ABSTRACT
Objective
This study aimed to investigate antinuclear antibody (ANA) staining patterns and titers in patients with juvenile idiopathic arthritis (JIA)-associated uveitis, idiopathic uveitis, and JIA without uveitis, in order to identify serologic profiles that may contribute to disease pathogenesis and guide clinical decision-making.
Methods
We analyzed patients with JIA and/or uveitis at our tertiary center with ANA titers ≥1/100. Patients were grouped as JIA-associated uveitis, JIA without uveitis, and idiopathic uveitis. Diagnoses followed International League of Associations for Rheumatology and standardization of uveitis nomenclature criteria. ANA testing was performed by indirect immunofluorescence on HEp-2 cells, and patterns and titers were evaluated per International Consensus on ANA Patterns guidelines. ANA profiles were compared across patient groups and JIA subtypes.
Results
Ninety-one patients were included: 21 (23%) with idiopathic uveitis, 12 (13.1%) with JIA-associated uveitis, and 58 (63.7%) with JIA without uveitis. The AC-1 pattern was present in all uveitis patients. The most common ANA patterns in JIA were AC-1 (65.7%), AC-4/5 (21.4%), and AC-2 (10%). ANA profiles differed across JIA subtypes (p<0.001), with AC-1 dominant in oligoarticular JIA (74.5%) and AC-4/5 in enthesitis-related arthritis (50%).
Conclusions
Our findings show that ANA pattern differences in JIA subtypes may provide significant clues regarding disease pathogenesis and clinical prediction. In particular, the prominence of the AC-1 pattern in JIA-associated uveitis may suggest a potential biomarker for the early identification of uveitis risk, which should be further explored in larger prospective studies
INTRODUCTION
Juvenile idiopathic arthritis (JIA) is a heterogeneous group of diseases characterized by chronic arthritis of unknown cause with onset before the age of 161, 2. Different clinical subtypes are defined by international classification criteria and antinuclear antibody (ANA) positivity stands out as a determinant serologic marker, especially in oligoarticular JIA3. Chronic anterior uveitis is a significant clinical manifestation of JIA, predominantly observed in the oligoarticular subtype, and in those with ANA positivity4. If not promptly identified and managed, this condition can result in irreversible vision impairment, thereby constituting a major health concern for individuals with JIA4-7.
The ANA test is a widely used method in the serologic evaluation of autoimmune diseases8, 9. This test, which is performed on HEp-2 cells by indirect immunofluorescence (IIF) method, defines not only autoantibody positivity and titer level, but also staining patterns determined according to the target structures of autoantibodies10. These patterns are mainly categorized into nuclear, cytoplasmic, and mitotic groups and provide important clues in clinical evaluation11. The diagnostic and prognostic value of ANA patterns has increasingly been investigated because they show characteristic associations with some autoimmune diseases12-14. A limited number of studies in the literature have evaluated the discrimination of specific ANA patterns in JIA and JIA-associated uveitis15, 16. However, the clinical reflection of specific patterns underlying ANA positivity and their relationship with the development of uveitis in JIA patients has not yet been fully elucidated. Accordingly, this study investigates ANA staining patterns and titers in patients with JIA-associated uveitis, idiopathic uveitis, and JIA without uveitis, aiming to delineate distinctive serologic markers that may inform the pathogenesis and guide clinical decision-making.
MATERIALS and METHODS
Patients and Data Collection
Patients admitted to our tertiary care center between June 2022 and June 2025 with a diagnosis of JIA and/or uveitis and a positive ANA test titer of 1/100 or higher were included in the study. Exclusion criteria included those with concomitant systemic autoimmune or autoinflammatory conditions or infectious uveitis, and those without sufficient clinical and laboratory data. Patients were divided into three groups according to their clinical diagnosis: JIA-associated uveitis, JIA without uveitis, and idiopathic uveitis. Patients with JIA were classified according to the International League of Associations for Rheumatology criteria17. Patients with uveitis were classified according to the Standardization of Uveitis Nomenclature criteria18. All uveitis diagnoses were established by ophthalmologists, and patients were followed jointly by pediatric rheumatology and ophthalmology clinics. In our cohort, all patients with JIA-associated uveitis were diagnosed with uveitis either at the time of JIA diagnosis or within the first year of follow-up. Demographic variables such as age at diagnosis and sex, laboratory parameters including acute phase reactants and ANA titers and patterns, clinical features such as JIA subtype and presence or type of uveitis, and as well as treatment data, were collected from patient records. In patients with JIA, disease activity at diagnosis was assessed using the juvenile arthritis disease activity score-71 (JADAS-71).
This study was conducted in compliance with the Helsinki Declaration as well as local laws and regulations. Informed consent was obtained from the patients and their legal caregivers. The ethics committee of Istanbul Medeniyet University Göztepe Training and Research Hospital tertiary center approved our study (approval number: 2023/0919, dated: 12023).
Evaluation of ANA Staining Patterns and Titers
ANA testing was performed using IIF assay on HEp-2 cell substrates with the EUROPLUS ANA mosaic (EUROIMMUN, Lübeck, Germany). HEp-2 IIF staining patterns and titer levels were evaluated according to the International Consensus on ANA Patterns guidelines19. ANA staining patterns and titer levels were compared between the three patient groups. In addition, patients with JIA were divided into subgroups according to JIA subtypes and ANA staining patterns, and titer levels were compared among these subgroups.
Statistical Analysis
We performed the statistical analysis using Statistical Package for the Social Sciences (SPSS) for Windows, version 26.0 (SPSS Inc., Chicago, IL). Kolmogorov-Smirnov test was used to assess the distribution of continuous variables. While variables with a normal distribution were presented as mean ± standard deviation, distributed abnormally were presented as median (minimum-maximum). The chi-square test or Fisher’s exact test was used to compare the categorical variables, which were expressed as numbers (percentages). For continuous variables, One-Way ANOVA or the Kruskal-Wallis test was used for comparisons across more than two groups, and the Mann-Whitney U test was applied for comparisons between two groups. Bonferroni adjustment was applied in post-hoc analyses for multiple comparisons. In addition, analysis of JADAS-71 scores across ANA staining pattern groups in JIA patients was performed using One-Way ANOVA (or Kruskal-Wallis test where appropriate). A p-value of <0.05 was considered statistically significant.
RESULTS
Baseline Characteristics of All Patients
A total of 91 patients were included in the study, of whom 21 (23%) had idiopathic uveitis, 12 (13.1%) had JIA-associated uveitis and 58 (63.7%) had JIA without uveitis. Gender distribution was 34% (n=31) male and 66% (n=60) female. The median age at diagnosis was 12.30 (3.50-16.50) years in the idiopathic uveitis group; 5.72 (1.50-13.50) years in the JIA-associated uveitis group; and 9.87 (1.00-16.42) years in the JIA without uveitis group. All patients with JIA-associated uveitis had chronic anterior uveitis and were classified in the oligoarticular JIA subtype. Among patients with JIA without uveitis, oligoarticular JIA was also the most common subtype (67.2%, n=39), followed by enthesitis-related arthritis (20.6%, n=12), while other subtypes were observed less frequently (Figure 1). In the idiopathic uveitis group, anterior uveitis was present in the majority of cases (85.7%, n=18), and panuveitis was seen in the remainder (14.2%, n=3). The AC-1 staining pattern was detected in all patients with JIA-associated uveitis. Among those without uveitis, AC-1 remained the most common pattern (58.6%), followed by AC-4/5 (22.4%) and AC-2 (12%). Patients with idiopathic uveitis most frequently exhibited AC-1 (52.3%), followed by AC-4/5 (28.5%) and AC-2 (19%) staining patterns (Figure 1). When the patients with idiopathic uveitis, JIA-associated uveitis, and JIA without uveitis were compared based on the presence/absence of the AC-1 pattern, the difference was statistically significant (Fisher’s exact test, p=0.007).
Comparison of Clinical and Laboratory Characteristics Among All Patient Groups
The median age at diagnosis was significantly younger in the JIA-associated uveitis group compared to the other groups (p=0.006). In addition, C-reactive protein and erythrocyte sedimentation rate were significantly lower in the idiopathic uveitis group than in the other groups (p=0.012, p=0.010). There was no statistically significant difference in ANA staining patterns and titer levels between the groups (Table 1). Additionally, JADAS-71 scores demonstrated no significant variation across both ANA staining pattern groups (p=0.522) and ANA titer groups (p=0.247).
Comparative Analysis of ANA Profiles in JIA Subtypes
The three most prevalent patterns identified were AC-1 (44, 62.8%), AC-4/5 (13, 18.5%), and AC-2 (7, 10%). The AC-1 pattern was notably more common in oligoarticular JIA, while the AC-4/5 pattern was significantly more prevalent in enthesitis-related arthritis (ERA) compared to the other groups (p<0.001) (Table 2). An ANA titer of 1/320-1/1000 was significantly more frequent in oligoarticular JIA than in other groups, whereas a titer of 1/100 was more commonly observed in patients with ERA than in other groups (p<0.001) (Table 3).
DISCUSSION
This study compared ANA profiles among patients with JIA-associated uveitis, idiopathic uveitis, and JIA without uveitis, revealing serological differences between the groups. The AC-1 staining pattern was significantly more common in oligoarticular JIA patients, while the AC-4/5 pattern predominated in patients with ERA. In contrast, all patients with JIA-associated uveitis exhibited the AC-1 pattern. When the patients with idiopathic uveitis, JIA-associated uveitis, and JIA without uveitis, were compared according to the presence or absence of the pattern, a statistically significant difference was observed, indicating a marked enrichment of AC-1 in JIA-associated uveitis. ANA patterns in the other patient groups were more heterogeneous. This finding suggests that the AC-1 pattern may serve as a potential serological marker for uveitis risk in JIA; however, given the limited sample size, this observation should be interpreted with caution and requires validation in larger, prospective studies.
Uveitis is the most common extraarticular involvement in patients with JIA and may lead to permanent vision loss or blindness if left untreated4. It is critical to understand the risk of uveitis in the follow-up of JIA patients, due to its significant contribution to prognosis. ANA positivity in oligoarticular JIA is known to be a risk factor for the development of uveitis5. In our study, all JIA-associated uveitis patients were observed to exhibit AC-1 positivity, which is a nuclear homogenous staining pattern. This pattern is usually associated with autoantibodies against nuclear antigens such as double-stranded DNA, nucleosomes, histones, and chromatin17. Indeed, one study has shown that JIA-associated uveitis is associated with antihistone antibodies20. A proteomic analysis to identify specific novel autoantigens for JIA-associated uveitis revealed that 17 autoantigens were associated with uveitis, and five of them were against nuclear components21. Moreover, studies in adult rheumatoid arthritis (RA) have reported more frequent pulmonary involvement in patients with nuclear homogeneous ANA staining, suggesting that this pattern may also be relevant to systemic organ involvement in other contexts.
The heterogeneous ANA staining patterns, we found in the idiopathic uveitis group suggest that this disease group differs immunologically from JIA-associated uveitis and that it may have more diverse autoantibody profiles, perhaps playing a secondary or limited role in the pathogenesis of the disease. Indeed, a previous study has shown that ANA positivity in pediatric patients with noninfectious uveitis is not an independent risk factor for the need for biologic therapy or the development of uveitis-related complications22. This finding suggests that the clinical predictive power of ANA in idiopathic uveitis may not be as strong as in JIA-associated uveitis. Additionally, the significantly lower acute phase reactant levels observed in the idiopathic uveitis group compared to the JIA groups suggest a more limited inflammatory process and further support the distinct immunological nature of these disease entities.
Previous studies have reported that the most frequently observed staining patterns in JIA are nuclear homogeneous and fine granular patterns10, 15, 16.
Consistently, the most common staining patterns in our JIA patients were AC-1 and AC-4/5. While AC-1 was the most common staining pattern in our patients with oligoarticular JIA, AC-4/5 was the most common one in those with ERA. This finding aligns with the results reported by Sener et al.15, who also identified similar AC-1 positivity rates in oligoarticular JIA, although they noted AC-4/5 as the most common pattern. However, the AC-4/5 pattern was also the most frequent in their ERA patients, consistent with our observations. As is well established, human leukocyte antigens-B27 rather than ANA positivity is typically expected in ERA, in line with its immunogenetic background. In addition, ANA titer distribution showed a significant difference between JIA subtypes in our study, with medium and high titers predominating in oligoarticular JIA and low titers in the ERA subtype. The nuclear homogeneous staining pattern has been reported to be a risk factor for RA23. In contrast, we observed a high variability of titers and staining patterns of ANA among our JIA patients. While adult RA exhibits a homogeneous disease profile with symmetric polyarthritis, JIA is a clinically and immunologically heterogeneous disease with several subtypes24, 25. Thus, we considered that our results may reflect the immunologic heterogeneity of JIA; and different autoantibody profiles may be at the forefront according to subtypes.
Study Limitations
The main limitations of our study are its retrospective design, the limited number of patients, and the fact that ANA testing was only evaluated at the time of diagnosis and not repeated during periods of disease remission or exacerbation. This may have resulted in missing serologic changes related to disease activity. However, the main strength of the study is it included not only patients with JIA but also patients with idiopathic uveitis, allowing comparison of serologic patterns and thus contributing to a better understanding of the pathogenesis of JIA and idiopathic uveitis. In addition, this is the second study in a limited number of studies, in which ANA staining patterns were evaluated in detail with AC codes, which is an important contribution to the literature.
CONCLUSION
Our findings show that ANA pattern differences in JIA subtypes may provide significant clues regarding disease pathogenesis and clinical prediction. In particular, the prominence of the AC-1 pattern in JIA-associated uveitis may suggest a potential biomarker for early identification of uveitis risk; however, this possibility requires confirmation in larger prospective studies before integration into clinical practice.
