Online

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Antinuclear antibodies: When to Habib U. Rehman, MB,

FRCPC, FRCPI, FRCP
(Glas), FACP

test and how to interpret findings Department of Medicine,

Regina General Hospital,
Saskatchewan, Canada

Order ANA assays only when clinical features suggest a habib31@sasktel.net

connective tissue disorder. Let ANA immunofluorescent The author reported no

potential conflict of interest
relevant to this article.
patterns direct additional testing decisions.

Practice
recommendation
› Reserve antinuclear
antibody testing for instances
A ntinuclear antibodies (ANA) are a spectrum of auto­
antibodies that react with various nuclear and cyto-
plasmic components of normal human cells. Their
detection is important in the diagnosis of some connective tis-
sue diseases (CTD)—eg, systemic lupus erythematosus (SLE),
of clinically suggestive Sjögren’s syndrome (SS), scleroderma, polymyositis, or mixed
connective tissue diseases connective tissue disease (MCTD). Unfortunately, ANA tests
(CTD) and for assessing
are often used indiscriminately in daily clinical practice.1
CTD prognosis. It can also
be useful in monitoring
disease progression. C
When is ANA testing warranted?
Strength of recommendation (SOR) Indiscriminate use of ANA testing can yield positive results that
A Good-quality patient-oriented falsely point to CTD in a high proportion of patients and thereby
evidence
lead to further inappropriate testing and errant management de-
B Inconsistent or limited-quality
patient-oriented evidence cisions. To wit: The presence of ANA in the serum can be associat-
C Consensus, usual practice, ed with any number of factors, such as genetic predisposition (eg,
opinion, disease-oriented
evidence, case series through histocompatibility locus DR3), environmental agents (vi-
ruses, 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 dilu-
tion 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 in-
formative antibody patterns. Five distinct patterns of fluores-
cence 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 phenom-

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 TABLE

Diagnostic significance of common

immunofluorescent ANA patterns3
ANA pattern Specificity Antigen Disease association
Homogenous Low dsDNA SLE
Histones DLE, RA
Rim High Centromere CREST, SLE
Speckled Low Ro/SS-A SS
La/SS-B SS
RNP MCTD
Sm SLE
Scl-70 Scleroderma
Nucleolar Low PM/Scl Scleroderma
Cytoplasmic Low tRNA synthetases, Jo-1 Poly/dermatomyositis
Mitochondria Primary biliary cirrhosis
When clinical Smooth muscle Autoimmune hepatitis
probability of
ANA, antinuclear antibody; CREST, calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia
connective tissue syndrome; DLE, discoid lupus erythematosus; dsDNA, double-stranded DNA; MCTD, mixed connective tissue disease; RA,
diseases is low, rheumatoid arthritis; RNP, ribonucleoprotein; SLE, systemic lupus erythematosus; SS, Sjögren’s syndrome; tRNA, transfer
ribonucleic acid.
the presence
of ANA in the
serum can
indicate chronic enon, esophageal dysmotility, sclero- highly characteristic profile of autoantibodies
infection, dactyly, and telangiectasia) syndrome to cellular antigens. A patient’s clinical fea-
neoplasm, or and SLE. tures and ANA fluorescence pattern should
advancing age. 3. Speckled, in which the nucleus fluo- direct additional testing.
resces in a speckled pattern, can be z Photosensitive butterfly rash, arthral-
seen in a variety of CTDs, including gias/arthritis, pleuritic chest pain, fever of
Sjögren’s syndrome, MCTD, SLE, and unknown cause, and urine sediment con-
scleroderma. sistent with nephritis point to a diagnosis of
4. Nucleolar, in which the nucleolus fluo- SLE. Order an assay for anti-double-stranded
resces, is associated with scleroderma. DNA (dsDNA) antibodies, which, if present,
5. Cytoplasmic, in which fluorescence confirm the diagnosis.4 Also order an assay
occurs outside the nucleus, typically for anti-Sm antibodies, which are highly spe-
occurs with poly/dermatomyositis, cific for SLE but found only in 30% to 40% of
primary biliary cirrhosis, or autoim- SLE patients.4
mune hepatitis. z  Raynaud’s phenomenon, skin hard-
ening or thickening, stiffness and tightening
of the skin on the fingers, hands and fore-
What is the next step arms, tight and mask-like skin on the face, dry
if ANA is positive? cough, shortness of breath, and difficulty in
A positive ANA result warrants additional swallowing are features of scleroderma. If you
studies to identify specific autoantibodies suspect this disorder, order an assay for anti-
suggested by the fluorescence pattern and by Scl-70 anti­bodies. These antibodies are highly
a patient’s signs and symptoms. specific for scleroderma, but sensitivity of the
assay is only 15% to 20%.5
Following up diagnostic clues z  Calcinosis, Raynaud’s phenomenon,
Most systemic autoimmune diseases have a esophageal dysmotility, sclerodactyly, and te-

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 antinuclear antibodies: when to test

langiectasia indicate CREST syndrome. Anti- thrombocytopenia.12

centromere antibodies are highly specific for • The presence of anti-Ro/SS-A in the cir-
CREST syndrome; sensitivity on assay is 50% culation of pregnant women with SLE
to 90%.6 confers a higher risk of neonatal lupus
z  MCTD combines features of rheumatoid erythematosus and of congenital heart
arthritis, SLE, myositis, and scleroderma. Or- block in their newborns.13
der an assay of anti-RNP (ribonucleoprotein) • Severe interstitial lung disease is fre-
antibodies. Although anti-RNP antibodies are quently found in scleroderma patients
also found in 25% to 30% of patients with SLE, who test positive for anti-Scl-70.14 An-
they typically appear in the company of anti- tibodies to aminoacyl-tRNA synthetas-
Sm antibodies.7 Isolated high titers of anti-RNP es—including anti–Jo-1, as mentioned
antibodies point to MCTD, and sensitivity on earlier—are associated with pulmonary
assay is 100%.8 Their absence on testing, there- involvement in polymyositis patients.11
fore, excludes the diagnosis of MCTD. • A positive ANA test result in Raynaud’s
RNP, anti-Ro/SS-A, La/SS-B, and Sm are phenomenon increases the likelihood
also referred to as extractable nuclear antigens that the patient will develop a systemic
(ENA). Assays of antibodies to ENA and anti- rheumatic disease; a negative result re-
dsDNA are warranted only if the ANA assay duces this likelihood.15
result is positive. It is rare to have a positive • While the ANA test is not useful for diag-
anti-ENA antibody test (with the exception of nosing juvenile chronic arthritis (JCA), it
antibodies to cytoplasmic antigens) in the ab- is useful to test for ANA in patients with Think
sence of a positive ANA test.9 known JCA. A positive test result should systemic lupus
z  Dry eyes, dry mouth, joint pain and prompt screening for uveitis.16 erythematosus
swelling, and swelling of parotid glands point to • An ANA test is not necessary for diagnos- when a
Sjögren’s syndrome. Anti-Ro/SS-A and La/SS-B ing antiphospholipid antibody syndrome patient has a
antibodies are associated with Sjögren’s syn- (APS). However, the presence of ANA in a photosensitive
drome, but are also found in seronegative SLE.10 patient with APS increases the likelihood butterfly rash,
Therefore, if patients with features suggestive of that APS is secondary to SLE.17 arthralgia,
SLE have a negative result on a dsDNA anti- pleuritic chest
body assay, test for anti-Ro/SS-A and La/SS-B Monitoring disease activity pain, fever, or
antibodies. Documenting titers of anti-dsDNA antibod- urine sediment
z Muscle weakness and soreness, pur- ies may help in monitoring the disease activity consistent with
plish discoloration of the upper eyelids, and of SLE in some patients. However, changes in nephritis.
purplish-red discoloration of the knuckles titers of anti-dsDNA should be interpreted in
suggest dermatomyositis. Muscle biopsy and the clinical context of the SLE Disease Activity
electromyography will clinch the diagnosis. Index.18 JFP
Also test for anti–Jo-1 antibodies, which are
associated with pulmonary involvement in Correspondence
Habib U. Rehman, MB, Department of Medicine, Regina
polymyositis.11 Qu’Appelle Health Region, Regina General Hospital,
1440–14th Avenue, Regina, SK, S4P 0W5, Canada;
habib31@sasktel.net
ANA’s continuing role—
prognosis and disease activity
Besides confirming a diagnosis of CTD in pa- References
tients with suggestive clinical features, ANA 1. Volkmann ER, Taylor M, Ben-Artzi A. Using the antinuclear
testing serves 2 additional purposes: to help antibody test to diagnose rheumatic disease: when does a posi-
tive test warrant further investigation? South Med J. 2012;105:
determine a patient’s prognosis and to monitor 100-104.
CTD activity. Consider the following: 2. Giannouli E, Chatzidimitriou D, Gerou S, et al. Frequency and
specificity of antibodies against nuclear and cytoplasmic anti-
• Patients with Sjögren’s syndrome who gens in healthy individuals by classic and new methods. Clin
Rheumatol. 2013;32:1541-1546.
test positive for anti-Ro/SS-A antibod-
3. O’Sullivan M, McLean-Tooke A, Loh RK. Antinuclear antibody
ies have aggressive, extra-glandular dis- test. Aust Fam Physician. 2013;42:718-721.
ease that can cause vasculitis, purpura, 4. Kurien BT, Scofield RH. Autoantibody determination in the
diagnosis of systemic lupus erythematosus. Scand J Immunol.
lymphadenopathy, leukopenia, and 2006;64:227-235.
c o nti nu ed

jfponline.com Vol 64, No 1 | JANUARY 2015 | The Journal of Family Practice E7

 5. Basu D, Reveille JD. Ant-scl-70. Autoimmunity. 2005;38: verse outcomes in primary Sjogren’s syndrome: identification
65-72. of prognostic factors. Rheumatology. 2007;46:1359-1362.
6. Caramaschi P, Biasi D, Manzo T, et al. Anticentromere anti- 13. Lindop R, Arentz G, Thurgood LA, et al. Pathogenicity and pro-
body—clinical associations. A study of 44 patients. Rheumatol teomic signatures of autoantibodies to Ro and La. Immunol Cell
Int. 1995;14:253-255. Biol. 2012;90:304-309.
7. Migliorini P, Baldini C, Rocchi V, et al. Anti-Sm and anti-RNP 14. Steen VD. Autoantibodies in systemic sclerosis. Semin Arthritis
antibodies. Autoimmunity. 2005;38:47-54. Rheum. 2005;35:35-42.
8. Alarcón-Segovia D, Cardiel MH. Comparison between 3 diag-
15. Spencer-Green G. Outcomes in primary Raynaud phenomenon:
nostic criteria for mixed connective tissue disease. Study of 593
a meta-analysis of the frequency, rates, and predictors of transi-
patients. J Rheumatol. 1989;16:328-334.
tion to secondary diseases. Arch Intern Med. 1998;158:595-600.
9. Phan TG, Wong RC, Adelstein S. Autoantibodies to extractable
nuclear antigens: making detection and interpretation more 16. Grassi A, Corona F, Casellato A, et al. Prevalence and outcome
meaningful. Clin Diagn Lab Immunol. 2002;9:1-7. of juvenile idiopathic arthritis-associated uveitis and relation to
articular disease. J Rheumatol. 2007;34:1139-1145.
10. Cross LS, Aslam A, Misbah SA. Antinuclear antibody-negative
lupus as a distinct diagnostic entity—does it no longer exist? 17. Petri M. Diagnosis of antiphospholipid antibody syndrome.
QJM. 2004;97:303-308. Rheum Dis Clin North Am. 1994;20:443.
11. Miller FW, Waite KA, Biswat T, et al. The role of an autoantigen, 18. Kavanaugh A, Tomar R, Reveille J, et al. Guidelines for clinical
histidyl-tRNA synthetase, in the induction and maintenance of use of the antinuclear antibody test and tests for specific auto-
autoimmunity. Proc Natl Acad Sci USA. 1990;87:9933-9937. antibodies to nuclear antigens. American College of Patholo-
12. Brito-Zerón P, Ramos-Casals M, Bove A, et al. Predicting ad- gists. Arch Pathol Lab Med. 2000;124:71-81.

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