Prevalence of antinuclear antibody in healthy young adults in Vietnam

To the editor, Antinuclear antibodies (ANA) are widely recognized as pivotal biomarkers for both the diagnosis and prognosis of systemic autoimmune diseases [1]. Despite their clinical significance, ANA positivity is not always disease-specific, as it may be detected in a certain proportion of asymptomatic individuals. The prevalence of ANA positivity varies according to the racial or ethnic background of the study population, as well as the analytical methods employed. Although several studies have investigated ANA prevalence in healthy populations, the available evidence remains limited, and considerable heterogeneity exists in the methodologies used [2]. According to the 2019 classification criteria for systemic lupus erythematosus (SLE) developed by the European League Against Rheumatism (EULAR) and the American College of Rheumatology (ACR), indirect immunofluorescence (IIF) on human epithelial type 2 (HEp-2) cells is considered the standard method for ANA screening in the diagnosis of SLE. In the present study, we aimed to evaluate the prevalence of ANA detected by indirect IIF on HEp-2 cells among young Vietnamese individuals without a diagnosis of autoimmune diseases, including SLE, and to compare these findings with results obtained using an enzyme-linked immunosorbent assay (ELISA)-based ANA screening method. We conducted a descriptive cross-sectional study involving 314 people aged 18 to 25 years without a medical record of autoimmune diseases at the University of Medicine and Pharmacy at Ho Chi Minh City. Participants were classified as healthy based on predefined inclusion and exclusion criteria, including the absence of prior autoimmune disease and any symptoms suggestive of an autoimmune disease. This approach was intended to minimize the inclusion of individuals with clinically apparent autoimmune conditions. However, subclinical or preclinical autoimmunity could not be completely excluded because autoantibodies may precede clinical disease onset in some individuals. A questionnaire was used to collect demographic and clinical characteristics of the study subjects. The original study questionnaire used for participant screening, including items on personal and family history of autoimmune diseases, has been provided in Supplementary File S1, Supplemental digital content 1, https://links.lww.com/PA9/A105. ANA were detected by IIF using HEp-20-10 cells and primate liver tissue as substrates (Mosaic HEp-20-10/Liver [Monkey]; EUROIMMUN, Lübeck, Germany). Serum samples were screened at an initial dilution of 1:80, and titers ≥1:80 were considered positive. This cutoff was selected in accordance with international recommendations, including the 2019 EULAR/ACR classification criteria, where a positive ANA at the titer of ≥1:80 is used as the entry criterion for SLE classification [3]. Because our study focused on healthy young individuals with a low pretest probability of autoimmune disease, we aimed to estimate the baseline ANA prevalence rather than define clinically significant titers. In addition, utilizing a cutoff at higher titers (eg, ≥1:160) could underestimate ANA prevalence in this low-risk population. Therefore, ANA positivity at the screening level should be interpreted cautiously in asymptomatic individuals. For references, ANA were evaluated using a commercially available solid-phase ELISA (ANA Screening IgG ELISA; DIA.PRO Diagnostic Bioprobes Srl, Milan, Italy), performed in accordance with the manufacturer’s instructions. A total of 314 subjects were enrolled in the study, of whom 187 (59.6%) were male, and 127 (40.4%) were female. The median age was 20 years (18–25 years). A family history of autoimmune disease was reported by 3 participants (1.0%), with the specified diagnoses being SLE, rheumatoid arthritis, and autoimmune thyroiditis (each n = 1). In addition, 9 participants (2.9%) reported a family history of chronic disease. Figure 1A shows the prevalence of ANA detection by IIF and ELISA. When IIF was utilized to detect ANA using Hep-20-10 cells, 29 subjects (9.2%) were positive, including 23 females (18.1%) and 6 males (3.2%) (P < 0.001). This finding revealed that females had a 5.6-fold higher risk of ANA positivity compared with males (prevalence ratio = 5.6; 95% confidence interval, 2.4–13.5), consistent with previous studies indicating a higher prevalence of ANA positivity in females [4,5]. However, when ELISA was utilized for ANA detection, only 5 cases (1.6%) were positive, and all of them were females. The above findings suggested that sex-related differences in producing autoimmune antibodies can derive from a complex interplay of multiple factors: (1) hormonal influences, (2) X chromosome-linked immune genes, and (3) epigenetic regulation of B-cell responses [6]. Figure 1B displays the patterns of ANA observed in IIF. Briefly, we recorded and classified the patterns according to the International Consensus on ANA Patterns. Our results revealed that speckled nuclear patterns are the most common among ANA-positive individuals (n = 19; 65.5%), including fine speckled (AC-4; n = 11; 38%) and dense fine speckled (AC-2; n = 8; 27.5%). This finding is consistent with previous studies showing that dense fine speckled patterns are common in healthy or low pretest probability populations [7,8]. These observations contrast with established IIF profiles in SLE, where homogeneous (AC-1) and coarse speckled (AC-5) patterns predominate, accounting for 35.2% to 42.1% and 46.1% to 52.1% of cases respectively [9]. This disparity reflects distinct autoantibody specificities: AC-1 was associated with anti-double-stranded DNA and anti-chromatin antibodies, which are central to EULAR/ACR classification criteria, whereas AC-5 was associated with anti-Sm and anti-U1RNP antibodies. Previous studies showed that ANA prevalence increased with age, particularly in elderly populations, where ANA positivity may reflect an IIF rather than a systemic autoimmune disease [10]. In addition, ANA was also found in several nonrheumatic immune conditions, including allergic diseases such as atopic dermatitis and asthma [11]. Taken together, these findings suggest that isolated ANA positivity in low-risk or asymptomatic individuals should be interpreted cautiously within an appropriate clinical context, as it may represent nonspecific immune activation rather than systemic autoimmunity.Figure 1.: ANA detection prevalence and pattern distributions. (A) Prevalence of ANA detection using IIF and ELISA. (B) IIF pattern distribution of ANA. (C) Comparison of ANA prevalence detected by indirect IIF and ELISA. ANA, antinuclear antibodies; ELISA, enzyme-linked immunosorbent assay; IIF, immunofluorescence.In addition, Fig. 1C shows that the agreement between IIF and ELISA for ANA detection was low (Cohen κ ≈ 0.28). ELISA identified only 5 of the 29 IIF-positive subjects and yielded no ELISA-positive/IIF-negative cases, indicating high specificity but low sensitivity relative to IIF. The ELISA cutoff used in this study was based on the manufacturer’s recommendation and has not been specifically validated in the Vietnamese population. The observed discordance likely reflects not only differences in analytical sensitivity but also fundamental differences in antigenic representation between the 2 platforms. Compared with ELISA, IIF on HEp-2 cells detects a broader spectrum of native conformational intracellular antigens, including nonclassical autoantibodies that may be present in asymptomatic or low pretest probability populations [12]. This distinction may be particularly relevant in our cohort, in which dense fine speckled (DFS70, AC-2) patterns predominated and may represent physiological or nonpathological autoimmunity rather than clinically significant disease. Consequently, ELISA may underestimate ANA prevalence in healthy individuals. The higher sensitivity of IIF observed in this study supports current recommendations that endorse IIF on HEp-2 cells as the reference method for ANA screening in systemic autoimmune diseases, including SLE. In conclusion, our findings indicated that serum ANA could be detected in a proportion of healthy young individuals. In addition, IIF was more sensitive than ELISA in ANA screening. Nevertheless, the limitations of this study include its single-center design, restriction to medical students, and lack of longitudinal follow-up to assess the possibility of the development of autoimmune diseases in ANA-positive individuals. Acknowledgements The authors thank the volunteers to donate their blood samples, and the valuable supports from the University of Medicine and Pharmacy at Ho Chi Minh City and the University Medical Center Ho Chi Minh City. This research was funded by the University of Medicine and Pharmacy at Ho Chi Minh City under contract number 106/2024/HĐ-ĐHYD, dated April 17, 2024.