Introduction to the Gold Standard Debate

Recent evidence suggests the ANA screening immunoassay (BioPlex 2200) has significant limitations compared to the gold standard indirect immunofluorescence assay (IIF). A massive population-based study analyzed nearly 400,000 individuals to determine if multiplex technology could replace traditional methods. Consequently, the findings have profound implications for primary care physicians who frequently order these tests for nonspecific symptoms.

Critical Findings on Assay Sensitivity

Specifically, the BioPlex 2200 multiplex immunoassay demonstrated a remarkably low sensitivity of 29.1%. This indicates that the test misses a substantial number of patients who would otherwise test positive via IIF. Moreover, the study reported a false-negative rate of 8.4% within the total population. These results suggest that relying solely on a multiplex platform for initial screening might lead to missed diagnoses of rheumatological diseases. Furthermore, the positive predictive value was only 24.5%, implying that many positive results do not correlate with autoimmune pathology.

Challenges with the ANA Screening Immunoassay in Clinical Practice

Primary care settings often use automated assays to handle high testing volumes. However, the American College of Rheumatology (ACR) continues to support HEp-2 IIF as the preferred screening method. This preference exists because HEp-2 cells contain over 100 autoantigens, whereas multiplex assays only target a limited panel. Therefore, clinicians must exercise caution when interpreting negative results from automated platforms if clinical suspicion remains high. In fact, following this study, the Clalit Health Services laboratory network discontinued simultaneous IIF and multiplex testing to avoid confusion and diagnostic errors.

Summary of Diagnostic Performance

Notably, while the specificity of the multiplex assay was relatively high at 87.8%, its inability to capture a broad range of autoantibodies limits its utility as a primary screening tool. Consequently, the researchers concluded that IIF remains essential for screening rheumatological diseases. Additionally, clinicians should always correlate laboratory findings with specific patient symptoms such as inflammatory arthritis or malar rashes.

Frequently Asked Questions

Why is IIF considered superior to the ANA screening immunoassay?

IIF utilizes HEp-2 cells which display a vast array of naturally occurring antigens. In contrast, multiplex assays only detect a pre-selected group of 8 to 13 specific autoantibodies, increasing the risk of false negatives for rarer conditions.

Should a negative multiplex result be followed by an IIF test?

Yes, if the clinical suspicion for an autoimmune disease is strong, guidelines recommend performing an IIF test even if an automated solid-phase assay is negative.

What are the risks of low sensitivity in ANA screening?

Low sensitivity leads to false negatives, which can delay the diagnosis and treatment of serious systemic autoimmune rheumatic diseases (SARDs) like lupus or scleroderma.

Disclaimer: This content is for informational and educational purposes only. It does not constitute medical advice or establish a doctor-patient relationship. Refer to the latest local and national guidelines for clinical practice.

References

Hijaze E et al. The Usefulness of Antinuclear Antibody Multiplex Immunoassay for Screening of Rheumatological Diseases: A Real-World Population-Based Study. J Clin Rheumatol. 2026 Jun 03. doi: 10.1097/RHU.0000000000002372. PMID: 42235123.

American College of Rheumatology Position Statement. Methodology of Testing for Antinuclear Antibodies.

Meroni PL, et al. Antinuclear antibody testing: the immunofluorescence assay as the gold standard. Ann Rheum Dis. 2010;69(8):1420-2.