Effect of antinuclear antibody positivity on antineutrophil cytoplasmic antibody results by indirect immunofluorescence assay.

BACKGROUND: The indirect immunofluorescence assay (IIFA) utilizing antineutrophil cytoplasmic antibodies (ANCA) is widely used as a diagnostic test for autoimmune vasculitis. The presence of antinuclear antibodies (ANA) might lead to a misleading interpretation of ANCA. This study aims to explore the impact of the presence of ANA on the interpretation of ANCA. METHODS: This retrospective research examined samples negative for antiMPO and antiPR3 ANCA by IIFA and explored correlations between the ANA-IIFA results and the ANCA interpretation frequencies. Our analysis involved the use of suitable statistical methods, including Chi-square and kappa statistics. RESULTS: Up to 75.2% of the ANCA-IIFA-positive samples exhibited a positive p-ANCA pattern when using the ethanol-fixed substrate, with c-ANCA positivity at 24.8%. In the ANA-IIFA-positive samples, ~77.3% displayed p-ANCA patterns on ethanol-fixed substrates. A comparison between the ANA-IIFA titers and the p-ANCA results revealed that p-ANCA positivity was notably more common in samples with higher titers, and this correlation was found to be statistically significant. CONCLUSION: Positive ANA results by IIFA tests are linked to a higher incidence of p-ANCA interpretation, particularly in cases with higher titer patterns. This insight aids laboratories in establishing effective workflows to investigate potential p-ANCA interference.

Neonatal lupus erythematosus as a rare trigger of gastrointestinal involvement in neonates.

Cutaneous and cardiac involvement in neonatal lupus erythematosus (NLE) has been extensively studied; however, gastrointestinal system involvement (GSI) remains unexplored. This study aimed to investigate the clinical features of GSI in patients with NLE with a particular focus on feeding intolerance (FI) and diarrhea. We conducted a retrospective analysis of the clinical data of patients diagnosed with NLE at the Children's Hospital of Soochow University between 2011 and 2022. In this study, of 39 patients diagnosed with NLE, 27 presented with GSI. 9 patients who presented with FI or diarrhea as the primary manifestation were positive for anti-SSA antibody, and 5 were dual positive for anti-SSA and anti-SSB antibodies. Among the mothers of the NLE patients with GSI, 18 had systemic lupus erythematosus, 3 had Sjogren's syndrome, 2 had mixed connective tissue disease, and one each had autoantibody abnormalities and photosensitivity symptoms; 4 mothers denied having any autoimmune disease. In this study, 69.23% of patients with NLE exhibited GSI, which was linked to hypocomplementemia and anti-SSA antibodies. Thus, clinicians should remain vigilant for NLE in neonates, particularly when accompanied with rash and other organ dysfunction and when the high-risk factors of FI and diarrhea have been excluded.

Clinical usefulness of anti-nuclear antibody in childhood: real-world experience at a tertiary care center : Usefulness of ANA in pediatric autoimmune diseases.

We evaluated the reasons for requesting anti-nuclear antibody (ANA) analysis in clinical practice at a tertiary center and the performance of ANA in pediatric autoimmune diseases. Patients under 18 years of age who underwent ANA testing for various symptoms between 2013 and 2017 were included. We retrieved data from medical records, including demographic and clinical characteristics, diagnoses, ANA results, titers, and staining patterns. The performance assessment tools were calculated according to the ANA titer for autoimmune diseases. Risk factors for autoimmune diseases in ANA-positive patients were evaluated using logistic regression analysis. Changes in ANA titer and seroconversion were evaluated using repeated ANA analyses. A total of 3812 patients underwent ANA. Medical records of 3320 patients were obtained. The rate of ANA positivity was 27.4%. ANA was requested most frequently because of musculoskeletal findings in 1355 patients (40.8%). Juvenile idiopathic arthritis (n = 174, 20.2%) was the most common diagnosis in ANA-positive patients, followed by systemic lupus erythematosus (n = 52, 6%). For autoimmune diseases, a titer of ≥ 1:100, a sensitivity of 40.1%, and a specificity of 77.1% were observed. At a titer ≥ 1:1000, the sensitivity and specificity were 24.1% and 89%, respectively. Homogeneous staining was an additional risk factor for autoimmune diseases in ANA-positive patients by multivariate logistic regression analysis (OR [95% CI]: 4.562 [3.076-6.766], p < 0.001).    Conclusion: Our results revealed that the performance of the ANA test in diagnosing autoimmune diseases in pediatric clinical practice was poor. Therefore, clinical findings should be carefully evaluated before ANA testing is performed. What is Known: • ANA can be detected in systemic autoimmune rheumatic diseases. • The diagnostic role of ANA is controversial, especially in childhood. What is New: • One in four patients who requested the ANA test had an autoimmune disease. • Less than half of patients with an autoimmune disease had ANA positivity.

Diagnostic Performance of Anti-dsDNA Tests by Indirect Immunofluorescence and Enzyme-linked Immunosorbent Assay in Patients with Systemic Lupus Erythematosus.

BACKGROUND: Several laboratory techniques for anti double-stranded (ds) DNA detection in systemic lupus erythematosus (SLE) are available, with variable diagnostic performance. We aimed to evaluate anti-dsDNA's diagnostic performance by indirect immunofluorescence (IIF) and enzyme-linked immunosorbent assay (EIA). METHODS: We conducted a single-center retrospective (2015 to 2020) study. Patients with anti-dsDNA tests by IIF and EIA were included. We evaluated the indications, applications, concordance, positive predictive value (PPV) of anti-dsDNA to confirm SLE diagnosis or flares, and associations of disease manifestations with positivity with each technique. RESULTS: A total of 1368 reports of anti-dsDNA tests by IIF and EIA and the corresponding medical records of the patients were analyzed. The main indication for anti-dsDNA testing was to help in the diagnosis of SLE in 890 (65%) of the samples, and the main application after obtaining the results was SLE exclusion in 782 (57.2%) cases. The combination with the highest frequency was the negativity result by both techniques in 801 (58.5%) cases (Cohen kappa 0.57). Both methods were positive in 300 patients with SLE (Cohen kappa 0.42). The PPVs of anti-dsDNA tests to confirm diagnosis/flare was 79.64% (95% CI, 75.35-83.35) by EIA, 78.75% (95% CI, 74.27-82.62) by IIF, and 82% (95% CI, 77.26-85.93) when both were positive. CONCLUSIONS: Anti-dsDNA detection by IIF and EIA are complementary and may indicate different clinical patterns in patients with SLE. The detection of anti-dsDNA antibodies by both techniques has a higher PPV than either separately for confirming SLE diagnosis or flares. These results highlight the need for evaluating both methods in clinical practice.

An automatic immunofluorescence pattern classification framework for HEp-2 image based on supervised learning.

Immunofluorescence patterns of anti-nuclear antibodies (ANAs) on human epithelial cell (HEp-2) substrates are important biomarkers for the diagnosis of autoimmune diseases. There are growing clinical requirements for an automatic readout and classification of ANA immunofluorescence patterns for HEp-2 images following the taxonomy recommended by the International Consensus on Antinuclear Antibody Patterns (ICAP). In this study, a comprehensive collection of HEp-2 specimen images covering a broad range of ANA patterns was established and manually annotated by experienced laboratory experts. By utilizing a supervised learning methodology, an automatic immunofluorescence pattern classification framework for HEp-2 specimen images was developed. The framework consists of a module for HEp-2 cell detection and cell-level feature extraction, followed by an image-level classifier that is capable of recognizing all 14 classes of ANA immunofluorescence patterns as recommended by ICAP. Performance analysis indicated an accuracy of 92.05% on the validation dataset and 87% on an independent test dataset, which has surpassed the performance of human examiners on the same test dataset. The proposed framework is expected to contribute to the automatic ANA pattern recognition in clinical laboratories to facilitate efficient and precise diagnosis of autoimmune diseases.

Antinuclear antibodies in childhood immune thrombocytopaenia: An increased risk for development of autoimmune disorders?

Antinuclear antibodies (ANA) have been detected in children with primary immune thrombocytopaenia (ITP), but the effect of ANA titres on clinical outcomes is unclear. Liu et al. retrospectively analysed a cohort of 324 children with primary ITP with a median follow-up time of 25 months and found that patients with high-ANA titres (≥1:160) had lower platelet counts at the onset with a higher subsequent platelet count recovery rate, and additionally were at an increased risk to develop autoimmune disease. These data highlight the possible predictive potential of ANA titres with respect to platelet counts and the development of autoimmunity in children with primary ITP. Commentary on: Liu, et al. The effect of antinuclear antibody titre and its variation on outcomes in children with primary immune thrombocytopaenia. Br J Haematol 2023;202:412-421.

Analytical aspects of the antinuclear antibody test by HEp-2 indirect immunofluorescence: EFLM report on an international survey.

OBJECTIVES: Detection of antinuclear antibodies (ANA) by indirect immunofluorescence assay using HEp-2 cells (HEp-2 IFA) is used to screen for various autoimmune diseases. HEp-2 IFA suffers from variability, which hampers harmonization. METHODS: A questionnaire was developed to collect information on HEp-2 IFA methodology, computer-assisted diagnosis (CAD) systems, training, inter-observer variability, quality assessment, reagent lot change control, and method verification. The questionnaire was distributed to laboratories by Sciensano (Belgium), national EASI groups (Italy, Croatia, Portugal, Estonia, Greece) and ICAP (worldwide). Answers were obtained by 414 laboratories. The results were analysed in the framework of the recent EFLM/EASI/ICAP ANA recommendations (companion paper). RESULTS: Laboratories used either HEp-2, HEp-2000, or HEp-20-10 cells and most laboratories (80%) applied the same screening dilution for children and adults. The conjugate used varied between laboratories [IgG-specific (in 57% of laboratories) vs. polyvalent]. Sixty-nine percent of CAD users reviewed the automatic nuclear pattern and 53% of CAD users did not fully exploit the fluorescence intensity for quality assurance. Internal quality control was performed by 96% of the laboratories, in 52% of the laboratories only with strongly positive samples. Interobserver variation was controlled by 79% of the laboratories. Limited lot-to-lot evaluation was performed by 68% of the laboratories. Method verification was done by 80% of the respondents. CONCLUSIONS: Even though many laboratories embrace high-quality HEp-2 IFA, substantial differences in how HEp-2 IFA is performed and controlled remain. Acting according to the EFLM/EASI/ICAP ANA recommendations can improve the global performance and quality of HEp-2 IFA and nurture harmonization.

Antinuclear antibodies in children: indirect immunofluorescence patterns, antigen targets and associated diagnoses / Anticorps antinucléaires chez l'enfant : aspects en immunofluorescence indirecte, cibles antigéniques et situations cliniques associées

Antinuclear antibodies tests are of a paramount importance in the diagnosis, classification, prognostic evaluation and management of autoimmune diseases in children. The present study aimed to describe the immuno-clinical profile of antinuclear antibodies tests in a pediatric population in order to guide the clinical practice of biologists and clinicians. Our study enrolled 268 children. Antinuclear antibodies screening was performed using the indirect immunofluorescence assay on HEp-2 cells. Identification of target antigens was conducted using separately or at one timepoint the following techniques: enzyme-linked immunosorbent assay, Immunodot and Chemiluminescence. The average age of patients was 9.6 ± 4.3 years, with a female predominance (sex-ratio = 1.9). Antinuclear antibodies screening was positive in 40.67% of cases. The most frequently observed antinuclear antibodies patterns were speckled (52.3%), homogeneous (13.8%) and mixed homogeneous-speckled (13.8%). Autoantibodies were detected in 4 patients (2.51%) for whom ANA testing using the indirect immunofluorescence assay was negative. Positive antinuclear antibodies specificities were detected in connective tissue diseases (44.03%; n = 48), organ-specific autoimmune diseases (10.09%; n = 11), and in non-autoimmune conditions (inflammatory diseases, infections, hematological diseases, vasculitis and Wilson's disease) (32.08%; n = 35). Our study revealed a high rate of positive antinuclear antibodies tests in the pediatric population, mainly related to autoimmune diseases (54.12%) besides non-autoimmune conditions (32.08%). Therefore, screening and interpretation of antinuclear antibodies testing in children require the consideration of clinical data and a close collaboration between clinicians and biologists.

ANA-specific antibodies, ANA patterns, anti-ds-DNA results, and clinical diagnosis: a laboratory and clinical audit.

The diagnosis of systemic autoimmune diseases (SAID) is challenging, due to overlapping features with other non-immune disorders. Anti-nuclear antibodies (ANA)/anti-cellular antibodies are the sensitive screening tests but anti-double-stranded-deoxyribonucleic acid-antibody (anti-ds-DNA) and ANA-specific antibodies are specific for SAID. We aimed to look at ANA-specific antibodies in our patients and correlated them with ANA patterns, anti-ds-DNA, and clinical diagnosis for proper interpretation and better patient management cost-effectively. A retrospective data analysis of 641 patients was done (1st of February 2019 to 31st of July 2021) who were tested for ANA-specific antibodies at the Immunology Department of Indus Hospital and Health Network. ANA and anti-ds-DNA results and clinical diagnosis were also analyzed for ANA-specific antibody-positive patients. Descriptive data were presented in mean ± standard deviation and frequency percentages whereas inferential data were analyzed with a chi-square test for association between ANA-specific antibodies status, ANA, anti-ds-DNA, and clinical features. ANA-specific antibodies test revealed positivity for at least one autoantibody in 245 (38.2%) patients. Of these, ANA was tested in 206 patients reactive for ANA-specific antibodies and found positive in 195 (95%) as compared to negative (< 0.001). Speckled and homogenous were predominant ANA patterns in ANA-specific antibody-positives (56% and 42% respectively). Multiple ANA patterns were found in 18 patients most commonly with systemic lupus erythematosus (SLE) and mixed connective tissue disorder (MCTD). Anti-SSA were the most common ANA-specific antibodies (50%) and were mostly found in sera with speckled (61/97) and homogenous (38/97) patterns and associated mostly with SLE (48%) and Sjogren's syndrome (86%). Among ANA-negative patients, anti-SSA were the most common antibodies (n = 5). Anti-ds-DNA was found in 66% of SLE patients along with another ANA-specific antibody. This study showed that testing for ANA-specific antibodies cannot be gated on ANA patterns. Also, there is a redundancy of these antibodies with various clinical diagnoses. Moreover, they are useful in making a diagnosis in ANA-negative patients as well with clinical suspicion.

Feasibility of Indirect immunofluorescence (IIF) alone as a screening method for antinuclear antibody in connective diseases in India's sub-Himalayan region.

Background: For the management of connective tissue disorders (CTDs), antinuclear antibody (ANA) testing is essential, both from diagnostic and prognostic points of view. Usually, patterns obtained by ANA-IIF testing correlates to specific autoantibodies as obtained from the test for ENA (by LIA/ELISA, etc.). But to apply these data from western studies, we may need validation in the local population like our subjects in sub-Himalayan (Garhwal region) area where CTDs are common. Also, suppose ANA-IFA pattern's correlation is reliably known in our population, it can minimize the cost of managing CTDs by limiting ENA testing, which is 10 times costlier than ANA-IIF. Hence, this study was undertaken to know the specific autoantibody targets (ENA by LIA) against ANA-IIF patterns in our local population. Materials and Methods: In this retrospective cross-sectional work, serum samples of CTDs were tested for ANA by IIF (Euroimmune AG) and ENA by LIA (Euroline ANA-3G) continuously for 36 months. The manufacturer's kit insert was followed, and results were analyzed applying appropriate statistical methods. Results: Major ANA-IIF patterns were found to be associated with specific autoantibodies, for example, Nuclear homogenous with dsDNA, nucleosomes, histones; speckled pattern with nRNP/Sm, Sm, SSA/Ro-52, SSB; nucleolar pattern with Scl-70, Pm-Scl 100 and centromere pattern with CENP-B. Anticytoplasmic (ACA) are found to be linked with some ANA negative (by IIF) samples, emphasizing the need for careful observation for ACA especially where ANA is not found. Conclusions: In most subjects, specific ENA targets correlated well with ANA-IIF patterns, implying effective cost minimization in CTD management. Similar future prospective studies (with clinical data) can provide a database and reference for our population.

The influence of demography and referral medical specialty on the detection of autoantibodies to HEP-2 cells in a large sample of patients.

BACKGROUND: The prevalence of anti-cell autoantibodies detected by indirect immunofluorescence assay on HEp-2 cells (HEp-2-IIFA) increases with age and is higher in female sex. The number of medical specialties that use HEp-2-IIFA in the investigation of autoimmune diseases has increased lately. This study aimed to determine the prevalence and patterns of autoantibodies on HEp-2-IIFA according to demographics variables and referring medical specialties. METHODS: A retrospective analysis of the HEp-2-IIFA carried out between January and June of 2017 was performed. The International Consensus on Antinuclear Antibodies Patterns (ICAP) and the Brazilian Consensus on Autoantibodies were used for patterns definition on visual reading of the slides. Anti-cell (AC) codes from ICAP and Brazilian AC codes (BAC) were used for patterns classification. RESULTS: From 54,990 samples referred for HEp-2-IIF testing, 20.9% were positive at titer ≥ 1/80. HEp-2-IIFA positivity in females and males was 24% and 12%, respectively (p < 0.0001). The proportion of positive results in the 4 age groups analyzed: 0-19, 20-39, 40-59, and ≥ 60 years was 23.3, 20.2, 20.1, and 22.8%, respectively (p < 0.0001). Considering all positive sera (n = 11,478), AC-4 nuclear fine speckled (37.7%), AC-2 nuclear dense fine speckled (21.3%), BAC-3 nuclear quasi-homogeneous (10%) and mixed/composite patterns (8.8%) were the most prevalent patterns. The specialties that most requested HEp-2-IIFA were general practitioner (20.1%), dermatology (15%), gynecology (9.9%), rheumatology (8.5%), and cardiology (5.8%). HEp-2-IIFA positivity was higher in patients referred by rheumatologists (35.7% vs. 19.6%) (p < 0.0001). Moderate (46.4%) and high (10.8%) titers were more observed in patients referred by rheumatologists (p < 0.0001). We observed a high proportion of mixed and cytoplasmic patterns in samples referred by oncologists and a high proportion of BAC-3 (nuclear quasi-homogeneous) pattern in samples referred by pneumologists. CONCLUSIONS: One-fifth of the patients studied were HEp-2-IIFA-positive. The age groups with more positive results were 0-19 and ≥ 60 years. AC-4, AC-2, BAC-3 and mixed/composite patterns were the most frequent patterns observed. Rheumatologists requested only 8.5% of HEp-2-IIFA. Positive results and moderate to high titers of autoantibodies were more frequent in patients referred by rheumatologists.

Autoantibody cluster analysis in juvenile lupus nephritis.

OBJECTIVE: Demographics, clinical features, and biomarkers do not consistently anticipate risk of end-stage renal disease (ESRD) in juvenile lupus nephritis (LN). Here, the existence of autoantibody clusters predictive of ESRD was explored in a cohort of biopsy-proven juvenile LN. METHODS: A retrospective chart review was performed of patients with juvenile systemic lupus erythematosus (jSLE) and biopsy-confirmed LN. Primary endpoints were ESRD and mortality. Patients were included for K-medians cluster analysis if they had a complete autoantibody profile, which included ANA titer, anti-dsDNA, anti-Smith, anti-RNP, anti-Ro/SSA, anti-La/SSB. Chi-square test was used for categorical variables and one-way ANOVA for continuous measures. Significance was p<0.05. RESULTS: Fifty-three met inclusion criteria, of which 45 were female and 37 were black. Over 80% developed LN within one year of jSLE onset and more than half (n=29) had LN at diagnosis of jSLE. Six developed ESRD. No mortalities were reported. Forty-six had a complete autoantibody profile, including four with ESRD. Three clusters were identified. Group 1 (n=8) was defined by anti-dsDNA; group 2 (n=28) by high-titer ANA (>1:1280), anti-Smith, anti-RNP, and anti-Ro/SSA; and group 3 (n=10) by anti-dsDNA and anti-Ro/SSA. There was no difference between the groups in demographics, jSLE manifestations, or markers of renal function. One in group 2 and three in group 3 developed ESRD. Those in group 3 were younger at diagnosis of LN (p=0.084) and had the highest frequency of ESRD (p=0.025). CONCLUSION: Cluster analysis revealed the highest frequency of ESRD in the group with LN defined by anti-Ro/SSA and anti-dsDNA co-positivity. Key Points • Lupus nephritis commonly is present at diagnosis of juvenile systemic lupus erythematosus or develops within the first year. • End-stage renal disease was more frequent in the cluster defined by anti-dsDNA and anti-Ro/SSA co-positivity; patients with this profile may benefit from more aggressive immunosuppression.

Fucosylation of anti-dsDNA IgG1 correlates with disease activity of treatment-naïve systemic lupus erythematosus patients.

BACKGROUND: Systemic Lupus Erythematosus (SLE) is a complex and heterogeneous autoimmune disease mediated by quantities of autoantibodies in which anti-double-stranded DNA (anti-dsDNA) antibodies are important. Besides, glycosylation is one of the most commonly post-translational modifications of antibodies. The association of anti-dsDNA antibodies glycosylation and SLE disease activity is still unknown. METHODS: We enrolled 101 consecutive treatment-naïve SLE patients with positive anti-dsDNA antibodies from the Department of Rheumatology and Immunology at Ruijin Hospital, Shanghai, between 2017 and 2019. Serum samples were used in this study. We analysed the glycosylation of anti-dsDNA IgG and total IgG subclasses according to systemic lupus erythematosus disease activity index (SLEDAI) scores. Statistical analysis and machine learning were performed to assess the correlation between glycosylation of anti-dsDNA IgG and total IgG with disease activity. FINDINGS: Serum samples from 86 patients could be detected with anti-dsDNA IgG glycopeptide and subclass of IgG glycoform. Cluster analysis showed that glycosylation of anti-dsDNA IgG and total IgG subclasses were different in SLE patients. Fucosylation, galactosylation, and sialylation levels of anti-dsDNA IgG1 were increased with SLEDAI scores (all p<0.05). The results of machine learning showed that all the glycoforms of anti-dsDNA IgG1 had better performance with lower standardised square error (SSE) than that of total IgG1, with anti-dsDNA IgG1 fucosylation level having the lowest SSE (0.009). INTERPRETATION: Our study indicated that glycosylation of anti-dsDNA IgG was different from that of total IgG and fucosylation of anti-dsDNA IgG1 correlated best with SLE disease activity. FUNDING: This work is supported by the National Key Research and Development Program of China (2018YFC0910303), National Natural Science Foundation of China (81801592, 82101876), Clinical Research Plan of SHDC (SHDC2020CR4011), Ruijin Hospital Youth Incubation Project (KY2021607) and Shanghai Pujiang Young Rheumatologists Training Program (SPROG202006).

Laboratory evaluation of anti-dsDNA antibodies.

Antibodies to dsDNA are an important laboratory parameter for diagnosis, monitoring and classification of systemic lupus erythematosus (SLE). In clinical laboratories, several techniques are used to detect and quantify anti-dsDNA antibodies. Each technique has its advantages and disadvantages regarding sensitivity, specificity, avidity and assay procedure. Assays differ with respect to the antigen source (native versus synthetic versus molecular biological) used and the way the antigen is presented (e.g. in solution, covalently linked to a solid phase,…). Consequently, correlation between assays can be poor and standardization of anti-dsDNA antibody tests is challenging. We here provide an overview of the currently available anti-dsDNA tests frequently used in clinical laboratories [Crithidia luciliae immunofluorescence test (CLIFT), Enzyme linked immune sorbent assay (ELISA), fluoroenzyme immunoassay (FEIA), chemiluminisence immunoassay (CIA), multiplexed bead-based assays and Farr-RIA] and their performance characteristics. From this literature study, we concluded that performance characteristics differ between assays. Often, a combination of techniques is necessary for the best result interpretation.

Longitudinal Tracking of Extractable Nuclear Antigen (ENA) Antibodies in a Quaternary Hospital Laboratory Cohort Reveals Dynamic Antibody Profiles.

BACKGROUND: Antiextractable nuclear antigens (anti-ENAs) are regarded as diagnostic tests with no established value for serial monitoring. We therefore sought to establish the stability over time of anti-ENAs in a large diagnostic immunopathology laboratory. METHODS: A retrospective review of all patients who had a serial anti-ENA ordered at the Westmead Hospital (Sydney, Australia) was performed over 24 months. Anti-ENA characterization was performed using line immunoassay, and historical data were available from 2013 onward. The earliest available densitometry readings were compared with the latest available to examine for a change in quantitation or qualitative (serostatus) result (from negative to positive, and vice versa). Medical records were examined for clinical correlations. RESULTS: A total of 283 patients (24.1%) had serial testing of anti-ENA in the audit period, with each patient having an average of 3.9 ± 2.9 tests each. Most patients were diagnosed with systemic lupus erythematosus or primary Sjögren's syndrome. About 25% and 58% of patients had a qualitative and quantitative change, respectively, in at least 1 anti-ENA in the study period. Changes in anti-ENA levels correlated with erythrocyte sedimentation rate and disease activity. Increasing duration between serial tests increased the probability of observing a change in anti-ENA levels. CONCLUSION: Certain anti-ENAs are dynamic autoantibodies that may have significance for monitoring disease activity. Laboratories may consider reporting quantitative results. Further disease- and autoantibody-specific studies are required to determine the clinical significance of changes in anti-ENAs.

Using two-step cluster analysis to classify inpatients with primary biliary cholangitis based on autoantibodies: A real-world retrospective study of 537 patients in China.

Background: A variety of autoantibodies have been detected in primary biliary cholangitis (PBC), while the presence of autoantibody clusters and their clinical significance have not been fully understood. We aimed at defining autoantibody clusters and to better understand the clinical features and prognosis of PBC patients based on autoantibody clusters under real-world conditions. Methods: We retrospectively analyzed 788 inpatients with PBC evaluated between October 2008 and July 2019, and included 537 patients. Nineteen autoantibodies which were measured routinely were investigated for cluster analysis. Two-step clustering, Kaplan-Meier survival, and Cox regression analyses were used. Results: Five clusters were defined. A cluster of antinuclear antibodies (ANA) and anti-gp210 positive patients were identified with a high rate of cirrhosis at baseline and low survival rate; a cluster of ANA, anti-centromere antibodies (ACA) and/or anti-CENP-B female dominant patients with older disease onset, low level of platelet count at baseline, high rate of hepatic decompensation, and low survival rate was also characterized; and another cluster of anti-mitochondrial antibodies (AMA) and/or AMA-M2, anti-Ro52 and a high rate of anti-gp210 positive patients were identified with a high proportion of male patients and low survival rate. A subgroup of patients with anti-SSA and/or anti-SSB coexists with SjS was also identified; patients with only AMA and/or AMA-M2-positive with a benign clinical outcome and relatively high complication of non-alcoholic fatty liver disease (NAFLD) were also identified. Only anti-gp210 was considered as a significant predictor for poor outcomes especially in patients with cirrhosis. Conclusion: Clustering methods allow the identification of distinct autoantibody profiles of PBC that form clinical subsets and can be useful for personalized approaches to diagnosis, clinical management, and the prediction of clinical outcomes. Anti-gp210 was the strongest predictive factor for poor outcomes especially in PBC patients with cirrhosis under real-world conditions.

Dense fine speckled immunofluorescence pattern in a Chinese population: Prevalence and clinical association.

OBJECTIVE: To provide information on the prevalence and possible clinical association in a Chinese population for medical practice of the dense fine speckled pattern (DFS pattern). METHODS: A retrospective study was conducted with patients who had the DFS pattern from June 2018 to December 2019 in West China Hospital. RESULTS: A total of 469 patients (1.27% of patients with positive anti-nuclear antibody indirect immunofluorescence (ANA IIF) test results) revealed the DFS pattern, of which 92.96% had isolated DFS pattern and 23.67% had titers above/equal to 1:320. The average age of patients with the DFS pattern was 43.45 years, and females accounted for 76.97% of them. Ten different kinds of diseases made up the vast majority of the disease spectrum, in which inflammatory or infectious diseases (46.11%), mental diseases (21.45%), and systemic autoimmune rheumatic diseases (SARDs) (18.23%) ranked in the top three. The most common SARDs were rheumatoid arthritis (RA), undifferentiated connective tissue disease (UCTD), and systemic lupus erythematosus (SLE). Forty-six patients (10.55%) had positive or suspicious extractable nuclear antigen (ENA) antibodies test results and a higher risk of suffering from SARDs. Forty-seven patients would be missed if the DFS pattern with negative ENA antibodies test result was considered as exclusion criterion of SARDs. CONCLUSIONS: The DFS pattern is basically isolated and with low titer. It is unwise to exclude the diagnosis of SARDs only depending on the appearance of the DFS pattern. Autoimmune diseases-related antibodies, clinical information of patients, and long-term follow-up are of great importance to avoid missed or delayed diagnosis of SARDs.

Antinuclear antibodies (ANAs) detected by indirect immunofluorescence (IIF) method in acute COVID-19 infection; future roadmap for laboratory diagnosis.

INTRODUCTION: As in other viral infections, anti-nuclear antibodies (ANAs) are observed in SARS-CoV-2 infection. We investigated the presence of autoantibodies in acute COVID-19 and the association with early laboratory findings. MATERIALS AND METHODS: We examined 50 sera (>18 years, 25 Female) from patients with acute COVID-19. ANAs (HEp-20-10 liver biochip), anti-neutrophil cytoplasmic antibody (ANCA, Europlus Granulocyte Mosaic 32) and anti-double stranded DNA were investigated with product of Euroimmune AG (Luebeck, Germany) by indirect immunofluorescence (IIF) method. Also, antibody against cyclic citrullinated peptide (anti-CCP) was examined by a chemiluminisens assay (Euroimmun AG, Luebeck, Germany). Samples from 50 blood bank donors collected before the COVID-19 pandemic were used as controls. RESULTS: The IIF-ANA test was positive in 18% (N = 9/50) of the patients. The median time of sample collection was 7 days (range: 1-28 days) after diagnosis. ANA was positive in only one (2%) control sample. Five (55.5%) patients were ANA positive with a strong titer (3+). There was no relationship between antibody titration and time of sample collection (p = 0,55). Anti-CCP was detected in a nucleolar (3+) positive patient (2%). ANA was detected in 14.28% (N = 1/7, rods-rings (±), p = 0,78) of patients in the intensive care unit(ICU). Patients treated in the clinic have more and higher titers of ANA, mostly in nucleolar patterns, than ICU patients. CONCLUSIONS: The variety of antibodies detected in acute COVID-19 and the uncertainty of how long they persist can lead to confusion, especially in the diagnosis of systemic autoimmune rheumatic diseases for IIF-ANA testing in immunology laboratories. Improvements in cell lines and methods will facilitate the diagnostic process.

Strong Association of the Myriad Discrete Speckled Nuclear Pattern With Anti-SS-A/Ro60 Antibodies: Consensus Experience of Four International Expert Centers.

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.

Clinical Experience of Proteasome Inhibitor Bortezomib Regarding Efficacy and Safety in Severe Systemic Lupus Erythematosus: A Nationwide Study.

As treatment options in advanced systematic lupus erythematosus (SLE) are limited, there is an urgent need for new and effective therapeutic alternatives for selected cases with severe disease. Bortezomib (BTZ) is a specific, reversible, inhibitor of the 20S subunit of the proteasome. Herein, we report clinical experience regarding efficacy and safety from all patients receiving BTZ as therapy for SLE in Sweden during the years 2014-2020. 8 females and 4 males were included with a mean disease duration at BTZ initiation of 8.8 years (range 0.7-20 years). Renal involvement was the main target for BTZ. Reduction of global disease activity was recorded by decreasing SLEDAI-2K scores over time and remained significantly reduced at the 6-month (p=0.007) and the 12-month (p=0.008) follow-up visits. From BTZ initiation, complement protein 3 (C3) levels increased significantly after the 2nd treatment cycle (p=0.05), the 6-month (p=0.03) and the 12-month (p=0.04) follow-up visits. The urine albumin/creatinine ratio declined over time and reached significance at the 6-month (p=0.008) and the 12-month follow-up visits (p=0.004). Seroconversion of anti-dsDNA (27%), anti-C1q (50%) and anti-Sm (67%) was observed. 6 of 12 patients experienced at least one side-effect during follow-up, whereof the most common adverse events were infections. Safety parameters (C-reactive protein, blood cell counts) mainly remained stable over time. To conclude, we report favorable therapeutic effects of BTZ used in combination with corticosteroids in a majority of patients with severe SLE manifestations irresponsive to conventional immunosuppressive agents. Reduction of proteinuria was observed over time as well as seroconversion of some autoantibody specificities. In most patients, tolerance was acceptable but mild adverse events was not uncommon. Special attention should be paid to infections and hypogammaglobinemia.