ANA Testing Dilemmas: Unravelling Discordant Results Between ELISA, IFA, and LIA Methods

Antinuclear antibodies (ANAs) are autoantibodies that target components of the cell nucleus. Their presence is associated with various autoimmune diseases, including systemic lupus erythematosus (SLE), Sjögren's syndrome, and rheumatoid arthritis, among others. Detecting ANAs is crucial for diagnosing these diseases, monitoring disease activity, and predicting clinical outcomes. Over the years, several methods have been developed to detect ANAs, each with its significance and limitations. Different methods of ANA detection may yield different results due to variations in antigen specificity, sensitivity, and interpretative criteria. 

ANA test by ELISA method:

The ELISA method offers a   semi-quantitative or quantitative determination of IgG antibodies to nuclear and cytoplasmic antigens in the bloodstream and is one of the commonly utilized techniques alongside the indirect immunofluorescence assay (IFA). While ELISA offers specificity and quantification, it is essential to interpret results cautiously, considering the clinical context, patient's symptoms, and other diagnostic findings. A positive ANA by ELISA does not confirm a specific diagnosis but suggests the need for further evaluation.

ANA by IFA method:

In ANA test by indirect immunofluorescence assay (IFA), patterns can be diverse, and interpreting the results requires expertise. These patterns can be suggestive but not definitive of specific autoimmune diseases. IFA is considered the gold standard for ANA detection due to its high sensitivity. However, false-positive results can occur, especially at lower dilutions, leading to potential misinterpretations.

ENA profile (ANA by LIA/ Immunoblot):

ENA profile is a subset of ANA testing that focuses on identifying specific autoantibodies against Extractable Nuclear Antigens (ENA) associated with particular autoimmune diseases. ENA testing is typically performed using specialized laboratory techniques such as immunoblotting, line immunoassay (LIA), or enzyme  immunosorbent assay (EIA or ELISA). The results identify the presence or absence of specific autoantibodies against particular ENAs, providing a more targeted and disease-specific analysis compared to ANA testing alone.  LIA offers high sensitivity and specificity, with the advantage of automation, reducing variability and improving reproducibility compared to manual methods like Immunoblotting.

Remember, the relationship between these tests can be described as follows: ANA by ELISA and ANA by IFA are used to screen for the presence of antinuclear antibodies (ANAs) in a patient's blood, while ENA profile is a subsequent test used to identify and differentiate specific extractable nuclear antigens (ENAs) targeted by the ANAs. Together, these tests help in the diagnosis and classification of autoimmune diseases, especially those with systemic involvement. However, it's important to note that the interpretation of these tests should always be done by experienced medical professionals in the context of the patient's clinical presentation and other relevant diagnostic information.

Limitations and Challenges:

• A positive ANA test does not necessarily indicate the presence of an autoimmune disease, as ANAs can also be present in healthy individuals or individuals with infections, malignancies, other non-autoimmune conditions and due to specific medications.
• In early stages of certain autoimmune diseases or in patients with low levels of ANAs, the concentration of antibodies may be below the detection limit of the kit, resulting in false negative results.
• While ANA by ELISA tests are sensitive, they lack specificity. Variability in antigen preparation, specificity, and assay conditions can lead to false-positive or false-negative results.
• ANA IFA patterns can be diverse, and interpreting the results requires expertise and can be subjective, leading to variability among observers. 
• Different methods of ANA detection may yield different results due to variations in antigen specificity, sensitivity, and interpretative criteria.
• Factors such as sample degradation, inadequate sample volume, or improper storage can affect the accuracy of LIA results, potentially leading to false negative outcomes.
• IFA might be more sensitive in detecting certain patterns of ANA, which could be missed by ELISA.

The discordance result between different methods of ANA tests can pose challenges in the diagnosis and management of autoimmune diseases. Here's a breakdown of potential reasons for discordance and their implications:

• The methodologies, reagents, and platforms used in ELISA, IFA, and LIA differ, potentially leading to variations in results.
• The sensitivity and specificity of each method may vary based on the antigens they detect and the techniques employed. Differences in assay sensitivity can result in positive results in one test and negative in another.

In all cases of discordant results, the interpretation should be done cautiously, considering the clinical presentation and other relevant diagnostic information. Sometimes, additional tests or repeat testing may be necessary to clarify the diagnosis. A sequential approach—starting with a broad test like ANA by IFA and then moving to more specific tests like ENA by LIA—can be beneficial to refine the diagnosis. In cases of clinical suspicion with initial discordant results, re-testing after some time or using alternative methods can help clarify the diagnosis.

In summary, understanding the strengths, limitations, and potential discrepancies associated with each method is essential for accurate interpretation, clinical correlation, and interdisciplinary collaboration. Standardization and ongoing research are essential to improve the accuracy and utility of ANA testing in clinical practice.

Dr Prashant Goyal.