Extractable Nuclear Antigen (ENA) Panel

The extractable nuclear antigen (ENA) panel identifies autoantibodies against soluble nuclear and cytoplasmic antigens that can be extracted from cells with saline: Sm, RNP (U1-ribonucleoprotein), Ro (SSA, Ro52 and Ro60), La (SSB), Scl-70 (topoisomerase I) and Jo-1 (histidyl-tRNA synthetase).

It is requested after a positive ANA, or in suspected connective tissue disease with a negative ANA, to define the disease subtype, to predict organ involvement and to direct management.

Methods include line immunoassay, ELISA, multiplex bead and chemiluminescent assays, and older double immunodiffusion.

Each antibody has a characteristic association: anti-Sm (highly specific for SLE), anti-RNP (mixed connective tissue disease), anti-Ro and anti-La (Sjögren syndrome, SLE, neonatal lupus), anti-Scl-70 (diffuse systemic sclerosis with lung fibrosis), and anti-Jo-1 (antisynthetase syndrome). The panel is useful when there is clinical suspicion, and it is not a screening test.

Indications. Positive ANA with symptoms suggesting connective tissue disease. Suspected Sjögren syndrome (sicca symptoms, parotid swelling), mixed connective tissue disease (Raynaud, swollen hands, myositis), systemic sclerosis, myositis with interstitial lung disease, SLE, or unexplained rash and arthritis. Pregnant women with autoimmune disease (anti-Ro and La). Infants with suspected neonatal lupus (heart block, rash, cytopenias).

Specimen. Serum 1 mL, no fasting. Stable for several days at 2 to 8 °C, or frozen for longer. Avoid haemolysis and microbial contamination.

Anti-Sm (Smith).

• Specific for SLE (specificity over 95%), but present in only 20 to 30% (30 to 40% in Black and Asian patients).
• Linked to renal and central nervous system involvement in some studies. Does not correlate well with disease activity.
• It is an immunological criterion in the SLE classification.

Anti-RNP (U1-RNP).

• High titres are the hallmark of mixed connective tissue disease (found in over 95%), with Raynaud phenomenon, puffy hands, arthritis, myositis, oesophageal dysmotility and pulmonary hypertension.
• Also present in 20 to 40% of SLE, usually without anti-Sm in mixed disease, and associated with milder renal involvement.
• Low titres occur in other connective tissue diseases.

Anti-Ro (SSA; Ro52 and Ro60).

• Present in Sjögren syndrome (60 to 70%), SLE (25 to 40%), subacute cutaneous lupus (70 to 90%), neonatal lupus, and some myositis and systemic sclerosis cases.
• Anti-Ro60 relates to SLE and Sjögren syndrome; anti-Ro52 alone is found in myositis (with anti-Jo-1 and interstitial lung disease), systemic sclerosis, primary biliary cholangitis and liver disease.
• Maternal anti-Ro/La cross the placenta from 16 to 24 weeks and cause congenital heart block in 1 to 2% of exposed pregnancies (recurrence 15 to 20%), neonatal lupus rash, cytopenias and hepatic disease.

Anti-La (SSB).

• Present in 40 to 50% of Sjögren syndrome and 10 to 15% of SLE, nearly always with anti-Ro. Isolated anti-La is rare and of uncertain significance.
• Associated with milder lupus nephritis, and with neonatal lupus.

Anti-Scl-70 (topoisomerase I).

• Specific for systemic sclerosis (present in 20 to 40%), associated with diffuse cutaneous disease, interstitial lung disease and poor prognosis. Seen in Black and Asian populations more frequently.
• Anti-centromere antibodies (limited disease and pulmonary hypertension) and anti-RNA polymerase III (renal crisis, malignancy) are tested separately in systemic sclerosis.

Anti-Jo-1 (histidyl-tRNA synthetase).

• Present in 15 to 30% of inflammatory myopathy and defines the antisynthetase syndrome (myositis, interstitial lung disease, arthritis, mechanic's hands, Raynaud, fever).
• Other antisynthetase antibodies (PL-7, PL-12, EJ, OJ) are on extended myositis panels. Anti-Jo-1 is associated with interstitial lung disease in 70 to 90%.

Critical values and findings needing immediate action.

• Anti-Ro or La positive woman who is pregnant or planning pregnancy: foetal echocardiography with PR interval surveillance weekly from 16 to 26 weeks, and hydroxychloroquine to reduce the risk of heart block.
• Neonate with heart block, rash or cytopenias and maternal anti-Ro/La: urgent paediatric cardiology.
• Anti-Scl-70 or anti-Jo-1 with dyspnoea or crackles: urgent high resolution CT and lung function.
• Anti-RNP positivity with signs of pulmonary hypertension: echocardiography and right heart catheterisation.
• Scleroderma renal crisis (hypertension, acute kidney injury) in patients with anti-RNA polymerase III: urgent ACE inhibitor therapy.

Interferences and pitfalls.

• ENA testing is requested after a positive ANA with a pattern or clinical suspicion, but antibodies to Ro and Jo-1 can occur with a negative ANA on HEp-2 cells.
• Different methods give different results: line immunoassay and multiplex assays are more sensitive than immunodiffusion, and low positive results in solid phase assays can be false positives. Confirmation by a second method may be needed.
• Anti-Ro60 and Ro52 should be reported separately, and isolated Ro52 has limited specificity.
• A positive antibody does not indicate disease activity. Titres are not used for monitoring.
• Antibodies can precede disease by years, and positive antibodies in asymptomatic people need clinical follow up rather than treatment.
• Anti-Sm is specific but insensitive. A negative result does not exclude SLE.
• Anti-histone antibodies (drug induced lupus) are not part of the ENA panel.
• Panels vary between laboratories, and some include Centromere B, Ribosomal P, PM-Scl and Mi-2, so the content of the local panel should be known.

Special groups.

• Pregnancy: anti-Ro/La management involves obstetric and paediatric cardiology teams. Women with a previous affected child have a 15 to 20% recurrence.
• Neonates: maternal antibodies clear by 6 to 8 months, and the rash resolves.
• Older adults: low titre antibodies are more frequent and less specific.
• Black patients: higher frequency of anti-Sm, anti-RNP and anti-Scl-70.
• Regions: availability of line assays differs, and dilution and confirmatory testing are done in reference laboratories.

Follow up tests. ANA with pattern, anti-dsDNA, complement (C3, C4), rheumatoid factor, anti-CCP, antiphospholipid antibodies, extended myositis and systemic sclerosis panels (anti-centromere, RNA polymerase III, PM-Scl, Mi-2, MDA5), creatine kinase, Schirmer test and salivary gland assessment, high resolution CT chest and lung function, echocardiography, nailfold capillaroscopy, urinalysis, and foetal echocardiography in pregnancy.