Antinuclear antibodies: When to test and how to interpret findings

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Applied Evidence

Antinuclear antibodies: When to test and how to interpret findings

J Fam Pract. 2015 January;64(1):E5-E8

Habib U. Rehman, MB, FRCPC, FRCPI, FRCP

(Glas), FACP

Order ANA assays only when clinical features suggest a connective tissue disorder. Let ANA immunofluorescent patterns direct additional testing decisions.

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References

1. Volkmann ER, Taylor M, Ben-Artzi A. Using the antinuclear antibody test to diagnose rheumatic disease: when does a positive test warrant further investigation? South Med J. 2012;105:100-104.

2. Giannouli E, Chatzidimitriou D, Gerou S, et al. Frequency and specificity of antibodies against nuclear and cytoplasmic antigens in healthy individuals by classic and new methods. Clin Rheumatol. 2013;32:1541-1546.

3. O’Sullivan M, McLean-Tooke A, Loh RK. Antinuclear antibody test. Aust Fam Physician. 2013;42:718-721.

4. Kurien BT, Scofield RH. Autoantibody determination in the diagnosis of systemic lupus erythematosus. Scand J Immunol. 2006;64:227-235.

5. Basu D, Reveille JD. Ant-scl-70. Autoimmunity. 2005;38:65-72.

6. Caramaschi P, Biasi D, Manzo T, et al. Anticentromere antibody—clinical associations. A study of 44 patients. Rheumatol Int. 1995;14:253-255.

7. Migliorini P, Baldini C, Rocchi V, et al. Anti-Sm and anti-RNP antibodies. Autoimmunity. 2005;38:47-54.

8. Alarcón-Segovia D, Cardiel MH. Comparison between 3 diagnostic criteria for mixed connective tissue disease. Study of 593 patients. J Rheumatol. 1989;16:328-334.

9. Phan TG, Wong RC, Adelstein S. Autoantibodies to extractable nuclear antigens: making detection and interpretation more meaningful. Clin Diagn Lab Immunol. 2002;9:1-7.

10. Cross LS, Aslam A, Misbah SA. Antinuclear antibody-negative lupus as a distinct diagnostic entity—does it no longer exist? QJM. 2004;97:303-308.

11. Miller FW, Waite KA, Biswat T, et al. The role of an autoantigen, histidyl-tRNA synthetase, in the induction and maintenance of autoimmunity. Proc Natl Acad Sci USA. 1990;87:9933-9937.

12. Brito-Zerón P, Ramos-Casals M, Bove A, et al. Predicting adverse outcomes in primary Sjogren’s syndrome: identification of prognostic factors. Rheumatology. 2007;46:1359-1362.

13. Lindop R, Arentz G, Thurgood LA, et al. Pathogenicity and proteomic signatures of autoantibodies to Ro and La. Immunol Cell Biol. 2012;90:304-309.

14. Steen VD. Autoantibodies in systemic sclerosis. Semin Arthritis Rheum. 2005;35:35-42.

15. Spencer-Green G. Outcomes in primary Raynaud phenomenon: a meta-analysis of the frequency, rates, and predictors of transition to secondary diseases. Arch Intern Med. 1998;158:595-600.

16. Grassi A, Corona F, Casellato A, et al. Prevalence and outcome of juvenile idiopathic arthritis-associated uveitis and relation to articular disease. J Rheumatol. 2007;34:1139-1145.

17. Petri M. Diagnosis of antiphospholipid antibody syndrome. Rheum Dis Clin North Am. 1994;20:443.

18. Kavanaugh A, Tomar R, Reveille J, et al. Guidelines for clinical use of the antinuclear antibody test and tests for specific autoantibodies to nuclear antigens. American College of Pathologists. Arch Pathol Lab Med. 2000;124:71-81.

PRACTICE RECOMMENDATION

› Reserve antinuclear antibody testing for instances of clinically suggestive connective tissue diseases (CTD) and for assessing CTD prognosis. It can also be useful in monitoring disease progression. C

Strength of recommendation (SOR)

A Good-quality patient-oriented evidence
B Inconsistent or limited-quality patient-oriented evidence
C Consensus, usual practice, opinion, disease-oriented evidence, case series

Antinuclear antibodies (ANA) are a spectrum of autoantibodies that react with various nuclear and cytoplasmic components of normal human cells. Their detection is important in the diagnosis of some connective tissue diseases (CTD)—eg, systemic lupus erythematosus (SLE), Sjögren’s syndrome (SS), scleroderma, polymyositis, or mixed connective tissue disease (MCTD). Unfortunately, ANA tests are often used indiscriminately in daily clinical practice.¹

When is ANA testing warranted?

Indiscriminate use of ANA testing can yield positive results that falsely point to CTD in a high proportion of patients and thereby lead to further inappropriate testing and errant management decisions.When clinical probability of connective tissue diseases is low, the presence of ANA in the serum can indicate chronic infection, neoplasm, or advancing age. To wit: The presence of ANA in the serum can be associated with any number of factors, such as genetic predisposition (eg, through histocompatibility locus DR3), environmental agents (viruses, drugs), chronic infections, neoplasms, and advancing age.¹ Therefore, the test should not be ordered in a patient with low pre-test probability of CTD. Moreover, higher titers of ANA are more clinically significant than lower titers. In one multicenter study, 31.7% of healthy individuals were ANA-positive at a serum dilution of 1:40, but only 5% were ANA-positive at a dilution of 1:160.²

What is the clinical significance of different immunofluorescent patterns?

Immunofluorescent ANA testing not only determines if such antibodies are present in a patient’s serum but also reveals informative antibody patterns. Five distinct patterns of fluorescence are possible and can help differentiate between various CTDs (TABLE³):
1. Homogenous, in which the entire nucleus fluoresces, is seen in SLE and discoid lupus erythematosus (DLE).
2. Rim, in which the nuclear perimeter fluoresces, is seen most often in CREST (calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia) syndrome and SLE.
3. Speckled, in which the nucleus fluoresces in a speckled pattern, can be seen in a variety of CTDs, including Sjögren’s syndrome, MCTD, SLE, and scleroderma.
4. Nucleolar, in which the nucleolus fluoresces, is associated with scleroderma.
5. Cytoplasmic, in which fluorescence occurs outside the nucleus, typically occurs with poly/dermatomyositis, primary biliary cirrhosis, or autoimmune hepatitis.

What is the next step if ANA is positive?

A positive ANA result warrants additional studies to identify specific autoantibodies suggested by the fluorescence pattern and by a patient’s signs and symptoms.

Following up diagnostic clues

Think systemic lupus erythematosus when a patient has a photosensitive butterfly rash, arthralgia, pleuritic chest pain, fever, or urine sediment consistent with nephritis. Most systemic autoimmune diseases have a highly characteristic profile of autoantibodies to cellular antigens. A patient’s clinical features and ANA fluorescence pattern should direct additional testing.

Photosensitive butterfly rash, arthralgias/arthritis, pleuritic chest pain, fever of unknown cause, and urine sediment consistent with nephritis point to a diagnosis of SLE. Order an assay for anti-double-stranded DNA (dsDNA) antibodies, which, if present, confirm the diagnosis.⁴ Also order an assay for anti-Sm antibodies, which are highly specific for SLE but found only in 30% to 40% of SLE patients.⁴

Raynaud’s phenomenon, skin hardening or thickening, stiffness and tightening of the skin on the fingers, hands and forearms, tight and mask-like skin on the face, dry cough, shortness of breath, and difficulty in swallowing are features of scleroderma. If you suspect this disorder, order an assay for anti-Scl-70 antibodies. These antibodies are highly specific for scleroderma, but sensitivity of the assay is only 15% to 20%.⁵

Calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia indicate CREST syndrome. Anti-centromere antibodies are highly specific for CREST syndrome; sensitivity on assay is 50% to 90%.⁶

MCTD combines features of rheumatoid arthritis, SLE, myositis, and scleroderma. Order an assay of anti-RNP (ribonucleoprotein) antibodies. Although anti-RNP antibodies are also found in 25% to 30% of patients with SLE, they typically appear in the company of anti-Sm antibodies.⁷ Isolated high titers of anti-RNP antibodies point to MCTD, and sensitivity on assay is 100%.⁸ Their absence on testing, therefore, excludes the diagnosis of MCTD.

RNP, anti-Ro/SS-A, La/SS-B, and Sm are also referred to as extractable nuclear antigens (ENA). Assays of antibodies to ENA and anti-dsDNA are warranted only if the ANA assay result is positive. It is rare to have a positive anti-ENA antibody test (with the exception of antibodies to cytoplasmic antigens) in the absence of a positive ANA test.⁹