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Mod Rheumatol. 2009 ; 19(3): 219–228. doi:10.1007/s10165-009-0155-3.

Clinical interpretation of antinuclear antibody tests in systemic

rheumatic diseases

Minoru Satoh,
Division of Rheumatology and Clinical Immunology, Department of Medicine, and Department of
Pathology, Immunology, and Laboratory Medicine, University of Florida, P.O. Box 100221,
Gainesville, FL 32610-0221, USA
Monica Vázquez-Del Mercado, and
Instituto de Investigación en Reumatología y del Sistema Músculo Esquelético Sierra Mojada 950,
Planta Baja, CP 44240 Edificio P Ala Oriente, Mexico. Centro Universitario de Ciencias de la Salud,
Universidad de Guadalajara, Guadalajara, Jalisco, Mexico. Divisiòn de Medicina Interna,
Reumatòloga, Departamento de Reumatologìa, Hospital Civil Juan I, Mechaca, Guadalajara,
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Jalisco, Mexico
Edward K. L. Chan
Department of Oral Biology, University of Florida, Gainesville, FL 32610-0424, USA
Minoru Satoh: Minoru.Satoh@medicine.ufl.edu

Abstract
Autoantibody tests have been used extensively in diagnosis and follow-up of patients in
rheumatology clinics. Immunofluorescent antinuclear antibody test using HEp-2 cells is still
considered the gold standard for screening of autoantibodies, and most of specific autoantibodies are
currently tested by ELISA as a next step. Among the many autoantibody specificities described, some
have been established as clinically useful diagnostic markers and are included in the classification
criteria of diseases. Despite a long history of routine tests and attempts to standardize such assays,
there are still limitations and problems that clinicians need to be aware of. Clinicians should be able
to use autoantibody tests more efficiently and effectively with a basic knowledge on the significance
of and potential problems in autoantibody tests.
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Keywords
Antinuclear antibodies; Autoantibodies; SLE; Scleroderma; Polymyositis

Introduction
Autoimmune disease is defined as a condition with tissue destruction or organ malfunction
caused by autoimmune mechanisms. It is often more generously interpreted as a disease
accompanied by an autoimmune phenomenon, since the direct role of the autoimmune reaction
in the disease pathogenesis is not always apparent. Systemic autoimmune diseases (as opposed
to organ-specific autoimmune diseases) are characterized by the presence of non-organ-
specific autoantibodies that target antigens that are present in virtually any type of cell.
Clinically, they are characterized by the systemic involvement of autoimmune tissue
destruction in various organs. Systemic autoimmune diseases, autoimmune rheumatic diseases,

Correspondence to: Minoru Satoh, Minoru.Satoh@medicine.ufl.edu.

 Satoh et al. Page 2

and systemic rheumatic diseases basically refer to the same category of diseases, and these
terms are used interchangeably. Systemic lupus erythematosus (SLE), scleroderma (SSc),
polymyositis/dermatomyositis (PM/DM), and rheumatoid arthritis (RA) are representative
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disorders in this category. Whether Sjögren’s syndrome (SjS) should be classified as organ-
specific (salivary glands, lacrimal glands) or systemic is arguable.

The presence of autoantibodies that react with cellular constituents, including proteins and
nucleic acids (dsDNA, RNA), can be screened for by observing mammalian cells on a slide
[1]. The incubation of human sera with fixed cells is followed by fluorochrome (such as
fluorescein isothiocyanate: FITC) conjugated antibodies against human IgG. Observing under
a fluorescent microscope allows one to determine the patterns as well as titers of autoantibodies.
This standard method of performing antinuclear antibody (ANA) tests by immunofluorescence
has been used for over 40 years as a first-step screening test for autoimmune diseases and is
still the standard method. Although the ANA test has a nearly 100% sensitivity for the diagnosis
of SLE, it is not specific for this diagnosis and is frequently positive in other systemic
autoimmune rheumatic diseases such as SSc, PM/DM, and SjS as well. ANA is also found in
organ-specific autoimmune diseases, and in other nonautoimmune diseases such as viral
infections [2]. In this review, we will focus on the clinical significance and interpretation of
autoantibody data for clinicians, and provide a practical guide on the use of autoantibody tests
for specific diseases.
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Patterns of antinuclear antibodies (ANA)

Although it is usually called the ANA test, the same procedure also exhibits reactivity against
all types of subcellular structures and cell organelles including cell surfaces, cytoplasm, nuclei,
or nucleoli [1]. The antigens recognized are mainly proteins, protein macromolecular
complexes, protein–nucleic acid complexes, and nucleic acids. In fact, most autoantibodies
that are clinically useful target RNA–protein or DNA–protein complexes. The staining may
be purely nucleolar, as seen in certain SSc patients, or purely cytoplasmic, as in anti-Jo-1
positive PM/DM patients. Thus, the ANA test is not just for “nuclear” staining.

The interpretation of most nuclear staining patterns is relatively straightforward, and they are
usually reported as being nuclear, centromere, or nucleolar. Cytoplasmic staining may not be
reported at all by some laboratories, and it is useful to know and distinguish whether it was
read as negative or it was simply not reported by the technical staff at the laboratory. A positive
nuclear staining result will usually come back with a more detailed staining pattern, such as
speckled (Fig. 1a), homogeneous, or peripheral. A homogeneous/peripheral pattern reflects
antibodies to histone/dsDNA/chromatin, whereas many other specificities found in systemic
rheumatic diseases show speckled patterns of various sizes and densities (fine speckled, large
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speckled, etc.). Thus, while this information is somewhat useful, it is relatively subjective, and
varies depending on the laboratory or individual; the pattern may also differ at different
dilutions. A centromere pattern is usually reported as distinct pattern, but they can also be
termed discrete speckled nuclear staining patterns (Fig. 1b). Unusual staining patterns in nuclei,
such as those for the nuclear mitotic apparatus (NuMA) [3] (Fig. 1c), Cajal body (p80-coilin),
or nuclear dots (Fig. 1d), may not be reported depending on the experience of the laboratory.
The cytoplasmic staining shown by anti-Jo-1 (histidyl tRNA synthetase) antibodies in PM/DM
or antimitochondrial antibodies in primary biliary cirrhosis (PBC) (Fig. 1d) is often either not
reported or incorrectly interpreted. The staining from cytoplasmic dots called GW bodies
(GWBs)/P-bodies (Fig. 1e) was ignored or unrecognized until recently [4], but this cytoplasmic
structure has become a hot area of research in molecular and cellular biology following the
discovery of the critical role it plays in the functions of siRNA and miRNA [5,6]. Furthermore,
anti-Su antibodies that recognize a component of GWB, Ago2 [7], are very common
autoantibodies found in 10–20% of various systemic autoimmune diseases in American and

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Japanese populations [8] and ~25% of Mexican SLE patients (Vázquez-Del Mercado,
manuscript in preparation). Thus, GWB staining from anti-Su antibodies is in fact a quite
common staining pattern if it is recognized by an experienced laboratory [1]. Golgi patterns
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[9] may also be overlooked or not reported properly in routine screening tests [1]. Another
example of unrecognized staining is a complete lack of reports of punctate nucleolar staining
by anti-RNA polymerase I from hospital laboratories [10].

Any of the staining patterns described above are considered evidence of non-organ-specific
autoimmunity. It is useful to know the significance of various staining patterns; however, in
practice, it is equally important for clinicians to know what will be reported from their clinical
laboratory, what may not be reported, and what may be incorrectly reported.

Titers of ANA
The introduction of human cancer cell lines (human laryngeal cancer cell line HEp-2 is the
standard) as a substrate for the ANA test significantly increased the sensitivity in the detection
of ANA in patients with systemic rheumatic diseases. However, a concomitant increase in
positive results in healthy individuals decreased the specificity. One multicenter study reported
that 31.7% of normal individuals were ANA positive at 1:40 dilution, which was decreased to
13.3% at 1:80 and 5.0% at 1:160 dilution. Since ~95% of SLE were still positive at 1:160
dilution, raising the negative cut-off titer from 1:40 to 1:160 may improve the distinction
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between a clinically significant ANA result and a positive ANA result occurring in a normal
individual [2]. The frequency of positive ANA detection using this cut-off was 95% in SLE,
87% in SSc, 74% in SjS, and 14% in RA [2]. It is generally true that the prevalence of positive
ANA among healthy individuals increases after switching the ANA substrate from frozen
tissues to human cell lines; however, the reactivity is significantly affected by the cell substrate,
fixation, secondary antibodies, buffer, fluorescent microscope, and other conditions. Thus, an
appropriate cut-off should be defined by each laboratory [1].

Based on these observations, there is some truth in saying that higher titers of ANA are more
clinically significant; ANA in healthy individual is generally in low titers. There is some
evidence that clinical manifestations associated with certain autoantibodies are more evident
among patients with high titers of that specificity. Sclerodactyly, Raynaud’s phenomenon and
vascular disease associated with anticentromere antibodies are more common in patients with
high titers of this specificity than in those with low titers [11]. Classic feature of mixed
connective tissue disease (MCTD) is associated with very high titers of anti-U1RNP antibodies
[12,13]. However, it should be noted that higher titers of ANA do not always mean that the
patient’s disease is more severe or active. Specificity of autoantibodies is one of the factors
that correlate strongly with titers of ANA. Certain autoantibodies such as anti-dsDNA usually
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show relatively low titers in ANA, while others such as anti-U1RNP and centromere may show
titers of 1:10,240 or even higher [11]. Individuals with high titers of these antibodies may lack
serious organ involvement, being classified as having undifferentiated connective tissue
disease (UCTD) or Raynaud’s disease, and may not require any medical treatment [14,15]. On
the other hand, patients with low titers of disease marker antibodies (see the next section) could
have a typical disease requiring attention and follow-up.

Autoantibodies associated with a certain diagnosis (disease marker

antibodies), particular symptoms, or disease activity
Several autoantibody specificities that are found almost exclusively in patients with a particular
diagnosis and are useful for diagnosing or predicting the development of disease are called
disease marker antibodies. Anti-Sm and anti-dsDNA antibodies are highly specific for the
diagnosis of SLE and are included in the classification criteria for SLE by the American College

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of Rheumatology (ACR) [16]. ANA is also included among these criteria, and nearly all SLE
patients are ANA positive; however, ANA is not specific for SLE. Antiphospholipid
antibodies, listed under immunological disorders in the SLE criteria, are also common in SLE
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but can also be found in various systemic rheumatic diseases and often in patients with anti-
phospholipid syndrome who do not fulfill the criteria for any rheumatic diseases.
Antiribosomal P antibodies found in ~10% of patients and anti-PCNA (proliferating cell
nuclear antigen) antibodies seen in ~2% of SLE patients are also considered to be disease-
specific, but the supporting data are not as extensive as those for anti-dsDNA or anti-Sm [1,
17,18]. An association between anti-ribosomal P antibodies and neuropsychiatric symptoms
has been suggested for many years, but a recent meta-analysis indicated that anti-ribosomal P
antibody testing has a negligible capacity to predict neuropsychiatric manifestations of SLE in
individual cases [19].

Antitopoisomerase I (topo I, Scl-70), found in 15–25% of patients, and anti-RNA polymerase

III (RNAP III) antibodies, found in 20–25% of patients, are highly specific for SSc [20–23].
Anticentromere antibodies were classically described in association with a subset of SSc,
CREST syndrome (calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly,
and telangiectasias) [24]; however, this specificity is also common in limited SSc without
typical features of CREST syndrome. It can also be found in other diagnoses [25,26] and in
UCTD [11,14]. Anti-U3RNP (fibrillarin) and anti-Th are also considered SSc markers,
although they may also be found in idiopathic interstitial lung disease (ILD) or primary
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pulmonary hypertension without apparent SSc at low frequency [27,28]. Antitopo I, RNAP
III, and U3RNP are associated with diffuse SSc, whereas anticentromere and Th are mainly
found in limited SSc. Severe ILD is frequently found in antitopo I positive patients, while it is
rare in anti-RNAP III positive cases. Anti-RNAP III is strongly associated with scleroderma
renal crisis. Isolated pulmonary hypertension is more common in patients with anticentromere,
U3RNP, and Th. Other SSc-related autoantibodies are also associated with unique clinical
features [23,29–31].

In PM/DM, the anti-Jo-1 antibodies found in ~20% of patients are classic disease marker
antibodies, and are associated with a unique subset of PM/DM called anti-synthetase syndrome,
characterized by symptoms such as myositis, ILD, arthritis, Raynaud’s phenomenon, and
mechanic’s hand. Autoantibodies to other aminoacyl tRNA synthetases including PL-7
(threonyl), PL-12 (alanyl), EJ (glycyl), OJ (isoleucyl), and KS (asparaginyl) are also associated
with similar clinical features [32,33]. Although either myositis or ILD can precede the other
symptoms [34], anti-PL-12 and anti-KS may be more frequent than the others in idiopathic
ILD without myositis [35]. Antibodies to SRP (signal recognition particle) are also specific
for PM and are associated with severe myositis that occurs without histopathological
inflammation and is resistant to treatment [36]; however, the unavailability of an anti-SRP test
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limits its clinical utility.

Other types of autoantibodies are found in various systemic rheumatic diseases, but they are
associated with certain clinical symptoms, regardless of the diagnosis [1]. Autoantibodies in
this category include anti-Ro/SS-A and La/SS-B (associated with SjS), anti-U1RNP
(associated with Raynaud’s phenomenon, swollen hands, leukopenia, overlapping features
such as sclerodactyly or myositis) [12], and anti-Ku (associated with muscle involvement)
[37].

Anti-dsDNA antibodies are the only autoantibodies that may be used to monitor the disease
activity of SLE. High levels of anti-dsDNA antibodies, often with hypocomplementemia,
correlate with clinical activity in a subset of SLE, such as patients with proliferative nephritis
[17]. Anti-dsDNA antibodies have been tested using various types of assays, including the Farr
assay, PEG (polyethylene glycol) assay, Crithidia lucilliae assay, and ELISA [38]. Each assay

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has its advantages and disadvantages. The Crithidia lucilliae assay and Farr assays are specific
but not particularly sensitive, whereas an ELISA can give a higher number of false positives,
in part because there can be ssDNA domains/epitopes in many preparations.
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Availability of testing for disease-related autoantibodies

Different groups of autoantibodies are strongly associated with SLE, PM/DM, and SSc, as
described above. In many cases, patients positive for these specificities have distinctive clinical
characteristics. However, despite detailed descriptions of these autoantibodies in virtually all
textbooks or review articles [18,30,32,33,39], many of these tests are still not commercially
available for clinicians [1].

In SLE, three autoantibody tests—anti-Sm, dsDNA, and phospholipid—included in the

classification criteria of SLE are commercially available in addition to standard
immunofluorescent ANA. Anti-dsDNA antibodies are detected at some point during the course
in ~70% of patients. Anti-Sm is found in ~15% of patients. Antiribosomal P, available as P-
peptide ELISA, is found in ~10% of patients (Table 1).

Among SSc-associated autoantibodies, only tests for anti-topo I (found in 15–25%, Fig. 2 left),
anticentromere (20–25% by ANA), and anti-U1RNP (10%, frequency depends on whether
MCTD is classified as a separate entity) were available until recently. Anti-RNAP III antibodies
seen in ~20% of SSc patients have been described for 20 years [20–22]. However, anti-RNAP
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III has not become standard or received as much clinical appreciation as that anti-topo I despite
its high specificity for SSc and tight link to scleroderma renal crisis [23,30,31]. This is mainly
because anti-RNAP III can only be detected by immunoprecipitation, which has been
performed at only a small number of institutes around the world. However, an anti-RNAP III
ELISA kit [40] was approved by the Food and Drug Administration (FDA) of the United States
in 2006 and has become widely available as a commercial test [10]. The high sensitivity and
specificity of this ELISA were confirmed by several independent reports [10,41–43]. With this
addition, ~70% of SSc patients will have identifiable antibodies that should help with predicting
prognosis and unique organ involvement. Tests for other autoantibodies including U3RNP
(fibrillarin), Th, and PM-Scl are not commercially available.

In PM/DM, anti-Jo-1, found in ~20% of patients, is the only commercially available test for
myositis-specific antibodies (Fig. 2 right). Anti-U1RNP, which is not specific for PM/DM, is
found in ~5% of patients. While anti-Jo-1 is the most common specificity found in PM/DM,
antibodies to other tRNA synthetases, such as PL-7 (threonyl), PL-12 (alanyl), EJ (glycyl), and
OJ (isoleucyl) are well described [32,33,39]. Typically, an individual patient is positive for
only one of these antibodies, so many individuals with anti-tRNA synthetase antibodies
(associated with the “antisynthetase autoantibody syndrome”) go undetected and may be
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clinically misclassified. Other clinically significant autoantibody tests, such as reactivity to

SRP [36], PM-Scl, are also unavailable. Unlike the pattern in SSc, in which three major
autoantibodies are detected in ~20% each, most PM/DM-related autoantibodies are each found
in only 1–5% of patients. This makes the development of commercial tests useful for many
patients difficult from a financial viewpoint.

Specialized autoantibody tests at research laboratories

Most of the disease-related autoantibodies that are unavailable commercially, as described
above, can be identified at research laboratory level by a combination of 35S-methionine-
labeled protein immunoprecipitation and analysis of immunoprecipitated RNAs by silver
staining (Fig. 3) [18,44]. Many autoantibody specificities can be confirmed definitively based
on protein analysis alone when they have a characteristic set of proteins such as snRNPs or
ribosomal P proteins (Fig. 3a). Identification of a single protein is more difficult unless it has

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a unique migration pattern, and may require additional tests for conclusive identification
[45]. Others, such as anti-Th, U3RNP, and SRP, may require confirmation by
immunoprecipitation of the associated RNA component (Fig. 3b). However, only a few
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institutes in the United States have the ability to analyze all of these autoantibodies in systemic
rheumatic diseases using a combination of various techniques. The characterization of
autoantibodies in samples from Mexican patients using a combination of these specialized
immunological assays is shown as an example of the systematic analysis of sera from a group
of patients (Fig. 3). As discussed above, some autoantibodies related to SSc or PM/DM are
still not widely available. Considering the high disease specificity and unique clinical
association, it will be necessary to make reliable assays for these autoantibodies available for
clinicians.

Autoantibody testing for clinicians

ANA by immunofluorescence has been used for more than 40 years as a screening method for
autoimmunity and is still the standard method. Several different types of alternative assays
based on ELISA or multiplex beads assay have been developed in an attempt to replace
immunofluorescent ANA. However, serious concerns have been raised about these new assays
among rheumatologists [46]. The ACR has established an ANA Task Force to address these
concerns and has been tracking members’ concerns on their website. The Autoantibody
Standardization Committee in Rheumatic and Related Disorders (http://www.AutoAb.org)
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organized a Study Group at the 2008 Annual ACR Scientific Meeting that included a
presentation entitled “Inaccurate results for ANA” referring to ANA screening using these
new, unconventional tests. Based on the prevalence of specific autoantibodies in patients with
rheumatic diseases, it is apparent that using mixtures of several recombinant or purified
autoantigens in these assays will not cover all the reactivities of immunofluorescent ANA-
positive patients. In addition, even if the target antigens of a particular patient’s autoantibodies
are included, recombinant or denatured proteins used in the assay may be poorly recognized
by human autoantibodies, similar to the false negatives found in a specific autoantibody ELISA
[1]. Although these new assays can be cost efficient and are somewhat comparable to
immunofluorescent ANA, to avoid confusion during interpretation by the clinician, laboratory
results derived from these assays should be reported along with exactly the type of assay used;
it should not be reported as an “ANA screening test.” Immunofluorescence ANA is still the
gold standard for the screening of ANA [1].

When an ANA result comes back positive, clinicians must decide what assay to order next to
confirm or follow-up on the initial finding. A list of potential diagnoses for the patient, clinical
symptoms, and laboratory findings will all help to make this decision. If the immunofluorescent
ANA pattern is read properly, this information should also help determine the next step. ANA
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staining patterns and corresponding common autoantibodies for different diagnoses are
summarized in Table 2. Although the ANA patterns are helpful for narrowing down the tests
for specific autoantibodies when performed and interpreted correctly, several potential pitfalls
should be noted. First, interpretation is somewhat subjective, and weaker staining may not be
reported. Since our eyes compare the fluorescent intensity of the area of interest to its
surrounding area (e.g., nucleoli vs. nuclei, nuclei vs. cytoplasm), only the stronger staining
may be reported [1,10]. For example, if strong nucleolar staining accompanies weaker nuclear
staining, as in certain anti-topo I-positive sera, it may be reported as nucleolar staining only.
Similarly, weak nuclear staining may be reported as negative in the presence of strong
cytoplasmic staining. Second, the report on the staining pattern may not always accurately
reflect the known cell biological location of the target antigen. Ro/SS-A antigens are present
in both nuclei and cytoplasm; however, anti-Ro/SS-A has been historically linked with ANA-
negative lupus, and the staining pattern from anti-Ro/SS-A has been controversial; nuclear
staining are reported by many, while some have reported cytoplasmic staining.

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Autoantibody testing in patients with a specific diagnosis

SLE
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If SLE is clinically suspected, three specific autoantibodies listed in the ACR SLE classification
criteria—anti-dsDNA, phospholipids, and Sm—should be tested for. Presence of active
nephritis is often accompanied by anti-dsDNA antibodies, and some patients with a history of
thrombosis or recurrent spontaneous abortions may have anti-phospholipid antibodies;
however, many unsuspected patients will also be found to be positive. This is mainly due to
the relatively low sensitivity of autoantibodies when used to detect their associated clinical
symptoms, as shown in a recent meta-analysis [19]. In some cases, this may be explained by
the observation that autoantibodies are often produced prior to clinical manifestation [14,47].
There are reports of the association of anti-Sm with certain clinical manifestations; however,
there is no way to predict or rule out the presence of anti-Sm. Antiribosomal P antibodies are
associated with neuropsychiatric symptoms of SLE in some studies, although the specificity
does not appear to be high [19]. Specific assays for anti-Ro/SS-A, La/SS-B, and U1RNP may
be ordered as well.

SSc
Autoantibodies to RNAP I always coexist with anti-RNAP III and RNAP I localizes to the
nucleoli; however, none of the anti-RNAP I/III-positive SSc sera were reported to be nucleolar
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staining positive from a hospital laboratory in one study; all sera were reported to be nuclear
speckled that reflected RNAP III staining [10]. Thus, despite earlier reports describing
nucleolar staining by anti-RNAP I antibodies in SSc [48], a report of nucleolar staining should
not be expected all the time [10]. It is reasonable to recommend that all SSc patients with
nuclear speckled and nucleolar patterns should be tested for anti-topo I and RNAP III [1].
Patients with anticentromere patterns may be excluded, because patients with SSc seldom have
more than one SSc-related autoantibody, and it is unlikely that they also have anti-topo I or
anti-RNAP III. Nucleolar staining by anti-U3RNP, Th, and PM-Scl is usually reported as such
from a hospital laboratory in our experience [10]. Anti-topo I may be reported as nucleolar
instead of nuclear, or nuclear plus nucleolar, due to strong nucleolar staining by some sera.
Inclusion of anti-topo I, RNAP III, and centromere will be considered in the classification
criteria of SSc in the future.

PM/DM
The targets of many myositis-specific autoantibodies such as anti-Jo-1 and other tRNA
synthetases and SRP are cytoplasmic antigens [32,33,39]. Unfortunately, they are often not
reported in clinical practice. Anti-Jo-1 for cytoplasmic and anti-U1RNP for nuclear speckled
are the only widely available tests for myositis-related autoantibodies. Nuclear speckled
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patterns with overlapping features of PM/DM and SLE or SSc may be similar in patients with
anti-U1RNP (MCTD) and anti-Ku-positive myositis patients.

RA
Although anti-CCP antibodies were first described just ten years ago, and the anti-CCP ELISA
test has only been widely available for a few years, it has rapidly become a standard test in
clinical practice. The frequencies of anti-CCP and rheumatoid factor (RF) are both ~70%;
however, anti-CCP is much more specific for RA. Nevertheless, anti-CCP may be positive in
up to 10–20% of other rheumatic diseases and associated with chronic arthritis or Jaccoud’s-
type arthritis in SLE [49]. Anti-CCP in nonrheumatic disease patients is less extensively
studied, and one may find unexpected positives in infections, such as in patients with
pulmonary tuberculosis [50]. Anti-CCP always needs to be interpreted carefully with other
clinical and laboratory features in the individual patient, particularly in non-RA patients.

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Sjögren’s syndrome
Anti-Ro/SS-A (60kD) and anti-La/SS-B are detected in ~70% and ~40%, respectively, by
standard tests [1]. These antibodies are included in the European Criteria [51] and should be
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tested for when the presence of SjS is suspected. Anti-Ro52 [52] and anti-NA14 [53] can be
found in patients with primary SjS without anti-Ro/SS-A or La/SS-B and may become useful
in the future.

Conclusion
Clinically useful information on common autoantibodies in rheumatic diseases was
summarized, emphasizing potential problems and pitfalls. Unfortunately there is a discrepancy
between the autoantibody tests described in textbooks or review articles and their availability
in clinical practice. Also, despite the long history of performing autoantibody assays using
standard methods, there are still many limitations and pitfalls that clinicians should be aware
of. Clinicians should be able to use autoantibody tests more efficiently and properly, and have
a basic knowledge of their significance and potential problems.

Acknowledgments
We thank Dr. Luis E. C. Andrade (Escola Paulista de Medicina, São Paulo, Brazil) for his critical reading of the
manuscript. This study was supported by CONACyT grant 51353, Universidad de Guadalajara agreement 25473, to
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MVDM. MS is supported by the Lupus Foundation of America, Inc. EKLC is supported in part by NIH AI47859.

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Fig. 1.
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Immunofluorescent antinuclear antibodies in patients with rheumatic diseases. a Nuclear

speckled pattern. b Centromere. c Mitotic spindle apparatus (NuMA). d Antimitochondria and
nuclear dots. e GW body (GWB) staining by anti-Su/Ago2 antibodies. f Anti-Golgi antibodies

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 Satoh et al. Page 13
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Fig. 2.
Prevalence of autoantibodies associated with scleroderma and polymyositis/dermatomyositis
(PM/DM). Commercially available tests (shaded) and other disease-related autoantibodies are
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indicated
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 Satoh et al. Page 14
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Fig. 3.
Characterization of various autoantibodies by immunoprecipitation. a Protein analysis by
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immunoprecipitation. 35S-methionine-labeled K562 cell lysate was immunoprecipitated by

sera from Mexican patients with SLE. Immunoprecipitated proteins were fractionated by SDS-
PAGE followed by autoradiography. b Analysis of RNA components immunoprecipitated by
sera from Mexican patients with PM/DM. K562 cell lysate was immunoprecipitated by sera
from patients with PM/DM (lanes 1–9) or control (NHS). Nucleic acid components were
extracted using phenol/chloroform and analyzed by urea-PAGE followed by silver staining
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Table 1
Autoantibody tests that are widely available and help diagnosis
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Specificity Frequency Disease specificity Currently tested by

SLE

dsDNA 50–80% High ELISA, Crithidia

Sm 15% High ELISA, (DID)

Ribosomal P 10% High ELISA

Scleroderma

Topo I 15% High ELISA, (DID)

Centromere 25% Moderate IF

RNA polymerase III (RNAP III) 20% High ELISA

PM/DM

Jo-1 (his-tRNA) 20% High ELISA (DID)

RA

CCP 70% Moderate ELISA

Rheumatoid factor (RF) 70% Low Laser nephelometry, ELISA, Latex agglutination
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Sjögren’s syndrome

Ro/SS-A 70% Low ELISA, (DID)

La/SS-B 40% Moderate ELISA, (DID)

MCTD (Mixed Connective Tissue Disease)

U1RNP 100% Low ELISA (DID)

ELISA enzyme-linked immunosorbent assay, DID double immunodiffusion

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Table 2
Common specificities of autoantibodies based on the ANA pattern and diagnoses
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ANA pattern Clinical diagnosis

SLE SSc PM/DM

Nuclear Homogeneous/peripheral dsDNA histones

Speckled Sm, U1RNP, Ro/SS-Aa, La/ Topo I (Scl-70), RNAP III, U1RNP, Mi-2, Ku
SS-B, RNA helicase A (RHA) U1RNP

Discrete speckled Centromere

Nucleolar (Ribosomal P)b U3RNP, Th, PM-Scl, (Topo PM-Scl
I)c
Cytoplasmic Diffuse Ribosomal P Jo-1 (histidyl tRNA
synthetase), Other ARSd,
SRP
GWBs Su/Ago2 Su/Ago2 Su/Ago2

a
Some sera show only cytoplasmic staining or negative ANA
b
Always with cytoplasmic, which may not be reported
c
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With nuclear staining

d
ARS: aminoacyl tRNA synthetases, PL-7 (threonyl), PL-12 (alanyl), EJ (glycyl), OJ (Isoleucyl), KS (asparaginyl)

Bold commercially available, nonbold research level, underlined disease marker antibodies
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