, Luebeck, Germany) were detected by immunoblot (IB) method. Demographic characteristics, clinical data, presence of systemic autoimmune rheumatic diseases (SARD), radiological and laboratory findings were determined from the medical records. Autoantibody tests were found to be negative in 461 (77.7%) of 593 patients (mean age= 53.3 ± 15.6, age range= 18-90), and were positive in 132 (22.3%) (86.4% female) of the patients. Of the patients with positive autoantibodies, 60.6% (80/132) were diagnosed as PBS and 1.5% (2/132) were diagnosed as AIH (positive anti-LC-1 and anti-LKM1 antibodies). Fourteen of the patients (10.6%) with centromere, nuclear membrane (NM), multiple nuclear dot (MND) staining patterns and elevated liver enzymes could not be diagnosed as a specific disease and were followed up. PBS (13/30) was detected in approximately half of the patients diagnosed with SARD. The most common accompanying SARD in PBC patients was Sjögren's syndrome (SS) (7.5%, 6/80), followed by rheumatoid arthritis (RA) (5.0%, 4/80), scleroderma (2.5%, 2/80), and systemic lupus erythematosus (SLE) (1.3%, 1/80) respectively. The most common pattern was the AMA staining pattern (34.8%, 46/132) among the autoantibody positive patients. AMA and ANA staining patterns were detected together in 31.1% (41/132) of the patients. In the ENA profile results of these patients, the most common profile detected was anti-Ro-52 (65.9%, 27/41), followed by anti-SSA (34.1%, 14/41), anti-SSB (22.0%, 9/41) and anti-CENP-B (12.2%, 5/41) autoantibodies , respectively. ANA patterns were detected in 32.6% (43/132) of the patients (NM 9.1%, centromere 9.1%, MND 6.8%, respectively). In our study, 87.5% (70/80) of the patients diagnosed as PBS were found to have AMA positivity and 12.5% (10/80) of them had ANA positivity (such as NM, CNN, centromere). The characteristics, laboratory and radiological findings of the patients with isolated AMA positivity alone (Group 1) and patients with multiple patterns/autoantibodies (Group 2) were compared. In patients with multiple patterns/autoantibodies (Group 2), the presence of cirrhosis and liver heterogeneity were found to be higher than Group 1 (p= 0.049). ALD associated autoantibodies can be detected before years from the clinical disease. ALD may be associated with various SARD. Detection of ALD-related autoantibodies in patients diagnosed with SARD can provide early diagnosis of these patients. These autoantibodies guide both diagnosis and prognosis as in PBC. Collaboration between the laboratory specialist and the clinician is critical in the diagnosis, management and early recognition of these patients before clinical disease.

The significance of cytoplasmic antinuclear antibody patterns in autoimmune liver disease.

We aimed to determine the significance of cytoplasmic antinuclear antibody (ANA) patterns using computer-aided immunofluorescence microscopy in patients with autoimmune liver diseases (AILD). ANA staining pattern was identified by treating cultured human epithelial type 2 (HEp-2) cells with the sera of the patients. Medical records of patients with suspected AILD who had positive cytoplasmic ANA patterns between February 2017 and November 2019 were retrospectively reviewed for clinical, laboratory, and immunological data. Cytoplasmic ANA patterns of AILD and non-AILD groups were compared. Further subgroup analysis of patients with AILD who had reticular or speckled cytoplasmic ANA patterns was conducted. We found that among the 196 patients with positive cytoplasmic ANA patterns, 113 (57.6%) were diagnosed with AILD. The percentage of reticular cytoplasmic pattern was higher in the AILD group than that in the non-AILD group (64.0% vs. 21.9%, p < 0.001). Furthermore, patients with AILD who exhibited a reticular ANA pattern demonstrated a higher positive rate for anti-mitochondrial antibodies (66.7% vs. 2.6%, p < 0.001) than those who exhibited the speckled ANA pattern. Moreover, AILD patients with the reticular ANA pattern displayed a lower positive rate for anti-smooth muscle antibodies (0% vs. 45%, p < 0.001) and nuclear ANA pattern (73.2% vs. 97.5%, p = 0.003) than those with the speckled ANA pattern. Therefore, cytoplasmic ANA patterns could be used to guide AILD characterization in suspected AILD cases, especially as the reticular ANA pattern is strongly associated with AILD. Thus, it is important to check cytoplasmic ANA patterns for AILD evaluation, even when nuclear ANA patterns are negative.

Staining of antinuclear antibodies and antibodies against removable nuclear antigens in connective tissue diseases

Introduction and objectivesConnective tissue diseases are inflammatory, autoimmune diseases and threaten quality of life. To determine the relationship between staining patterns of antinuclear antibodies and antibodies against extractable nuclear antigens in patients with connective tissue disease. Materials and methodsObservational, basic, analytical and transversal study. Study conducted in the Immunology Service of the Arzobispo Loayza National Hospital between January 2017 and June 2017. We analyzed 291 samples of patients with CTD and for the detection of anti-nuclear antibody staining patterns, the immunological kit and observation with microscope of at 40X Immunofluorescence and for the detection of the antibodies against extractable nuclear antigens. The Immunoblot method was employed. Statistical analyses were carried out with the statistical package SPSS version 21 for Windows. We used the Pearson Chi-square test for the categorical variables, a value of p<0.05 was considered significant. ResultsThere was a significant relationship p<0.05 of the homogeneous pattern, the mottled pattern with Anti-histones (p=0.000), Anti-nucleosomes (p=0.000), Anti-Ro 52 (p=0.000), Anti-SSA (p=0.001), Anti-SSB (p=0.003), Anti-dsDNA (p=0.000) with the Pearson Chi-square test. There was a significant relationship of p<0.05 of the centromeric pattern with Anti-Cenp B (p=0.000) with Fisher⿿s exact statistic. ConclusionsThere was a significant relationship between the anti-nuclear antibody staining patterns and the antibodies to the core extractable antigens in patients with systemic lupus erythematosus, Sjögren⿿s syndrome, Calcinosis, Raynaud⿿s phenomenon, esophageal Dysmotility, sclerodactyly and Telangiectasia (CREST), Scleroderma and Polymyositis.

Undifferentiated Connective Tissue Disease and Interstitial Lung Disease: Trying to Define Patterns

ObjectivesTo identify clinical or immunological features in patients with undifferentiated connective tissue disease (UCTD) associated interstitial lung disease (ILD), in order to group them and recognize different functional and high resolution computed tomography (HRCT) behavior. MethodsRetrospective cohort study. Patients meeting Kinder criteria for UCTD were included. We defined the following predictive variables: ‘highly specific’ connective tissue disease (CTD) manifestations (Raynaud's phenomenon, dry eyes or arthritis), high antinuclear antibody (ANA) titer (above 1:320), and ‘specific’ ANA staining patterns (centromere, cytoplasmic and nucleolar patterns). We evaluated the following outcomes: change in the percentage of the predicted forced vital capacity (FVC%) during the follow-up period, and HRCT pattern. ResultsSixty-six patients were included. Twenty-nine (43.94%) showed at least one ‘highly specific’ CTD manifestation, 16 (28.57%) had a ‘specific’ ANA staining pattern and 29 (43.94%) high ANA titer. Patients with ‘highly specific’ CTD manifestations were younger (mean [SD] 52 years [14.58] vs 62.08 years [9.46], P<.001), were more likely men (10.34% vs 48.65%, P<.001) and showed a smaller decline of the FVC% (median [interquartile range] 1% [−1 to 10] vs −6% [−16 to −4], P<.006). In the multivariate analysis, the presence of highly specific manifestations was associated with improvement in the FVC% (B coefficient of 13.25 [95% confidence interval, 2.41 to 24.09]). No association was observed in relation to the HRCT pattern. ConclusionThe presence of ‘highly specific’ CTD manifestations was associated with female sex, younger age and better functional behavior. These findings highlight the impact of the clinical features in the outcome of patients with UCTD ILD.

Anti-centromere antibody exhibits specific distribution levels among anti-nuclear antibodies and may characterize a distinct subset in rheumatoid arthritis

Anti-centromere antibody (ACA) is one of the classical anti-nuclear antibody (ANA) staining patterns. However, characteristics of ACA in comparison with the other ANA patterns and clinical features of ACA-positive subjects have not been elucidated. Here, we examined all ANA patterns by indirect immunofluorescence for 859 rheumatoid arthritis (RA) patients. Together with the ANA data of 9,575 healthy volunteers, we compared distributions of the ANA levels. ACA was the only ANA that demonstrated a definite bimodal distribution of levels. ACA showed significantly higher levels than the other ANA staining patterns in both RA and healthy population (p < 0.0001). ACA-positivity was associated with old age and was observed more in females. We further recruited another cohort of 3,353 RA patients and confirmed the findings. ACA was also associated with Raynaud’s phenomenon (p = 6.8 × 10−11) in RA. As a conclusion, ACA displays a specific ANA staining pattern with a bimodal distribution, and ACA-positive RA may constitute a distinct subset with specific clinical features.

Serum antinuclear antibody in adult Thais.

This study investigated the presence of antinuclear antibody (ANA) in older Thais compared with middle-age and younger participants. Antinuclear antibody represents the first step in the diagnostic testing for lupus erythematosus (LE) and other autoimmune diseases. Due to the lack of reference ANA levels in older, middle-age and younger Thais healthy participants, this study will be useful for determining the proper diagnostic and treatment criteria. There were 28 older (60-76 years), 17 middle-age (41-59 years) and 13 younger (24-40 years) participants in this study. Immunofluorescence was performed to analyse the ANA staining pattern and titre levels in the participants' blood samples. The presence of serum ANA was found in 18 of 28 cases (64.3%), four of 17 (23.5%) and one of 13 cases (7.7%) of the older, middle-age and younger participants, respectively. The difference in the number of serum ANA-positive participants between the older, middle-age and younger groups was statistically significant (p < 0.05). Interestingly, the ANA positive in older participants presented more than one staining pattern. The speckled pattern was the most commonly detected ANA staining pattern in the older group, being found in 12 cases followed by cytoplasmic pattern (10 cases), homogeneous pattern (nine cases) and nucleolar pattern (five cases). In the middle-age group, the speckled pattern was found in four cases, whereas one younger participant presented a nucleolar pattern. Serum ANA positive was significantly higher in the older group compared with the middle-age and younger groups. There were variations of the serum ANA staining patterns in the older group.

Lupus erythematosus cells in vivo (pleural fluid): An unusual presentation of systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease in which organs and cells undergo damage mediated by tissue-binding autoantibodies and immune complexes. In earlier times, the diagnosis of SLE included the lupus erythematosus (LE) cell assay. Though the LE cell test has now been declared obsolete, its presence carries importance especially when one finds LE cells in vivo. We report an unusual case of SLE in a 27-year-old female who presented with acute shortness of breath, fever, and cough. On initial outpatient basis investigations, the patient was detected with anemia, bilateral pleural effusion, synovitis, and juxta-articular swelling of the soft tissues. Her chest radiograph effusion was tapped and sent to the cytopathology laboratory. The cytological examination of the pleural fluid revealed numerous LE cells that prompted the diagnosis of SLE. Autoimmune serology techniques such as antinuclear antibody staining have replaced the LE cell assay. However, as presented in this report and found in a review of the literature, the in vivo finding of LE cells by cytopathology can provide an important clue to the diagnosis of SLE. This case is interesting because although pleural effusion is common, very rarely LE Cells are encountered in vivo. There are very few case reports of SLE diagnosed in a cytopathology laboratory. Moreover, our finding of LE cells in the pleural fluid of the patient led to swift diagnosis, helped rule out the differential diagnosis of rheumatoid arthritis and prompted the immediate initiation of treatment. Furthermore, our case highlights the fact that no matter the advances in diagnostic testing methods, practitioners must always keep an eye open for the basic pathognomonic findings of diseases.

Alterations in nuclear structure promote lupus autoimmunity in a mouse model.

ABSTRACTSystemic lupus erythematosus (SLE) is an autoimmune disorder characterized by the development of autoantibodies that recognize components of the cell nucleus. The vast majority of lupus research has focused on either the contributions of immune cell dysfunction or the genetics of the disease. Because granulocytes isolated from human SLE patients had alterations in neutrophil nuclear morphology that resembled the Pelger–Huet anomaly, and had prominent mis-splicing of mRNA encoding the nuclear membrane protein lamin B receptor (LBR), consistent with their Pelger–Huet-like nuclear morphology, we used a novel mouse model system to test the hypothesis that a disruption in the structure of the nucleus itself also contributes to the development of lupus autoimmunity. The lupus-prone mouse strain New Zealand White (NZW) was crossed with c57Bl/6 mice harboring a heterozygous autosomal dominant mutation in Lbr (B6.Lbric/+), and the (NZW×B6.Lbric)F1 offspring were evaluated for induction of lupus autoimmunity. Only female (NZW×B6.Lbric)F1 mice developed lupus autoimmunity, which included splenomegaly, kidney damage and autoantibodies. Kidney damage was accompanied by immune complex deposition, and perivascular and tubule infiltration of mononuclear cells. The titers of anti-chromatin antibodies exceeded those of aged female MRL-Faslpr mice, and were predominantly of the IgG2 subclasses. The anti-nuclear antibody staining profile of female (NZW×B6.Lbric)F1 sera was complex, and consisted of an anti-nuclear membrane reactivity that colocalized with the A-type lamina, in combination with a homogeneous pattern that was related to the recognition of histones with covalent modifications that are associated with gene activation. An anti-neutrophil IgM recognizing calreticulin, but not myeloperoxidase (MPO) or proteinase 3 (PR3), was also identified. Thus, alterations in nuclear structure contribute to lupus autoimmunity when expressed in the context of a lupus-prone genetic background, suggesting a mechanism for the development of lupus autoimmunity in genetically predisposed individuals that is induced by the disruption of nuclear architecture.

Anti-dsDNA antibodies in systemic lupus erythematosus: A combination of two quantitative methods and the ANA pattern is the most efficient strategy of detection

Several methods have been used to measure anti-double-stranded DNA auto-antibody (anti-dsDNA). Our aim was to determine the most efficient strategy to test anti-dsDNA in systemic lupus erythematosus (SLE). In this study, anti-dsDNA and anti-nuclear antibody (ANA) tests were requested for 644 patients. Anti-dsDNA was tested by RIA, ELISA and CLIA in all patients. The results indicated that 78 patients had a positive anti-dsDNA test according to at least one of the methods. After a 3-year follow-up period only 26 patients were diagnosed with SLE. We evaluated each method and combination of methods. Specificity and positive predictive value (PPV) increased with the number of assay methods used (p=0.002 for trend), and PPV was 100% in patients whose results were positive by all three anti-dsDNA assay methods. The proportion of anti-dsDNA-positive patients who had SLE was highest (82%; p<0.001) among those with a homogeneous pattern of ANA staining, followed by those with a speckled pattern. In ANA positive patients, when only RIA was considered, 59% of anti-dsDNA-positive patients had SLE, but when RIA and CLIA were both considered, all patients with positive results on both tests had SLE. The combination of RIA+CLIA in patients with homogeneous and speckled ANA staining showed a similar cost and higher sensitivity than RIA alone in ANA positive patients (p<0.001). We conclude that the most efficient strategy was to combine simultaneously two quantitative and sensitive methods but only in patients with a homogeneous or speckled pattern of ANA staining. This approach maximized specificity and PPV, and reduced costs.

Systemic lupus erythematosus onset in lupus-prone B6.MRL/lpr mice Is influenced by weight gain and Is preceded by an increase in neutrophil oxidative burst activity

In this study, we assessed whether weight gain influenced the systemic lupus erythematosus (SLE) onset and/or outcome, and examined the role that reactive oxygen species (ROS) production by neutrophils played in the SLE onset and/or outcome. Female control (C57BL/6) and lupus-prone B6.MRL/lpr mice (CM and LPM, respectively) at 4 weeks old were fed standard diet or standard diet plus cafeteria diet during 12 weeks. SLE diagnosis relied on the presence of both antinuclear antibodies (ANA) and renal abnormalities. We found that the percentage of weight gain in CM and LPM increased as a function of the length of cafeteria diet feeding period, but it was not associated with energy intake. Cafeteria diet-fed CM and LPM at 8 and 12 weeks old were overweight, while CM and LPM at 16 weeks old were obese. Compared with standard diet-fed CM and LPM, cafeteria diet-fed CM and LPM exhibited elevated glucose and total cholesterol levels, and diminished triglycerides levels. Standard diet-fed 16-week-old LPM and cafeteria diet-fed 12-week-old LPM had nephritis, characterized by the increased interstitial infiltration of leukocytes. Cafeteria diet-induced weight gain rose the frequency of homogeneous and speckled ANA staining patterns in the 12- and 16-week-old LPM groups. Together, these results indicated that weight gain anticipated the SLE onset. In addition, neutrophils from cafeteria diet-fed 8-week-old LPM exhibited augmented ROS production capacity; in standard diet-fed LPM, such rise occurred only in the 16-week-old group. Thus, the neutrophil ROS production capacity was increased before the SLE onset and during its outcome. Overweight and obese CM and LPM displayed elevated levels of kidney, liver, heart, and spleen lipid peroxidation. In conclusion, cafeteria diet-induced weight gain is associated with the increased production of ANA and neutrophil-derived ROS, which may contribute to accelerate the SLE onset.

Oral lichenoid lesions and serum antinuclear antibodies in Thai patients

The aim of this study was to investigate the presence of serum antinuclear antibodies (ANA) in Thai oral lichenoid drug reaction (OLDR) and oral lichen planus (OLP) patients. This study comprised 20 patients diagnosed with OLDR, 23 patients with OLP, and 24 healthy control subjects. Participants' blood samples were assayed for ANA staining patterns and serum ANA titer levels by immunofluorescence using human epithelial type 2 (HEp-2) as a substrate. The serum ANA titer levels were defined as low (1:40-1:80), medium (1:160-1:320), and high (>1:640). Serum ANA were detected in 73.9%, 70%, and 25% of OLP, OLDR, and control subjects, respectively. There was a statistically significant difference between the number of serum-ANA-positive subjects in the OLP or OLDR groups and the control group (P < 0.01), but no significant difference between the OLP and OLDR groups. The speckled pattern was the most commonly found staining pattern, present in 60.9%, 55.0%, and 20.8% of the OLP, OLDR, and control subjects, respectively. The number of subjects with low ANA titers in the OLP and OLDR groups was significantly higher than that of the control group (P < 0.01). Medium ANA titers were found in 15%, 4.4%, and 4.2% of the OLDR, OLP, and control subjects, respectively, while high ANA titers were not found in any group. The number of serum-ANA-positive OLP and OLDR patients was significantly higher than the control group. Speckled pattern and low titer levels were the most common findings in both OLP and OLDR groups.

AB0513 The Clinical Significance of Rarely Seen Staining Patterns of anti Nuclear Antibodies

BackgroundAnti nuclear antibodies (ANA) are one of the key diagnostic analyse for autoimmune diseases in rheumatological practice. The staining patterns of ANA include two main subgroups; nuclear and cytoplasmic staining...

Anti-ENA profiles related with anti-SS-A/Ro. The detection of Ro52 and Ro60 according to the presence of SS-B/La, and ANA pattern and titer

Anti-Ro52 (Ro52) and anti-Ro60 (Ro60) antibodies are associated with different clinical entities. We investigated their relationship with the presence of anti-SS-B/La (SSB) antibody, the pattern and titer of antinuclear antibody (ANA), and the variations in antibody profiles related with anti-SS-A/Ro (SSA) positivity. Our aim was to develop a strategy to increase the efficiency of anti-extractable nuclear antigen (ENA) determinations. Statistical analyses were based on the Chi-squared test for categorical variables, the Mann–Whitney U test to compare profiles, and the odds ratio (OR) and 95% confidence interval (95% CI) to estimate the risk of variability. We analyzed 800 SSA-positive samples with Ro52 or Ro60 reactivity. The most frequent profiles were Ro52+Ro60+SSB (n=349, 43.6%); Ro52+Ro60 (n=126, 15.8%); Ro52 (n=121, 15.1%) and Ro60 (n=71, 8.9%). In samples positive only for SSA and an ANA titer ≤1:640, the most likely profile was positivity for either Ro52 or Ro60, whereas when the ANA titer was >1:640, positivity for both Ro52 and Ro60 simultaneously was more likely (p<0.001). In samples positive for both SSA and SSB, the most likely profile was Ro52+Ro60+SSB regardless of the ANA titer (p=0.001). When only SSA was positive and the ANA staining pattern was nucleolar, centromeric or cytoplasmic, Ro52 positivity was most likely (p<0.001). When both SSA and SSB were positive, both Ro52 and Ro60 were likely to be positive regardless of the ANA staining pattern. In 28.7% of the patients the profile was variable. Variability was significantly greater in those with the SSA profile (23/67) than with the SSA+SSB profile (15/105; OR=1.9, 95% CI=1.1–3.3; p=0.025), and the difference in variability was greatest between the Ro52+Ro60 profile (8/23) and the Ro52+Ro60+SSB profile (8/68; OR=4.2, 95% CI=1.9–9.5; p<0.001). We conclude that to increase efficiency in the immunology laboratory, positivity for Ro52 and Ro60 individually or simultaneously can be deduced from SSB status and the ANA pattern and titer. In general, for the most frequent anti-ENA findings, priority should be given to retesting autoantibodies not detected in the initial analysis.

Associations between antinuclear antibody staining patterns and clinical features of systemic lupus erythematosus: analysis of a regional Swedish register

ObjectiveAntinuclear antibody (ANA) analysis by immunofluorescence (IF) microscopy remains a diagnostic hallmark of systemic lupus erythematosus (SLE). The clinical relevance of ANA fine-specificities in SLE has been addressed repeatedly, whereas...