ANA Multiplex, with Reflex to dsDNA
The ANA Multiplex with Reflex to dsDNA combines immunofluorescence ANA screening with automatic reflex to anti-dsDNA and extractable nuclear antigen testing when positive. It evaluates systemic lupus erythematosus, Sjögren syndrome, systemic sclerosis, and other connective tissue diseases, balancing sensitivity with specificity through targeted antigen identification.
3
Biomarkers
Blood (serum)
Sample
3–5 business days
Turnaround
No
Fasting
What is the ANA Multiplex test?
The ANA Multiplex with Reflex to dsDNA combines screening immunofluorescence with targeted antigen-specific testing to evaluate suspected systemic autoimmune rheumatic disease. An initial positive or equivocal ANA triggers reflex to anti-dsDNA (specific for systemic lupus erythematosus) and an extractable nuclear antigen panel that includes anti-Smith, anti-RNP, anti-SSA/Ro, anti-SSB/La, anti-Scl-70, anti-centromere, and anti-Jo-1, each associated with a distinct clinical phenotype. This cascade design balances sensitivity with cost and clinical utility: the sensitive ANA screen captures nearly all cases of SLE, mixed connective tissue disease, Sjögren syndrome, systemic sclerosis, and inflammatory myositis, while reflex testing narrows the differential and estimates prognosis only when screening is abnormal. Practitioners order the multiplex to investigate presentations such as unexplained arthritis, photosensitive rash, serositis, cytopaenias, or multisystem inflammation, recognising that a positive result must be interpreted alongside clinical criteria and that low-titre ANA positivity in isolation does not establish disease.
What does the ANA Multiplex test measure?
Antinuclear antibodies bind nuclear antigens and are detected by indirect immunofluorescence on HEp-2 cells. Low-titre positivity (1:40–1:80) is found in approximately 10–15% of healthy individuals, increasing with age and female sex. Higher titres and specific patterns correlate more strongly with systemic autoimmune rheumatic diseases, though pattern alone does not diagnose a particular condition.
ANA screening is the entry test for systemic lupus erythematosus, mixed connective tissue disease, Sjögren syndrome, systemic sclerosis, and polymyositis/dermatomyositis. A negative screen at high-quality laboratory threshold effectively excludes SLE in the absence of treatment. Positive results require correlation with clinical features and are followed by extractable nuclear antigen and dsDNA testing to narrow the differential.
Anti-dsDNA antibodies are directed against the sugar-phosphate backbone of native double-stranded DNA. They form immune complexes that deposit in glomeruli and other tissues, contributing directly to tissue injury in lupus nephritis. Levels fluctuate with disease activity in many patients, particularly those with renal involvement.
Anti-dsDNA is highly specific for systemic lupus erythematosus (present in approximately 60–70% of patients, lower in those without nephritis) and is one of the immunological criteria in classification systems. Rising titres often herald flare, especially nephritis, making serial measurement useful for monitoring. Transient positivity can occur with infection or medication but sustained elevation in the appropriate clinical context strongly supports SLE.
Extractable nuclear antigens are a family of nuclear and cytoplasmic proteins recognised by autoantibodies in systemic rheumatic diseases. Each target antigen associates with a distinct clinical phenotype: anti-Smith with SLE, anti-RNP with mixed connective tissue disease, anti-SSA/Ro and anti-SSB/La with Sjögren and neonatal lupus, anti-Scl-70 with diffuse systemic sclerosis, anti-centromere with limited cutaneous systemic sclerosis, and anti-Jo-1 with antisynthetase syndrome.
ENA testing refines the differential when ANA is positive, guiding diagnosis and prognostication. Anti-Smith is specific for SLE but insensitive; anti-SSA/Ro carries neonatal lupus and congenital heart block risk in pregnancy; anti-centromere predicts limited scleroderma; anti-Scl-70 signals diffuse scleroderma and lung fibrosis risk; anti-Jo-1 identifies antisynthetase syndrome. These results shape monitoring, counselling, and specialist referral.
Why is the ANA Multiplex test ordered?
- Investigation of suspected systemic lupus erythematosus in patients with arthritis, malar or discoid rash, photosensitivity, serositis, cytopaenia, or glomerulonephritis
- Evaluation of Raynaud phenomenon, scleroderma-pattern skin changes, or dysphagia to screen for systemic sclerosis and identify prognostic antibodies (anti-centromere, anti-Scl-70)
- Work-up of sicca symptoms (dry eyes, dry mouth) or parotid swelling suggestive of Sjögren syndrome, where anti-SSA/Ro and anti-SSB/La add diagnostic and prognostic information
- Assessment of unexplained inflammatory arthritis or myositis when a systemic autoimmune rheumatic disease is in the differential, particularly with multisystem features
- Screening before conception or in early pregnancy when autoimmune disease is suspected, since anti-SSA/Ro and anti-dsDNA carry foetal and neonatal risks
- Monitoring known SLE with serial anti-dsDNA to detect rising titres that may herald flare, especially lupus nephritis
- Clarification of overlap syndromes when clinical features span more than one connective tissue disease
- Exclusion of systemic autoimmune disease in patients with non-specific symptoms, recognising that a negative ANA at high-quality laboratory effectively rules out most SLE cases not on immunosuppression
Sample collection and turnaround
Sample type
Blood (serum)
Fasting required
No
Collection method
Venipuncture at patient service centre
Turnaround
3–5 business days
Collection notes
Phlebotomy at Quest patient service centre; no special preparation. Separate serum from cells promptly. Note that serial results are best compared within the same laboratory owing to method and threshold variation.
Specimen requirements
Minimum 1 mL serum (gel separator acceptable). Stable refrigerated for up to one week; frozen serum acceptable for reflex testing if initial screen is positive.
What can affect ANA Multiplex results?
Low-titre ANA positivity increases with age and is more common in women; approximately 10–15% of healthy adults over 65 may have a titre of 1:40–1:80 without clinical disease.
Drug-induced lupus and drug-induced ANA positivity are well recognised; anti-histone antibodies are more common in drug-induced lupus, while anti-dsDNA and anti-Smith remain rare, helping distinguish drug-induced from idiopathic SLE.
Acute and chronic viral infections can trigger transient or persistent low-titre ANA and occasionally specific ENA, so clinical and serological correlation is essential.
ANA positivity is reported in some patients with solid and haematological malignancies; paraneoplastic syndromes occasionally mimic autoimmune disease, and concurrent malignancy should be considered when the clinical picture is atypical.
Corticosteroids, hydroxychloroquine, and other disease-modifying drugs can lower antibody titres, so a negative result on treatment does not exclude prior or underlying disease; baseline testing is preferable when feasible.
Immunofluorescence on HEp-2 cells is more sensitive than older substrates; titre cut-offs and pattern interpretation vary between laboratories, so serial results should be compared within the same laboratory when possible.
What do ANA Multiplex results mean?
Elevated
A positive ANA (titre ≥1:40, often ≥1:80 in clinical laboratories) warrants reflex testing. Anti-dsDNA positivity is highly specific for SLE and correlates with nephritis and disease activity. Anti-Smith is specific for SLE but less sensitive. Anti-SSA/Ro and anti-SSB/La suggest Sjögren or lupus with sicca features and confer neonatal lupus risk. Anti-centromere points to limited cutaneous systemic sclerosis, anti-Scl-70 to diffuse disease with lung involvement, and anti-Jo-1 to antisynthetase syndrome with myositis and interstitial lung disease. High ANA titres (≥1:160–1:320) and specific patterns (homogeneous, speckled, nucleolar, centromere) guide further testing but do not diagnose in isolation — clinical criteria are required.
Normal
A negative ANA screen effectively excludes SLE in untreated patients (sensitivity ~95–98%), as well as mixed connective tissue disease and most systemic sclerosis. It does not exclude anti-SSA/Ro-positive lupus (a small subset can be ANA-negative by standard immunofluorescence), late-stage 'burnt-out' disease, or non-systemic autoimmune conditions. A negative screen means reflex testing is not performed.
Reduced
Not applicable; ANA and specific antibodies are reported as negative or positive, not as low values.
Note: ANA positivity is common in healthy individuals, especially at low titre, and does not establish disease without supporting clinical features. Titre and pattern inform probability but are not diagnostic. Specific antibodies (dsDNA, ENA) add specificity and guide prognosis. Drug-induced and infection-associated positivity must be excluded. Serial anti-dsDNA is useful for SLE flare monitoring in some patients but not all. Always interpret in the clinical context and consider rheumatology referral for positive results with compatible symptoms.
Frequently asked questions
Reflex testing means that if the initial ANA screen is positive or equivocal, the laboratory automatically performs anti-dsDNA and extractable nuclear antigen testing on the same specimen without requiring a second order or draw, making the cascade efficient and cost-effective.
How to access the ANA Multiplex test
The ANA Multiplex reference page is free and open to all practitioners. Create a free practitioner account to see pricing, save tests to your list, and generate requisition forms. Verification takes 1–2 business days.
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Published: October 7, 2026
Last reviewed: October 7, 2026
- Aringer M, et al. 2019 European League Against Rheumatism/American College of Rheumatology classification criteria for systemic lupus erythematosus. Arthritis Rheumatol. (2019)
- Agmon-Levin N, et al. International recommendations for the assessment of autoantibodies to cellular antigens referred to as anti-nuclear antibodies. Ann Rheum Dis. (2014)
- Pisetsky DS, et al. ANA as an entry criterion for the classification of SLE. Autoimmun Rev. (2019)
- Mahler M, et al. Autoantibodies to extractable nuclear antigens: making detection and interpretation more reliable. Clin Chem Lab Med. (2020)
- Meroni PL, Schur PH. ANA screening: an old test with new recommendations. Ann Rheum Dis. (2010)
This content is for licensed healthcare practitioners only and does not constitute medical advice. Clinical decisions should be based on the full clinical picture and not on any single test result.