European Journal of Cardiovascular Medicine

Systemic autoimmune disorders, such as systemic lupus erythematosus, systemic sclerosis, Sjögren’s syndrome, and mixed connective tissue disease, are characterized by autoantibody-mediated tissue injury. Detection of antibodies against nuclear antigens (ANA) is central to diagnosis. IIFA enables sensitive screening and pattern recognition, whereas LIA allows targeted identification of disease-specific autoantibodies.

When immune tolerance breaks down, the body’s defenses turn against its own tissues, causing autoimmune diseases. These disorders can target a single organ—such as Hashimoto’s thyroiditis or Graves’ disease—or involve multiple systems, as seen in systemic lupus erythematosus (SLE), rheumatoid arthritis, and Sjögren’s syndrome. Antinuclear antibodies (ANA) form when the immune system loses the ability to distinguish self from non-self, contributing to disease development.

A positive ANA result helps confirm an autoimmune disorder, while a negative result makes an autoimmune cause less likely. To further refine diagnosis, the Line Immunoassay (LIA) was developed as a confirmatory profiling tool, providing greater sensitivity and specificity than traditional assays ⁽¹⁾

This prospective observational study included 50 patients (above 18 years) with clinical features suggestive of systemic autoimmunity in pathology department (July 2023–Dec 2024). Serum ANA testing was performed by: 1) IIFA on HEp-2 cells (1:80 dilution) for ANA detection and pattern classification; 2) LIA using 18-antigen strip for specific autoantibody profiling. Data on patterns, titers, and band intensities were recorded. LIA performance was evaluated against IIFA for sensitivity and specificity.

Inclusion Criteria

Adults (≥18 years) of any gender, with clinical features suggestive of systemic autoimmune disease (e.g., fatigue, arthralgia, cutaneous changes, lymphadenopathy).

Exclusion Criteria

Previously diagnosed systemic autoimmune disorder. Age under 18 years.

Indirect immunofluorescence assay: Indirect immunofluorescence was performed using the Immuno Concepts Hep-2000 HEp-2 cell system. Sera were diluted 1:80 in manufacturer’s diluent, and 30 µL was added to each well. After a 30-minute incubation at room temperature, unbound antibodies were washed away with PBS. Wells were then incubated with FITC-conjugated anti-human IgG for 30 minutes, followed by a 10-minute PBS wash in the dark. Slides were mounted and examined under a Nikon ECLIPSE Ni-U - Florescent microscope at 40× magnification. Nuclear staining equivalent to or below the negative control was scored as ANA-negative; samples showing bright, pattern-specific nuclear fluorescence in the majority of interphase cells were considered positive. Fluorescence intensity was graded on a three-point scale (+, ++, +++). Positive and negative controls accompanied each run.

Autoantibodies detection with LIA: The patient serum was then processed with LIA using ANA LIA XL kit (Cat. No. ITC92007). Following the kit protocol, sera were diluted 1:100 and incubated with 18 antigen–coated strips for 30 min on a shaker. After three 5-min washes, strips were incubated with conjugate for 30 min, washed again, then reacted with substrate for 10 min and stopped for 5 min. Strips were dried and read only if functional and cut-off controls were valid; bands weaker than, equal to, or stronger than the cut-off were scored as negative, equivocal, or positive and graded + to ++.

A 13-month prospective study was conducted at tertiary-care center. Approximately 50 patients with suspected autoimmune disease—recruited from both outpatient and inpatient Medicine services—had blood samples collected and processed in the Pathology Department for analysis.

Out of the 50 serum samples tested for antinuclear antibodies (ANA) using Indirect Immunofluorescence Assay (IIFA), 34 samples (68%) showed a positive ANA result, while the remaining 16 samples (32%) were found to be ANA negative. Out of 34 positive samples, speckled (50%) is most common pattern on IIFA followed by Nucleolar (29.4%).

Clinical subtyping by LIA identified SLE (31.25%), MCTD (25%), Sjögren’s syndrome (25%), systemic sclerosis (6.25%), PBC (6.25%), and myositis (6.25%) as shown in Table II.

Table I: Results with ANA Indirect immunofluorescence assay with Line immunoassay in study population.

Elevated ESR occurred in 60% of cases. LIA detected specific autoantibodies in 16/50 (32%): anti-SSA/Ro-52 (7), anti-U1-snRNP (7), smD1 (5), among others.

Table II: Clinical profile of suspected cases of systemic autoimmune disorders

Compared to IIFA, LIA sensitivity was 44.12% and specificity 93.75% as shown in Table III.

Table III: Sensitivity and Specificity of Line immunoassay in comparison with Indirect immunofluorescence assay

Figure 1– Image showing LIA ANA XL strip with result and score sheet.

CENTROMERE

NUCLEOLAR

HOMOGENOUS

The observed increase in prevalence to 68.0% may be explained, in part, by improved awareness and medical understanding of these diseases over the past two to three decades, leading to more frequent identification and diagnosis. The greater availability and reduced cost of ANA testing have also played a role, enabling more individuals to undergo screening. This has contributed to the earlier and more frequent diagnosis of autoimmune disorders.

The high ANA positivity and speckled pattern distribution align with SARD epidemiology. IIFA’s broad sensitivity facilitates initial screening but lacks specificity. LIA’s targeted autoantibody detection supports precise disease classification, though with moderate sensitivity. Combined use of IIFA and LIA leverages the strengths of both methods and is recommended for comprehensive ANA evaluation.

Different ANA patterns point to specific autoimmune diseases, so identifying exact autoantibodies helps guide diagnosis. The line immunoassay (LIA) is a rapid, user-friendly test with high sensitivity and specificity, detecting antibodies linked to SLE, systemic sclerosis, MCTD, dermatomyositis/polymyositis, and Sjögren’s syndrome

 In the present study we assessed the Gender wise distribution among the study subjects which showed female preponderance (68%) with female to male ratio of ANA positive cases. High levels of interferon-alpha (IFN-α), produced by the placenta, are thought to play a role in the development of SLE. The genes for IFN-α have evolved significantly in placental mammals. Also, having two active X chromosomes—due to sex, duplication, or translocation—may increase the risk of SLE compared to having only one.

Majority of patients were in the age group of 31-40 yrs (11 cases) followed by 41-50yrs (09 cases). Women in this age group tend to have higher levels of hormones, especially estrogen, which significantly influences immune system function. Strong evidence supports the role of estrogen in regulating immune responses.

SLE emerged as the most frequently occurring autoimmune disease in the study population, followed by mixed connective tissue disease. The high prevalence of SLE highlights the need for effective diagnostic strategies. Since several clinical criteria are available for diagnosing SLE, combining clinical judgment with reliable laboratory tests can improve the accuracy and timeliness of diagnosis. Similarly, Pandey et al⁽²⁾ and Kaur et al⁽¹⁾ also reported SLE as the most frequently identified condition by LIA. Present study shows dsDNA; SmD1; Nucleosome linked to SLE similar to the studies of Sarojini Raman et al⁽³⁾

 The autoantibody profile observed in patients diagnosed with Sjögren’s syndrome showed a high prevalence of SSA/Ro-60, SSA/Ro-52, and SS-B (La), matching with Pandey et al ^(2), Jaspreet Kaur et al^(1). The current profile strongly validates the diagnostic reliability of LIA in confirming MCTD. This is fully aligned with findings from Jaspreet Kaur et al^(1).

In the study conducted by Pandey et al^(2), both Jo-1 and Ku antibodies were identified in patients with Myositis. Pandey et al⁽²⁾ also observed AMA-M2 positivity, highlighting its status as a hallmark serological marker of classical PBC similar to what has been found in our present study.

In this study, the ANA test showed a sensitivity of 44.12%, which is lower than that reported in many other investigations. However, it achieved a notably high specificity of 93.75%. This indicates that although the test may not detect all true positive cases, it is highly reliable in correctly identifying individuals who do not have the condition, thereby minimizing the chance of false-positive outcomes.

IIFA remains the gold standard for sensitive ANA screening, while LIA offers confirmatory specificity and fine autoantibody profiling. An integrated diagnostic algorithm employing both assays enhances accuracy and informs targeted management of systemic autoimmune disorders. This study is helpful in further sub-classification of the type of Systemic autoimmune disease (SAIDs) by studying the various pattern shown by ANA positive samples on Indirect Immunofluorescence assay (IIFA), followed by confirmation on Line immunoassay so this is a very useful method for confirmation of Autoimmune disease.

Ethical Clearance status: The Institutional Ethics Committee of MGM Medical College & Hospital, Navi Mumbai, 28/06/23, DHR-EC/SC/2023/06/111

Declaration of competing interests: None

Funding: Nil

Author’s contribution:  Dr. Purvi Rupala – Concepts, Designs, Definition of intellectual content, Literature search, Clinical studies, Experimental studies, Data acquisition, Data analysis, Statistical analysis, Manuscript preparation, Manuscript editing.  Dr. Shilpi Sahu – Concepts, Design, Definition of intellectual content, Data analysis. Dr. Arpita Singh – Concepts, Experimental study

Acknowledgement

The authors thank the patients and the technical staff of the Pathology Department for their assistance in sample processing and assay performance.

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