Autoimmune diseases are a heterogeneous group of chronic conditions in which autoimmune attack of self-tissues occurs because of the failure of immunological tolerance [1]. They involve a variety of organ systems, such as musculoskeletal, endocrine, gastrointestinal, dermatological, neurological and renal systems, and can cause significant morbidity and impaired quality of life [2]. Examples of autoimmune diseases are systemic lupus erythematosus, rheumatoid arthritis, autoimmune thyroid disease, autoimmune hepatitis, inflammatory myopathies, vasculitis, and Sjögren's syndrome [3].

The clinical diagnosis of autoimmune diseases is still difficult due to the overlapping clinical features, the different clinical manifestations and the lack of a specific diagnostic test in most cases [4]. Nonspecific symptoms like arthralgia, myalgia, fever, rash, organ dysfunction or unexplained inflammatory markers can be present in patients, making diagnosis challenging [5]. Thus, a combination of clinical examination, serological evaluation, imaging studies and histopathological examination is often necessary for an accurate diagnosis [6]. Autoantibodies are the most important laboratory markers in clinical practice and are a key part of diagnosing and classifying autoimmune diseases [7].

Antinuclear antibodies, anti double stranded DNA antibodies, rheumatoid factor, anti cyclic citrullinated peptide antibodies, anti-neutrophil cytoplasmic antibodies, anti-thyroid antibodies and disease specific autoantibodies are useful for diagnosis and prognosis [8]. There is, however, some variation in the sensitivity and specificity of autoantibodies according to the type and clinical stage of disease and false positives occur in healthy people or in other inflammatory conditions [9].

Imaging modalities have come to play an ever greater role in the assessment of the various autoimmune conditions, as they are useful for detecting damage, inflammation and organ involvement [10]. The assessment of joint inflammation, interstitial lung disease, organ enlargement, vasculitis-related changes and/or tissue fibrosis are commonly performed using ultrasound, computed tomography, magnetic resonance imaging and/or radiographic imaging [11].

Imaging is also helpful in the assessment of disease activity and response to treatment, particularly in systemic autoimmune diseases [12]. In some autoimmune diseases, such as those that require tissue confirmation, histopathological examination remains an important diagnostic test [13]. Biopsy results can give direct evidence of inflammation, immune-mediated injury, vasculitis, fibrosis, granulomatous changes or organ-specific changes, and thus enhance the likelihood of diagnosis [14]. Histopathology is especially helpful in the diagnosis of autoimmune hepatitis, lupus nephritis, vasculitis, inflammatory bowel disease, autoimmune skin diseases, and connective tissue diseases that involve organs [15].

A few studies have investigated serologic markers, imaging, and tissue pathology as diagnostic tools in autoimmune diseases, and their correlation with the clinical picture is variable between disease categories [16]. The use of several diagnostic modalities may enhance the diagnostic accuracy, allow early diagnosis, and help to decide the treatment plan for each patient [17].

Objective

To evaluate the role of autoantibodies, imaging, and histopathological findings in the diagnosis and clinical correlation of autoimmune diseases.

This was a cross-sectional analytical study conducted at KRL Hospital, Islamabad, from July 2024 to June 2025, including 192 patients with suspected or confirmed autoimmune diseases. The study included patients ≥ 18 years of age with a clinically suspected or known autoimmune disease. Patients included were those who were eligible for autoantibody testing during their diagnosis and for whom imaging and/or histopathology were available, as indicated by the clinical situation. Patients were enrolled if they were willing to participate and provided informed consent. Patients were excluded with acute infectious diseases with autoimmune-like features or malignancies with paraneoplastic autoimmune-like features. Patients that did not have serological, imaging or histopathological records were also not included. Further, patients who received immunosuppressive treatment prior to the diagnostic evaluation were excluded as were patients with overlap syndromes who were not definitively classified. Data Collection Ethical approval was obtained, and the data were collected using a structured pro forma. Baseline demographic and clinical data collected were age, sex, presenting symptoms, duration of illness, presence of comorbidities, and provisional clinical diagnosis. Assays were performed to detect autoantibody markers, including antinuclear antibody (ANA), anti-double-stranded DNA, rheumatoid factor, anti-cyclic citrullinated peptide, anti-neutrophil cytoplasmic antibodies, anti-thyroid antibodies, and other disease-specific markers. Imaging data from ultrasound, X-ray, computed tomography (CT) scan, or magnetic resonance imaging (MRI) were recorded based on organ involvement and/or the imaging indication. Relevant tissue biopsy was performed, and histopathological examination was reviewed for inflammatory, autoimmune, and/or organ-specific pathological changes. Correlations with the final diagnosis were carried out. Statistical Analysis Data analysis was carried out using SPSS version 26.0. Data for continuous variables were presented as mean ± standard deviation and data for categorical variables were presented as frequency and percentage. Associations between categories of autoimmune disease and types of diagnostic modalities were evaluated by using chi-square testing and independent t testing. Logistic regression was used to determine predictors of confirmed autoimmune diagnosis and clinically significant organ involvement. A p-value ≤0.05 was considered statistically significant.

The study included 192 patients with a mean age of 41.6 ± 13.8 years, with female predominance noted in 136 (70.8%) cases. The mean symptom duration was 11.4 ± 6.7 months. Fatigue was the most common symptom, reported in 118 (61.5%) patients, followed by arthralgia or joint pain in 104 (54.2%). Organ-specific symptoms were present in 77 (40.1%) patients, while family history of autoimmune disease was reported in 29 (15.1%) cases.

Table 1: Baseline Demographic and Clinical Characteristics of Patients with Autoimmune Diseases (n = 192)

Autoantibody positivity was common among the study population. ANA was positive in 126 (65.6%) patients, followed by rheumatoid factor in 54 (28.1%), anti-dsDNA in 48 (25.0%), anti-CCP in 39 (20.3%), and ANCA in 22 (11.5%). Abnormal imaging findings were observed in 117 (60.9%) patients. Histopathology was performed in 86 (44.8%) cases, and diagnostic histopathological findings were present in 68 (35.4%), supporting the role of combined diagnostic evaluation.

Table 2: Autoantibody, Imaging, and Histopathological Findings (n = 192)

Confirmed autoimmune disease was strongly associated with positive diagnostic findings. ANA positivity was significantly higher in confirmed cases compared with unconfirmed cases (113; 75.8% vs. 13; 30.2%; p<0.001).

Table 3: Clinical Correlation of Diagnostic Findings with Confirmed Autoimmune Disease (n = 192)

Logistic regression showed that positive ANA was a strong predictor of confirmed autoimmune disease (aOR 4.12; 95% CI: 1.98–8.56; p<0.001). Disease-specific antibodies also showed strong predictive value (aOR 3.76; p=0.001), followed by diagnostic histopathology (aOR 3.28; p=0.005), abnormal imaging findings (aOR 2.94; p=0.004), and organ-specific symptoms (aOR 2.41; p=0.02).

Table 4: Logistic Regression Analysis for Predictors of Confirmed Autoimmune Disease (n = 192)

In autoimmune diseases, this study was aimed at assessing the value of autoantibodies, imaging and histopathological examinations, and it revealed that multimodal diagnostic approach helps greatly in terms of diagnosis accuracy and clinical correlation. The results underline the relevance of the use of serological, radiological and tissue-based assessment, especially if there are complex or overlapping autoimmune patient presentations. The mean age of the study population was 41.6 ± 13.8 years and there was a definite predominance of females, with nearly three-quarters of the cases being female. This result is in line with the fact that autoimmune disorders are more common in females, which could be attributed to hormonal, genetic and immunological influences. Another study found a similar trend of a higher prevalence among women for all systemic autoimmune diseases, especially connective tissue and endocrine autoimmune diseases [18].

Fatigue and arthralgia were the most frequently presenting symptoms, emphasizing the non-specific and often overlapping clinical manifestations of autoimmune diseases. Organ-specific manifestations were also common, indicating that multiple organ systems are involved in autoimmune diseases at the time of diagnosis. Fatigue, musculoskeletal symptoms, and organ-specific complaints were also common initial symptoms in a previous study, which may lead to diagnostic delay because they are non-specific [19]. This study confirmed that autoantibody testing has good diagnostic value. ANA positivity was found in 65.6% of patients and was significantly higher among those with confirmed autoimmune disease. There was also excellent clinical relevance of disease-specific antibodies, in line with their well-established diagnostic use in systemic lupus erythematosus, rheumatoid arthritis, vasculitis and autoimmune thyroid disease. ANA and disease-specific antibodies were also found to have high diagnostic sensitivities in a previous study, along with clinical findings [20].

In 60.9% of the patients, imaging findings were abnormal, and this was significantly correlated with a confirmed autoimmune disease. Imaging plays a vital role in

diagnosing organ involvement, inflammatory changes, and structural damage that may not be clinically evident in the early phase of the disease. Objective evaluation of the diagnosis and disease staging may be obtained from ultrasound, CT, MRI, and X-rays. Another study confirmed that imaging markedly improved diagnostic confidence, especially in autoimmune diseases of joints, lungs, kidneys, and vascular structures [21].

The histopathological examination also played an important role in diagnosis. Consistent with this, the diagnostic biopsy results were significantly more frequent in confirmed autoimmune cases and were independent predictors of confirmed autoimmune disease in regression analysis. In specialized cases like autoimmune hepatitis, lupus nephritis, vasculitis, inflammatory dermatoses, and organ-specific autoimmune disorders, tissue diagnosis is especially important as it helps to confirm the diagnosis. Histopathology was also shown to significantly enhance diagnostic specificity in complicated cases of autoimmune disease having indeterminate serology in a previous study. Results of logistic regression corroborate the utility of a comprehensive diagnostic approach. Disease-specific antibodies, histopathology and imaging abnormalities were the other strongest predictors of confirmed autoimmune disease, with positive ANA following with OR of 4.12. These results suggest that serological testing is very useful for screening and diagnosis, but adding imaging and tissue diagnosis greatly increases diagnostic certainty. Diagnostic accuracy was also found to improve significantly when multiple modalities were used compared with single laboratory markers in a previous study [22].

Despite advances in diagnostic technology, organ-specific symptoms were independently associated with confirmed autoimmune disease, indicating that pattern recognition in the clinical setting remains necessary. Rather than having a single, consistent pattern, autoimmune diseases can present in a variety of ways, and no one test will give universal certainty of diagnosis. Thus, the role of clinical judgement will remain critical when evaluating laboratory and imaging results. Another study previously noted the importance of clinical context when interpreting serology tests for autoimmunity, given overlapping syndromes and the possibility for false positive results. These are some practical implications of this study. Early and correct diagnosis of auto-immune diseases enables the early start of immunomodulatory therapy, the prevention of irreversible organ damage and better long-term results. In tertiary care centers, the multimodal diagnostic approach may prove to be especially beneficial in patients with complex autoimmune diseases where multisystem involvement is common.

Limitations

This study has several limitations. Being a single-center cross-sectional study, the findings may have limited generalizability to broader populations and different healthcare settings. The heterogeneity of autoimmune diseases included in the study may have introduced variability in diagnostic performance across disease categories. Not all patients underwent histopathological evaluation, as biopsies were performed only when clinically indicated, which may have affected uniform comparison between diagnostic modalities. Some imaging findings may have been operator-dependent, introducing interpretive variability.

It is concluded that autoantibody testing, imaging, and histopathological examination each play significant and complementary roles in the diagnosis of autoimmune diseases. Positive ANA, disease-specific autoantibodies, abnormal imaging findings, and diagnostic histopathological changes were significantly associated with confirmed autoimmune disease. A combined multimodal diagnostic approach improves diagnostic accuracy, strengthens clinical correlation, and supports early identification of organ involvement. Integrating serological, radiological, and tissue-based evaluation with clinical assessment may facilitate timely diagnosis and improve management outcomes in patients with autoimmune diseases.

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