Clinical Significance of Positive ANA by IFA with Negative Multiplex
A positive ANA by immunofluorescence assay (IFA) with a negative multiplex result indicates the presence of autoantibodies not included in the multiplex panel and requires clinical correlation with specific follow-up testing based on the IFA pattern and clinical suspicion.
Understanding the Discrepancy
IFA remains the gold standard for ANA detection and can identify a broader range of autoantibodies than multiplex assays, which test only for specific extractable nuclear antigens (ENAs) 1
Multiplex assays typically screen for common ENAs (SSA/Ro, SSB/La, Sm, RNP, Scl-70, Jo-1) but miss other clinically significant antibodies such as anti-histone, anti-nucleosome, anti-ribosomal P, and certain pattern-specific antibodies 1, 2
False negatives occur with automated multiplex methods in approximately one-third of patients because these assays may not detect antibodies to antigens whose specificity has not been determined or are not included in the panel 1, 3
Clinical Approach Based on IFA Pattern
If Homogeneous Pattern:
- Order anti-dsDNA antibodies using both Crithidia luciliae immunofluorescence test (CLIFT) for specificity and solid phase assay for sensitivity 1
- Test for anti-histone antibodies, particularly if drug-induced lupus is suspected 1
- Consider anti-nucleosome antibodies if SLE is clinically suspected 1
If Speckled Pattern:
- Request specific ENA testing regardless of multiplex results, as the multiplex may have missed less common specificities 1, 4
- Consider anti-ribosomal P if neuropsychiatric SLE is suspected 1
- Test for anti-Jo-1 if inflammatory myopathy symptoms are present, as this can be positive even with negative ANA by some methods 1
If Nucleolar Pattern:
- Order anti-PM/Scl, anti-RNA polymerase III, anti-U3-RNP, and anti-Th/To antibodies for systemic sclerosis evaluation 1
If Centromere Pattern:
- Test for specific anti-centromere antibodies (CENP-A, B, C, F) which may not be included in standard multiplex panels 1
Titer Considerations
Titers ≥1:160 have significantly higher clinical specificity (86.2%) and warrant aggressive follow-up regardless of multiplex results 4, 5
Low titers (1:40-1:80) occur in 13.3-31.7% of healthy individuals, but should still be investigated if clinical suspicion exists 4, 5
The same IFA method should be used for serial monitoring to ensure comparability 1
Critical Management Principle
In cases of high clinical suspicion, determination of specific antibodies should be pursued regardless of any prior negative test results, including negative multiplex 1, 4
Specific autoantibodies like anti-Jo-1, anti-ribosomal P, or anti-SSA/Ro may be present in patients with positive IFA but negative multiplex 1
Consider referral to a specialized reference laboratory for comprehensive autoantibody evaluation if the clinical picture strongly suggests autoimmune disease 1
Common Pitfalls to Avoid
Do not dismiss a positive IFA based solely on negative multiplex results - the IFA detects a broader antigen repertoire 1, 2
Different laboratories use different methods and cutoffs, affecting result interpretation - always specify the method used 1, 4
Multiplex assays using ELISA can result in false negatives in about one-third of patients because they target limited antigen specificities 1
Some clinically significant antibodies (anti-DFS70, anti-PCNA, anti-proliferating cell nuclear antigen) are rarely included in standard multiplex panels but are visible on IFA 1
When to Repeat Testing
If initial multiplex was negative but clinical suspicion remains high, request individual specific antibody testing rather than repeating the multiplex 1
ANA testing is primarily for diagnosis, not monitoring - do not repeat ANA/multiplex for disease activity assessment 4
For diagnosed SLE, use quantitative anti-dsDNA by the same method consistently for monitoring, not repeat ANA testing 1, 4