Clues
- The antinuclear antibodies of 1:160 are very specific for collagen diseases, with less than 5% yield positive result in healthy population
- There are stains and patterns of antibodies; stains: nuclear, nucleolar, cytoplasmic, mitotic, golgi bodies stains; and the patterns varies according to the staining (e.g. in the nuclear staining, are three types of patterns: homogeneus, peryphery and speckled)
- The more specific Antibodies for SLE are: ANA with nuclear staining with homogeneus, speckled and peripheral pattern, Anti-dsDNA and Anti-Sm
- The more specific Antibodies for Scleroderma are: ANA with nucleolar staining, Anti-topoI, Anti-RNAPIII and Anti-centromere.
- The more specific Antibodies for Dermatomiositis-Polymiositis are: ANA with cytoplasmic staining, Anti-Jo1.
- The more specific Antibodies for Sjogren Disease are: ANA with nuclear staining in a speckled pattern and Anti-Ro/SS and Anti La-SS
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 [
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
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 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
[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
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
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
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
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.
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
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
but can also be found in various systemic rheumatic diseases and often in patients with antiphospholipid
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 diseasespecific,
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
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
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.
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
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)
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 Ppeptide
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
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)
In PM/DM, anti-Jo-1, found in ~20% of patients, is the only commercially available test for
myositis-specific antibodies
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
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 ANAnegative
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.
SLE: 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
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
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.
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
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’stype
arthritis in SLE
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
Source
-Antinuclear antibodies in rheumatology
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