Clinical Significance of Antinuclear Antibody Staining Patterns and Associated Autoantibodies

24/10/2018 Clinical significance of antinuclear antibody staining patterns and associated autoantibodies - UpToDate
Official reprint from UpToDate®

www.uptodate.com ©2018 UpToDate, Inc. and/or its affiliates. All Rights Reserved.
Clinical significance of antinuclear antibody staining patterns and associated autoantibodies
Author: Donald B Bloch, MD

Section Editor: Robert H Shmerling, MD
Deputy Editor: Monica Ramirez Curtis, MD, MPH
All topics are updated as new evidence becomes available and our peer review process is complete.
Literature review current through: Sep 2018. | This topic last updated: Sep 15, 2017.
INTRODUCTION — The indirect immunofluorescence (IIF) test for antinuclear antibodies (ANA), using the
human cell line HEp-2 as substrate, is a commonly used assay to detect human autoantibodies. The results
of ANA testing are reported in two parts: the titer of the antibodies and the staining pattern produced by the
antibodies. The titer of the antibodies refers to the highest dilution of serum that produces visible
fluorescence. The ANA pattern refers to the distribution of staining produced by autoantibodies reacting with
antigens in the HEp-2 cell nucleus and cytoplasm. The measurement and clinical significance of ANA titer is
reviewed elsewhere. (See "Measurement and clinical significance of antinuclear antibodies".)
Unfortunately, there is limited agreement among laboratories as to which ANA staining patterns should be
identified and reported to clinicians. The patterns to be reported are determined by individual laboratory
directors. The directors also choose from among HEp-2 cell slides prepared by different companies. These
companies may use different fixative and permeabilization techniques. Depending on the preparation of the
HEp-2 cell substrate, some autoantibodies may or may not be detected by IIF. The clinician should know
which staining patterns are recognized and reported by their reference laboratory.
An international workshop attempted to arrive at a consensus on the nomenclature of ANA staining patterns
[1]. The participants suggested that there are 11 staining patterns that "must" be reported by all "competent-
level" laboratories. An additional 22 ANA staining patterns should be reported by "expert-level" laboratories.
Whether or not the recommendations of the workshop will become widely accepted remains to be
determined.
This topic review will cover three broad categories of ANA staining patterns: nuclear, cell cycle-associated,
and cytoplasmic. Within each of these categories, individual patterns will be defined and autoantibodies that
produce the staining patterns will be identified. The disease associations of autoantibodies producing the
staining patterns will be described as well as additional laboratory tests that may be used to further
characterize the autoantibodies.
ANA STAINING PATTERNS INVOLVING THE NUCLEUS
Homogeneous
● Definition – The homogenous antinuclear antibody (ANA) pattern refers to diffuse staining of the
nucleus in resting cells. There is also diffuse staining of the chromosome region in dividing cells. The
homogenous staining pattern was reported in 36 percent of more than 9200 ANA-positive serum
samples tested at the University Hospitals in Leuven, Belgium [2].
● Targets of antibodies – Histone proteins, double-stranded (ds)DNA, single-stranded DNA.
● Disease association – Homogeneous nuclear staining may be seen in patients with many different
systemic autoimmune diseases including systemic lupus erythematosus (SLE), drug-induced lupus,
https://www.uptodate.com/contents/clinical-significance-of-antinuclear-antibody-staining-patterns-and-associated-autoantibodies/print?search=Cl… 1/13
Sjögren's syndrome (SS), scleroderma (SSc), and rheumatoid arthritis (RA), as well as organ-specific
autoimmune diseases including Hashimoto's thyroiditis, primary biliary cholangitis (PBC), and
autoimmune hepatitis.
Specific antibodies responsible for homogeneous staining, such as anti-dsDNA antibodies, may be
detected by indirect immunofluorescence (IIF) using Crithidia luciliae assay, enzyme-linked
immunosorbent assay (ELISA), or fluorescent bead assay and may be helpful to support the diagnosis of
SLE. Anti-dsDNA antibodies are present in approximately 40 to 60 percent of patients with SLE and are
highly specific for the disease (75 to 99 percent, depending on the assay used to detect the antibodies)
[3]. In addition, anti-dsDNA antibodies may develop in patients who are treated with inhibitors of tumor
necrosis factor (TNF)-alpha [4]. These patients may or may not have symptoms of drug-induced lupus.
(See "Diagnosis and differential diagnosis of systemic lupus erythematosus in adults", section on
'Laboratory testing' and "Drug-induced lupus", section on 'Causative drugs' and "Tumor necrosis factor-
alpha inhibitors: An overview of adverse effects", section on 'Autoimmunity and autoantibodies'.)
In patients suspected of having drug-induced lupus that is not associated with anti-TNF-alpha therapy,
ELISA or fluorescent bead assays (hereafter referred to as "solid phase assays") for antihistone
antibodies may be helpful; a negative test for antihistone antibodies would argue against a diagnosis of
drug-induced lupus. (See "Drug-induced lupus", section on 'Anti-histone antibodies'.)
The homogeneous ANA pattern may obscure other staining patterns, such as nuclear speckled.
Therefore, additional testing for specific autoantibodies will be required to fully characterize the spectrum
of autoantibodies that may be present in serum that produces the homogeneous ANA staining pattern.
Nuclear speckled — Three types of nuclear speckled patterns ("fine," "coarse," and "fine dense") have been
described. Individual laboratories may or may not distinguish between these different patterns.
Fine speckled
● Definition – Fine speckled staining refers to hundreds to thousands of dots present throughout the
nucleus. Nucleoli may or may not be stained, depending on the specific autoantibody. Except for
antibodies directed against Scl70 (topoisomerase I), there is no staining of the chromosome region in
dividing cells. The fine nuclear speckled pattern was reported in 20 percent of more than 9200 ANA-
positive serum samples [2]
● Targets of antibodies – Ro60, La, Topoisomerase I (also known as Scl70), Ku, Mi-2, transcriptional
intermediary factor (TIF)-1, and melanoma differentiation-associated protein-5 (MDA-5; also known as
CADM-140 and RNA helicase).
● Disease association – As is true with the homogeneous pattern, the fine speckled pattern may be
present in patients with a broad spectrum of autoimmune diseases. Additional testing is required to
further characterize the antibodies in patient serum that produce this staining pattern.
Antibodies directed against Ro60 are present in approximately 75 percent of patients with subacute
cutaneous lupus erythematosus, 60 percent of patients with SS, 30 percent of patients with SLE, 30
percent of patients with PBC, 20 percent of patients with polymyositis (PM), and 15 percent of patients
with RA [5]. Anti-Ro60 and/or anti-Ro52 antibodies (see below) are present in nearly all women who
have a child with neonatal lupus syndrome (NLS). It is critical to note that anti-Ro60 antibodies may not
be detected in Hep-2 cell substrate if the cells are fixed with methanol (as opposed to acetone) [6].
Because anti-Ro60 antibodies are not detected using some HEp-2 cell substrates, if anti-Ro60 is the
only autoantibody present in a patient with SLE or SS, the patient will appear to have ANA-negative
disease. Anti-Ro60 antibodies may be the only autoantibody present in 25 to 50 percent of all patients
with "ANA-negative" SLE [7]. (See "The anti-Ro/SSA and anti-La/SSB antigen-antibody systems" and
"Diagnosis and differential diagnosis of systemic lupus erythematosus in adults", section on 'ANA-
negative lupus' and "Neonatal lupus: Epidemiology, pathogenesis, clinical manifestations, and
diagnosis", section on 'Autoantibodies'.)
Antibodies directed against La are present in approximately 20 percent of patients with SS and are
considered relatively specific for this disease. Anti-La antibodies may also be detected in patients with
SLE and in women who have a child with NLS. (See "Diagnosis and classification of Sjögren's
syndrome", section on 'Antibodies to Ro/SSA and La/SSB' and "Diagnosis and differential diagnosis of
systemic lupus erythematosus in adults", section on 'Laboratory testing' and "Neonatal lupus:
Epidemiology, pathogenesis, clinical manifestations, and diagnosis", section on 'Autoantibodies'.)
Antibodies directed against Scl70 are present in approximately 20 to 40 percent of patients with diffuse
SSc and are rarely detected in patients with other autoimmune diseases or in healthy individuals. In
patients with diffuse SSc, anti-Scl70 antibodies are associated with the presence of interstitial pulmonary
fibrosis [8]. (See "Diagnosis and differential diagnosis of systemic sclerosis (scleroderma) in adults",
section on 'Laboratory testing'.)
Anti-Ku antibodies are rare, with an estimated prevalence of 0.5 percent of all ANA-positive sera. Anti-Ku
antibodies are present in approximately 2.5 percent of patients with SSc and are associated with the
presence of myositis and arthritis [9]. Anti-Ku antibodies may also be detected in patients with SLE and
PM. (See "Clinical manifestations of dermatomyositis and polymyositis in adults", section on
'Autoantibodies and overlap syndromes'.)
Antibodies directed against Mi-2 (a nuclear DNA helicase involved in transcriptional activation) are a rare
cause of a speckled ANA. Anti-Mi-2 antibodies are present in approximately 15 percent of patients with
DM [10]. In general, patients with these antibodies have skin findings characteristic of DM, including
heliotrope rash and Gottron's papules, but do not develop lung disease and respond well to
corticosteroids [11]. (See "Clinical manifestations of dermatomyositis and polymyositis in adults", section
on 'Anti-Mi-2 antibodies'.)
Antibodies directed against TIF-1 proteins (as detected by immunoprecipitation) may be present in 15 to
25 percent of patients with dermatomyositis (DM) and may be associated with the presence of an
underlying malignancy [12]. (See "Malignancy in dermatomyositis and polymyositis", section on 'Serum
autoantibodies'.)
Anti-MDA-5 antibodies may produce speckled staining in either the nucleus or cytoplasm of HEp-2 cells.
These antibodies were detected (by immunoprecipitation) in 25 percent of patients with DM and are
associated with amyopathic disease and rapidly progressive interstitial lung disease (ILD) [11,13]. (See
"Clinical manifestations of dermatomyositis and polymyositis in adults", section on 'Autoantibodies and
overlap syndromes'.)
Coarse speckled
● Definition – The coarse nuclear speckled pattern refers to hundreds to thousands of nuclear dots,
variable in size, but generally larger than the dots seen in the fine speckled pattern. There is no staining
of nucleoli or dividing chromosomes.
● Targets of antibodies – Sm, U1 ribonuclear protein (RNP).
● Disease association – Antibodies directed against Sm are relatively specific for the diagnosis of SLE.
Antibodies directed against Sm may be detected in 10 to 50 percent of SLE patients, and estimates of
the specificity of anti-Sm antibodies for the diagnosis of SLE range from 55 to 100 percent [14-17]. Anti-
Sm antibodies may also be detected in patients with mixed connective tissue disease (MCTD), but are
rare in other autoimmune diseases.
Antibodies directed against U1RNP antigens are present in all patients with MCTD because the
antibodies are a critical component of the diagnosis of this disease. Anti-U1RNP antibodies may also be
present in 30 to 40 percent of patients with SLE, as well as some patients with RA, SS, SSc, and PM.
(See "Diagnosis and differential diagnosis of systemic lupus erythematosus in adults", section on
'Laboratory testing' and "Definition and diagnosis of mixed connective tissue disease", section on
'Autoantibodies' and "Diagnosis and differential diagnosis of systemic sclerosis (scleroderma) in adults",
section on 'Laboratory testing' and "Clinical manifestations of dermatomyositis and polymyositis in
adults", section on 'Autoantibodies'.)
Dense fine speckled pattern
● Definition – In the dense fine speckled pattern, the speckles are distributed throughout the nucleus of
interphase cells, excluding nucleoli. This pattern differs from the fine and coarse speckled patterns in that
the speckles associate with chromosomes in dividing cells. Estimates of the prevalence of the dense fine
speckled pattern ranges from 0.8 [18] to 37 percent [19].
● Antibody target – Dense fine speckled, 70 kD (DFS70; also known as lens epithelium-derived growth
factor protein 75 kD [LEDGFp75]).
● Disease association – In a study of 597 healthy hospital workers, 20 percent were found to have a
positive ANA at a dilution of >1:40. More than half of these ANA-positive individuals had antibodies that
produced the dense fine speckled pattern [20]. Other studies have confirmed that patients with
antibodies producing this staining pattern have a low prevalence of underlying autoimmune disease [21-
23]. Anti-DFS70 antibodies were found to be the only detectable autoantibody in only 1.1 percent of
patients with SLE in a larger, international cohort. These results further suggest that anti-DFS70
autoantibodies are not associated with systemic rheumatic disease [24]. In a patient population with a
low prior probability of having an autoimmune disease, the presence of antibodies producing the DFS70
pattern may be considered a reassuring result, even when these autoantibodies are present in high
(>1:640) titer [23]. However, the DFS70 staining pattern may obscure patterns produced by co-occurring
autoantibodies. In patients with a high prior probability of autoimmune disease (as determined by history,
physical examination, and other laboratory findings), additional testing using solid phase assays may be
required to detect disease-associated autoantibodies.
Nucleolar
● Definition – Three distinct types of nucleolar staining patterns have been described ("homogeneous,"
"clumped," and "speckled"). However, most clinical laboratories do not distinguish between these
subtypes. The prevalence of antinucleolar antibodies in serum samples varies widely, from 1.4 percent of
all samples submitted for ANA testing to a hospital in the United Kingdom [25] to 17 percent of more than
9200 ANA-positive sera [2].
● Targets of autoantibodies – Components of the PM/Scl complex (PM-Scl-75, PM-Scl-100), U3RNP

(also known as fibrillarin); the 7-2RNP complex (Th/To); RNA polymerases I, II, and III; Topoisomerase I
(Scl70).
● Disease association – Antinucleolar antibodies may be detected in patients with SLE, RA, SS, SSc,
PM, DM, MCTD, and the Raynaud phenomenon. Antibodies producing the nucleolar staining pattern
have been reported in 15 to 40 percent of patients with SSc [8] and the presence of these antibodies has
important implications for diagnosis and prognosis. Therefore, in the appropriate clinical setting,
additional testing using solid phase or immunoprecipitation assays may be required to identify the
autoantibodies that are responsible for the nucleolar staining pattern.
The PM/Scl complex, also known as the exosome, is a multi-protein structure that degrades RNA.
Antibodies directed against one or both of two components of the exosome, PM-Scl-75 and PM-Scl-100,
are present in 5 to 10 percent of SSc patients and are associated with limited skin disease and
decreased risk of pulmonary and renal disease, but increased risk of associated inflammatory myositis
[8]. (See "Clinical manifestations of dermatomyositis and polymyositis in adults", section on

'Autoantibodies and overlap syndromes'.)
U3-RNP (fibrillarin) is involved in the first step of processing pre-ribosomal RNA. Anti-U3-RNP antibodies
are present in 5 to 10 percent of patients with SSc and are associated with diffuse skin disease,
pulmonary artery hypertension, pulmonary fibrosis, and myositis [26]. (See "Clinical manifestations,
evaluation, and diagnosis of interstitial lung disease in systemic sclerosis (scleroderma)", section on
'Laboratory findings'.)
The Th/To complex functions as a ribonuclease that removes the 5' leader sequences from precursor
tRNA molecules. Antibodies directed against components of the Th/To complex are present in 2 to 5
percent of patients with SSc and are associated with limited skin disease but increased risk of pulmonary
fibrosis and renal crisis [26]. (See "Clinical manifestations, evaluation, and diagnosis of interstitial lung
disease in systemic sclerosis (scleroderma)", section on 'Laboratory findings'.)
Antibodies directed against RNA polymerases are detected in approximately 20 percent of patients with
SSc and have a high degree of specificity for this disease. The presence of anti-RNA polymerase
antibodies is associated with diffuse skin involvement and increased risk of renal crisis. Pulmonary
fibrosis is uncommon in patients with anti-RNA polymerase antibodies [27]. (See "Diagnosis and
differential diagnosis of systemic sclerosis (scleroderma) in adults", section on 'Laboratory testing'.)
Antibodies directed against Scl70 (topoisomerase I) produce nuclear speckled (see 'Fine speckled'
above) as well as nucleolar staining patterns.
Nuclear dot staining patterns — Three types of nuclear dot staining patterns have been described:
centromere, PML-Sp100 nuclear body, and Cajal body.
Centromere
● Definition – The centromere staining pattern is characterized by the presence of 30 to 60 discrete, large
speckles present in the nucleus of resting cells. The speckles are larger and fewer in number than those
seen in the fine and coarse speckled patterns. The speckles align with the chromosome region in
dividing cells. Failure to pay attention to the uniform size and limited number of speckles may lead to
confusion with the fine dense speckled pattern (see 'Dense fine speckled pattern' above). Antibodies
directed against centromeres were detected in 3 percent of more than 9200 ANA-positive sera [2].
● Targets of antibodies – Centromere proteins A, B, and C.
● Disease association – Anticentromere antibodies are present in approximately 30 percent of patients

with limited SSc. The antibodies are associated with calcinosis and pulmonary hypertension.
Anticentromere antibodies are also present in approximately 15 percent of patients with PBC; such
patients tend to have a worse outcome than anticentromere antibody-negative patients [28].
Anticentromere antibodies were present in 4 to 15 percent of patients with SS [29,30] and approximately
4 percent of SLE patients [31]. Anticentromere antibodies may be also detected in patients with isolated
Raynaud phenomenon who do not have evidence of a systemic autoimmune disease [32]. (See
"Diagnosis and differential diagnosis of systemic sclerosis (scleroderma) in adults", section on
'Laboratory testing' and "Clinical manifestations, diagnosis, and prognosis of primary biliary cholangitis
(primary biliary cirrhosis)", section on 'Laboratory tests' and "Clinical manifestations and diagnosis of the
Raynaud phenomenon", section on 'Primary Raynaud phenomenon'.)
PML-Sp100 nuclear body
● Definition – The PML-Sp100 nuclear body staining pattern is characterized by the presence of 5 to 20
discrete, large speckles present in the nucleus of resting cells. The pattern can be distinguished from the
centromere pattern because there are fewer dots in each cell and there is no staining of chromosomes in
dividing cells. Antibodies directed against PML-Sp100 nuclear bodies were detected in 0.2 percent of
more than 9200 ANA-positive sera [2].
● Target of antibodies – Speckled-100 kD (Sp100), promyelocytic leukemia protein (PML).
● Disease association – The PML-Sp100 nuclear body is a multi-protein cellular structure that is involved
in a variety of cellular functions including gene transcription, cellular apoptosis, cell-cycle control, and
DNA repair. Antibodies directed against components of the PML-Sp100 nuclear body (PML, Sp100, or
usually both proteins) are present in 20 percent of patients with antimitochondrial antibody (AMA)-
positive PBC [28]. Anti-PML-Sp100 nuclear body antibodies are also present in approximately 40
percent of PBC patients who do not have AMA as detected by IIF [33] and may be the only antibody
marker in some PBC patients. Anti-PML-Sp100 nuclear body antibodies are rarely detected in patients
with other autoimmune diseases. Because the PML-Sp100 nuclear body staining pattern is similar to that
of the Cajal body staining pattern (see 'Cajal body' below) and because anti-PML-Sp100 nuclear body
antibodies are specific for the diagnosis of PBC, it is critical to confirm the presence of these antibodies
using a solid phase assay. (See "Clinical manifestations, diagnosis, and prognosis of primary biliary
cholangitis (primary biliary cirrhosis)".)
Cajal body
● Definition – The Cajal body staining pattern is characterized by the presence of 0 to 8 discrete, large
speckles in the nucleus of resting cells. There is no staining of chromosomes in dividing cells. Antibodies
directed against Cajal bodies were present in 1.3 percent of more than 9200 ANA-positive sera [2].
● Target of antibodies – p80-coilin.
● Disease association – Autoantibodies directed against Cajal bodies are rarely present in patients with
SS and PBC [28]. These antibodies may also be detected in patients with a variety of other autoimmune
diseases, including SLE, SSc, and PM [34-36], as well as in healthy individuals. Because antibodies
directed against Cajal bodies are rarely detected and are not disease-specific, the clinical significance of
these antibodies is uncertain.
Nuclear envelope — Two types of nuclear envelope staining patterns have been reported. The first is
characterized by smooth, continuous, linear staining of the nuclear envelope; the second by discontinuous
linear staining of the nuclear envelope. Autoantibodies directed against components of the nuclear envelope
were detected in 0.5 percent of more than 9200 ANA-positive sera [2].
Nuclear envelope pattern
● Targets of antibodies – Nuclear lamin proteins, lamin-associated proteins.
● Disease association – Autoantibodies directed against lamins and lamin-associated proteins have been
reported in a broad spectrum of diseases, including SLE, SS, antiphospholipid syndrome (APS), and
autoimmune liver disease [37]. Because of the uncertain sensitivity of detecting these antibodies in any
autoimmune disease and the lack of disease specificity, the clinical significance of these antibodies is
uncertain.
Nuclear pore complex pattern
● Targets of antibodies – Glycoprotein-210 kD (Gp210), nucleoporin-62 kD (Nup62).
● Disease association – Antibodies directed against the nuclear pore complex are detected by IIF in
approximately 20 percent of patients with AMA-positive PBC [28]. Using an ELISA assay and either
recombinant Gp210 or a Gp210 peptide fragment as substrate, anti-Gp210 antibodies were detected in
25 percent of PBC patients and were more than 95 percent specific for this diagnosis. The prevalence of
anti-Nup62 antibodies in patients with PBC was approximately 20 percent [38]. (See "Clinical
manifestations, diagnosis, and prognosis of primary biliary cholangitis (primary biliary cirrhosis)", section
on 'Laboratory tests'.)
Not all clinical laboratories distinguish between nuclear envelope and nuclear pore complex staining
patterns. Because of the specificity of anti-Gp210 antibodies for the diagnosis of PBC, it is critical to test
for these antibodies by ELISA in patients with autoantibodies reacting with the nuclear envelope.
CELL CYCLE-ASSOCIATED STAINING PATTERNS
PCNA
● Definition – Autoantibodies producing the proliferating cell nuclear antigen (PCNA) pattern produce
nuclear speckled staining during the G1 phase of the cell cycle, when cells are increasing in size. During
S phase, when DNA replication occurs, there is dense homogeneous nuclear staining. There is no
staining in dividing cells. Autoantibodies producing the PCNA staining pattern were detected in 0.3
percent of more than 9200 antinuclear antibody (ANA)-positive sera [2].
● Target of antibodies – Delta chain of DNA polymerase.
● Disease association – Autoantibodies producing the PCNA staining pattern were originally identified in
a small percentage of patients with systemic lupus erythematosus (SLE) [39]. Subsequent studies
reported the presence of anti-PCNA antibodies in a variety of disorders including other autoimmune
diseases, viral hepatitis, and malignancies [40]. In a retrospective study, only 1 of 28 patients with
antibodies producing the PCNA staining pattern was diagnosed with SLE, while six patients were
diagnosed with autoimmune thyroid disease [2]. Based on these results, the clinical significance of
antibodies producing the PCNA staining pattern is uncertain.
CENP-F
● Definition – The centromere protein-F (CENP-F) staining pattern is characterized by fine speckled
staining of the nucleus in resting cells; staining spares the nucleolus. The strongest staining cells are in
the G2 phase of cell division, during the "gap" between DNA synthesis and mitosis. There is centromere-
associated staining of dividing cells. In addition, during the late stage of cell division, there is staining of
the middle region of separating spindles, known as the spindle "midzone." In cells that have progressed
further in cell division, there may be staining of a small structure between the two dividing cells known as
the mid-body. The CENP-F staining pattern was detected in 0.3 percent of more than 9200 ANA-positive
sera [2].
● Target of antibodies – CENP-F.
● Disease association – Approximately half of the patients with autoantibodies producing the CENP-F
staining pattern have an underlying malignancy. Associated malignancies include breast, lung, and
prostate cancer, as well as lymphoma [41].
Mitotic spindle apparatus — Two types of nuclear mitotic apparatus staining patterns have been described:
nuclear mitotic apparatus-1 (NuMA1) and NuMa2.
NuMA1 staining pattern
● Definition – The NuMA1 staining pattern is characterized by speckled nuclear staining in resting cells. In
dividing cells, there is staining of the spindles near the centrosomes, but the staining does not extend to
the centromeres. There is no staining of spindles between the dividing chromosomes. The NuMA1
staining pattern was detected in 0.7 percent of more than 9200 ANA-positive sera [2].
● Target of antibodies – NuMA1.
● Disease association – In a review of the diagnoses in patients whose serum produced the NuMA1
staining pattern, 20 percent had Sjögren's syndrome (SS), 12 percent SLE, and 40 percent other
autoimmune diseases [42]. (See "Diagnosis and classification of Sjögren's syndrome".)
NuMA2 staining pattern
● Definition – Antibodies producing the NuMA2 staining pattern do not stain the nuclei of resting cells. In
dividing cells, there is staining of the spindle fibers that extends from the centrosomes to the
centromeres. Later in cell division, there is also staining of spindles between the centromeres. The
NuMA2 staining pattern was detected in 0.06 percent of 9200 ANA-positive sera [2].
● Target of antibodies – Human kinesin-like protein (HsEg5; also known as "kinesin family member 11
[KIF11]").
● Disease association – Antibodies that produce the NuMA2 staining pattern are not associated with any
specific autoimmune disease [42].
CYTOPLASMIC STAINING PATTERNS
Antimitochondrial antibody
● Definition – The antimitochondrial antibody (AMA) staining pattern, as seen using the HEp-2 cell
substrate, is characterized by the presence of granular and filamentous staining throughout the
cytoplasm. On a substrate that is rich in mitochondria, such as rat kidney, intense cytoplasmic staining
may be seen in the distal tubular cells. The AMA staining pattern was detected in 74 of 8300 serum
samples submitted for testing to the Clinical Immunology Laboratory at the Massachusetts General
Hospital in 2012 [43].
● Targets of antibodies – E2 component of pyruvate dehydrogenase complex (also known as

dihydrolipoamide S-acetyltransferase [DLAT]), 2-oxo-glutarate dehydrogenase (dihydrolipoamide
dehydrogenase [DLD]), and branched-chain 2-oxo-acid dehydrogenase (BCKDC), as well as other
mitochondrial components.
● Disease association – Antibodies directed against DLAT, DLD, and BCKDC are highly specific for the
diagnosis of primary biliary cholangitis (PBC). Autoantibodies directed against other components of
mitochondria may be detected in a broad spectrum of diseases, including infections, myocarditis,
systemic lupus erythematosus (SLE), and drug-induced hepatitis [44]. Because only antibodies directed
against DLAT, DLD, and BCKDC are specific for PBC, if the AMA staining pattern is detected by indirect
immunofluorescence (IIF), then solid phase assays should be used to confirm the presence of antibodies
directed against DLAT, DLD, and/or BCKDC [45]. (See "Clinical manifestations, diagnosis, and prognosis
of primary biliary cholangitis (primary biliary cirrhosis)".)
Cytoplasmic fine speckled
● Definition – In this staining pattern, fine speckles are distributed throughout the cytoplasm of resting
HEp-2 cells. Antibodies producing the cytoplasmic fine speckled staining pattern were present in 1.2
percent of more than 9200 antinuclear antibody (ANA)-positive sera [2].
● Target of antibodies – Jo-1 (histidine tRNA synthetase) or any of seven other tRNA synthetases,
ribosomal P proteins, Ro52, signal recognition particle (SRP).
● Disease association – Antibodies directed against aminoacyl-tRNA synthetases are present in 25 to 35

percent of patients with dermatomyositis (DM) or polymyositis (PM). These antibodies are rarely
detected in patients with other autoimmune diseases. In general, anti-tRNA synthetase antibodies are
markers of a subtype of DM or PM characterized by interstitial lung disease (ILD), myositis, nonerosive
arthritis, Raynaud phenomenon, and "mechanic's hands." The term "mechanic's hands" refers to
hyperkeratosis at the ulnar aspect of the thumb and radial aspect of the index finger. Antibodies directed
against Jo-1 (histidyl tRNA synthetase) are the most common anti-tRNA synthetase autoantibody, found
in 20 to 30 percent of patients with PM and in 60 to 70 percent of PM patients with ILD [12]. (See
"Clinical manifestations of dermatomyositis and polymyositis in adults", section on 'Antisynthetase
antibodies'.)
Antibodies directed against ribosomal P antigens are found in 10 to 20 percent of patients with SLE and
are rare in other autoimmune diseases. (See "Antiribosomal P protein antibodies".)
Anti-Ro52 antibodies are present in approximately 40 percent of patients with SLE, 75 percent of
patients with Sjögren's syndrome (SS), 35 percent of patients with PM, and 20 percent of patients with
systemic sclerosis (SSc) [5]. Anti-Ro52 and/or anti-Ro60 antibodies are present in nearly all women who
have children with neonatal lupus syndrome (NLS). (See "Diagnosis and differential diagnosis of
systemic lupus erythematosus in adults", section on 'Laboratory testing' and "Diagnosis and classification
of Sjögren's syndrome", section on 'Antibodies to Ro/SSA and La/SSB' and "Neonatal lupus:
Epidemiology, pathogenesis, clinical manifestations, and diagnosis", section on 'Autoantibodies'.)
The signal recognition particle is a cytoplasmic, macromolecular structure that binds to an amino
terminal signal sequence that is present in some newly synthesized proteins and directs the proteins to
the endoplasmic reticulum. Anti-SRP antibodies are detected in approximately 10 percent of patients
with inflammatory muscle disease and identify a subset of patients with rapidly progressive, necrotizing
disease [46,47]. (See "Clinical manifestations of dermatomyositis and polymyositis in adults", section on
'Anti-SRP antibodies'.)
Cytoskeletal staining
● Definition – The cytoskeletal staining pattern is characterized by staining of a network of fibers in the
cytoplasm.
● Targets of antibodies – Actin and actin-associated proteins, cytokeratin, tropomyosin, vimentin.
● Clinical significance – Anti-actin antibodies are relatively specific markers for the diagnosis of Type I
autoimmune hepatitis, but may also be detected in patients with viral hepatitis [48]. Antibodies directed
against other components of the cytoskeleton are not disease-specific. Therefore, in a patient with
suspected autoimmune hepatitis, the presence of anti-actin antibodies should be sought by enzyme-
linked immunosorbent assay (ELISA) using purified actin antigen.
Golgi apparatus
● Definition – The Golgi apparatus pattern is characterized by irregular staining of the cytoplasm adjacent
to the nucleus. The Golgi apparatus staining pattern was detected in 0.4 percent of more than 9200
ANA-positive sera [2].
● Targets of antibodies – A large number of components of the Golgi apparatus have been found to be
targets of autoantibodies, including gigantin, golgin 245, golgin 110, and others.
● Clinical significance – Anti-Golgi apparatus antibodies were initially identified using serum from a
patient with SS. Antibodies that produce this staining pattern were subsequently detected in patients with
SLE, rheumatoid arthritis (RA), sarcoidosis, idiopathic cerebellar ataxia, and viral infections [49-51].
Because anti-Golgi apparatus autoantibodies are rare and there is no consistent disease association, the
clinical significance of these antibodies is uncertain.
Rods and rings
● Definition – The rods and rings staining pattern is characterized by staining of one or a few circular
and/or cylindrical structures in the cytoplasm of HEp-2 cells. This staining pattern can be detected using
some, but not all, commercial HEp-2 cell slides [52].
● Targets of antibodies – Inosine monophosphate dehydrogenase 2 (IMPDH2) and cytidine triphosphate

synthase 1 (CTPS1).
● Clinical significance – This cytoplasmic staining pattern may be produced by serum from a subset of
patients with hepatitis C who have received antiviral therapy with pegylated interferon-alpha and
ribavirin. Antibodies directed against rods and rings are rarely detected in patients with autoimmune
diseases [53].
SOCIETY GUIDELINE LINKS — Links to society and government-sponsored guidelines from selected
countries and regions around the world are provided separately. (See "Society guideline links: Antinuclear
antibodies".)
SUMMARY
● The indirect immunofluorescence (IIF) test for antinuclear antibodies (ANA), using the human cell line
HEp-2 as substrate, is a commonly used assay to detect human autoantibodies. The results of ANA
testing are reported in two parts: the titer of the antibodies and the staining pattern produced by the
antibodies. The titer of the antibodies refers to the highest dilution of serum that produces visible
fluorescence. The ANA pattern refers to the distribution of staining produced by autoantibodies reacting
with antigens in the HEp-2 cell nucleus and cytoplasm. (See 'Introduction' above.)
● There is limited agreement among laboratories as to which ANA staining patterns should be identified
and reported to clinicians. The patterns to be reported are determined by individual laboratory directors.
The directors also choose from among HEp-2 cell slides prepared by different companies, which may
use different fixative and permeabilization techniques. Depending on the preparation of the HEp-2 cell
substrate, some autoantibodies may or may not be detected by IIF. The clinician should know which
staining patterns are recognized and reported by his or her reference laboratory. (See 'Introduction'
above.)
● There are three broad categories of ANA staining patterns: nuclear, cell cycle-associated, and
cytoplasmic. Within each of these categories there are individual staining patterns produced by specific
autoantibodies. The disease associations, or lack thereof, for each of the staining patterns are discussed
above. (See 'ANA staining patterns involving the nucleus' above and 'Cell cycle-associated staining
patterns' above and 'Cytoplasmic staining patterns' above.)
● ANA staining patterns involving the nucleus include homogeneous, nuclear speckled, nucleolar, nuclear
dot, and nuclear envelope. Three types of nuclear speckled patterns ("fine," "coarse," and "dense fine")
have been described. Three types of nuclear dot staining patterns have been described: centromere,
PML-Sp100 nuclear body, and Cajal body. (See 'ANA staining patterns involving the nucleus' above and
'Homogeneous' above and 'Nuclear speckled' above and 'Nucleolar' above and 'Nuclear envelope'
above.)
● Cell cycle-associated staining patterns include proliferating cell nuclear antigen (PCNA) pattern,
centromere protein-F (CENP-F), and mitotic spindle apparatus. Two types of nuclear mitotic apparatus
staining patterns have been described: nuclear mitotic apparatus-1 (NuMA1) and NuMa2. (See 'Cell
cycle-associated staining patterns' above and 'PCNA' above and 'CENP-F' above and 'Mitotic spindle
apparatus' above.)
● Cytoplasmic staining patterns include antimitochondrial antibody (AMA), cytoplasmic fine speckled,
cytoskeletal staining, Golgi apparatus, and rods and rings. (See 'Cytoplasmic staining patterns' above
and 'Antimitochondrial antibody' above and 'Cytoplasmic fine speckled' above and 'Cytoskeletal staining'
above and 'Golgi apparatus' above and 'Rods and rings' above.)
https://www.uptodate.com/contents/clinical-significance-of-antinuclear-antibody-staining-patterns-and-associated-autoantibodies/print?search=… 10/13
ACKNOWLEDGMENT — The editorial staff at UpToDate would like to acknowledge Peter Schur, MD, who
contributed to an earlier version of this topic review.
Use of UpToDate is subject to the Subscription and License Agreement.
REFERENCES
1. Chan EK, Damoiseaux J, Carballo OG, et al. Report of the First International Consensus on
Standardized Nomenclature of Antinuclear Antibody HEp-2 Cell Patterns 2014-2015. Front Immunol
2015; 6:412.
2. Vermeersch P, Bossuyt X. Prevalence and clinical significance of rare antinuclear antibody patterns.
Autoimmun Rev 2013; 12:998.
3. Haugbro K, Nossent JC, Winkler T, et al. Anti-dsDNA antibodies and disease classification in
antinuclear antibody positive patients: the role of analytical diversity. Ann Rheum Dis 2004; 63:386.
4. Eriksson C, Engstrand S, Sundqvist KG, Rantapää-Dahlqvist S. Autoantibody formation in patients with
rheumatoid arthritis treated with anti-TNF alpha. Ann Rheum Dis 2005; 64:403.
5. Schulte-Pelkum J, Fritzler M, Mahler M. Latest update on the Ro/SS-A autoantibody system.
6. Mimori T. Atlas of Antinuclear Antibodies. Nagoya: Medical and Biological Laboratories Co, LTD, 1999.
7. Blomberg S, Ronnblom L, Wallgren AC, et al. Anti-SSA/Ro antibody determination by enzyme-linked
immunosorbent assay as a supplement to standard immunofluorescence in antinuclear antibody
screening. Scand J Immunol 2000; 51:612.
8. Ho KT, Reveille JD. The clinical relevance of autoantibodies in scleroderma. Arthritis Res Ther 2003;
5:80.
9. Rigolet A, Musset L, Dubourg O, et al. Inflammatory myopathies with anti-Ku antibodies: A prognosis
dependent on associated lung disease. Medicine (Baltimore) 2012; 91:95.
10. Ghirardello A, Zampieri S, Iaccarino L, et al. Anti-Mi-2 antibodies. Autoimmunity 2005; 38:79.
11. Ghirardello A, Bassi N, Palma L, et al. Autoantibodies in polymyositis and dermatomyositis. Curr
Rheumatol Rep 2013; 15:335.
12. Ghirardello A, Borella E, Beggio M, et al. Myositis autoantibodies and clinical phenotypes. Auto Immun
Highlights 2014; 5:69.
13. Koga T, Fujikawa K, Horai Y, et al. The diagnostic utility of anti-melanoma differentiation-associated
gene 5 antibody testing for predicting the prognosis of Japanese patients with DM. Rheumatology
(Oxford) 2012; 51:1278.
14. Barada FA Jr, Andrews BS, Davis JS 4th, Taylor RP. Antibodies to Sm in patients with systemic lupus
erythematosus. Correlation of Sm antibody titers with disease activity and other laboratory parameters.
Arthritis Rheum 1981; 24:1236.
15. Beaufils M, Kouki F, Mignon F, et al. Clinical significance of anti-Sm antibodies in systemic lupus
erythematosus. Am J Med 1983; 74:201.
16. Phan TG, Wong RC, Adelstein S. Autoantibodies to extractable nuclear antigens: making detection and
interpretation more meaningful. Clin Diagn Lab Immunol 2002; 9:1.
17. Arroyo-Ávila M, Santiago-Casas Y, McGwin G Jr, et al. Clinical associations of anti-Smith antibodies in
PROFILE: a multi-ethnic lupus cohort. Clin Rheumatol 2015; 34:1217.
18. Bizzaro N, Tonutti E, Visentini D, et al. Antibodies to the lens and cornea in anti-DFS70-positive
subjects. Ann N Y Acad Sci 2007; 1107:174.
19. Dellavance A, Viana VS, Leon EP, et al. The clinical spectrum of antinuclear antibodies associated with
the nuclear dense fine speckled immunofluorescence pattern. J Rheumatol 2005; 32:2144.
20. Watanabe A, Kodera M, Sugiura K, et al. Anti-DFS70 antibodies in 597 healthy hospital workers.
21. Miyara M, Albesa R, Charuel JL, et al. Clinical phenotypes of patients with anti-DFS70/LEDGF
antibodies in a routine ANA referral cohort. Clin Dev Immunol 2013; 2013:703759.
22. Mahler M, Parker T, Peebles CL, et al. Anti-DFS70/LEDGF antibodies are more prevalent in healthy
individuals compared to patients with systemic autoimmune rheumatic diseases. J Rheumatol 2012;
39:2104.
23. Mariz HA, Sato EI, Barbosa SH, et al. Pattern on the antinuclear antibody-HEp-2 test is a critical
parameter for discriminating antinuclear antibody-positive healthy individuals and patients with
autoimmune rheumatic diseases. Arthritis Rheum 2011; 63:191.
24. Choi MY, Clarke AE, St Pierre Y, et al. The prevalence and determinants of anti-DFS70 autoantibodies
in an international inception cohort of systemic lupus erythematosus patients. Lupus 2017; 26:1051.
25. Khan S, Alvi A, Holding S, et al. The clinical significance of antinucleolar antibodies. J Clin Pathol 2008;
61:283.
26. Steen VD. Autoantibodies in systemic sclerosis. Semin Arthritis Rheum 2005; 35:35.
27. Hamaguchi Y, Fujimoto M, Matsushita T, et al. Common and distinct clinical features in adult patients
with anti-aminoacyl-tRNA synthetase antibodies: heterogeneity within the syndrome. PLoS One 2013;
8:e60442.
28. Yang WH, Yu JH, Nakajima A, et al. Do antinuclear antibodies in primary biliary cirrhosis patients
identify increased risk for liver failure? Clin Gastroenterol Hepatol 2004; 2:1116.
29. Bournia VK, Diamanti KD, Vlachoyiannopoulos PG, Moutsopoulos HM. Anticentromere antibody
positive Sjögren's Syndrome: a retrospective descriptive analysis. Arthritis Res Ther 2010; 12:R47.
30. Lee KE, Kang JH, Lee JW, et al. Anti-centromere antibody-positive Sjögren's syndrome: A distinct
clinical subgroup? Int J Rheum Dis 2015; 18:776.
31. Hanly JG, Thompson K, McCurdy G, et al. Measurement of autoantibodies using multiplex methodology
in patients with systemic lupus erythematosus. J Immunol Methods 2010; 352:147.
32. Hossny E, Hady HA, Mabrouk R. Anti-centromere antibodies as a marker of Raynaud's phenomenon in
pediatric rheumatologic diseases. Pediatr Allergy Immunol 2000; 11:250.
33. Bizzaro N, Covini G, Rosina F, et al. Overcoming a "probable" diagnosis in antimitochondrial antibody
negative primary biliary cirrhosis: study of 100 sera and review of the literature. Clin Rev Allergy
Immunol 2012; 42:288.
34. Onouchi H, Muro Y, Tomita Y. Clinical features and IgG subclass distribution of anti-p80 coilin
antibodies. J Autoimmun 1999; 13:225.
35. Vázquez-Talavera J, Ramírez-Sandoval R, Esparza Ibarra E, et al. Autoantibodies against Cajal bodies
in systemic lupus erythematosus. Med Sci Monit 2004; 10:BR130.
36. Andrade LE, Chan EK, Raska I, et al. Human autoantibody to a novel protein of the nuclear coiled body:
immunological characterization and cDNA cloning of p80-coilin. J Exp Med 1991; 173:1407.
37. Nesher G, Margalit R, Ashkenazi YJ. Anti-nuclear envelope antibodies: Clinical associations. Semin
38. Duarte-Rey C, Bogdanos D, Yang CY, et al. Primary biliary cirrhosis and the nuclear pore complex.
39. Fritzler MJ, McCarty GA, Ryan JP, Kinsella TD. Clinical features of patients with antibodies directed
against proliferating cell nuclear antigen. Arthritis Rheum 1983; 26:140.
40. Vermeersch P, De Beeck KO, Lauwerys BR, et al. Antinuclear antibodies directed against proliferating
cell nuclear antigen are not specifically associated with systemic lupus erythematosus. Ann Rheum Dis
2009; 68:1791.
41. Rattner JB, Rees J, Whitehead CM, et al. High frequency of neoplasia in patients with autoantibodies to
centromere protein CENP-F. Clin Invest Med 1997; 20:308.
42. Szalat R, Ghillani-Dalbin P, Jallouli M, et al. Anti-NuMA1 and anti-NuMA2 (anti-HsEg5) antibodies:
Clinical and immunological features: A propos of 40 new cases and review of the literature. Autoimmun
Rev 2010; 9:652.
43. Bloch D. Unpublished data. 2015.
44. Berg PA, Klein R. Mitochondrial antigens and autoantibodies: from anti-M1 to anti-M9. Klin Wochenschr
1986; 64:897.
45. Dähnrich C, Pares A, Caballeria L, et al. New ELISA for detecting primary biliary cirrhosis-specific
antimitochondrial antibodies. Clin Chem 2009; 55:978.
46. Dalakas MC. Inflammatory muscle diseases. N Engl J Med 2015; 372:1734.
47. Casciola-Rosen L, Mammen AL. Myositis autoantibodies. Curr Opin Rheumatol 2012; 24:602.
48. Aubert V, Pisler IG, Spertini F. Improved diagnoses of autoimmune hepatitis using an anti-actin ELISA. J
Clin Lab Anal 2008; 22:340.
49. Fritzler MJ, Etherington J, Sokoluk C, et al. Antibodies from patients with autoimmune disease react
with a cytoplasmic antigen in the Golgi apparatus. J Immunol 1984; 132:2904.
50. Hong HS, Morshed SA, Tanaka S, et al. Anti-Golgi antibody in rheumatoid arthritis patients recognizes a
novel antigen of 79 kDa (doublet) by western blot. Scand J Immunol 1992; 36:785.
51. Bizzaro N, Pasini P, Ghirardello A, Finco B. High anti-golgi autoantibody levels: an early sign of
autoimmune disease? Clin Rheumatol 1999; 18:346.
52. Seelig HP, Appelhans H, Bauer O, et al. Autoantibodies against inosine-5'-monophosphate dehydrogen
ase 2--characteristics and prevalence in patients with HCV-infection. Clinical Lab 2011; 57:753.
53. Calise SJ, Keppeke GD, Andrade LE, Chan EK. Anti-rods/rings: a human model of drug-induced
autoantibody generation. Front Immunol 2015; 6:41.
Topic 1823 Version 15.0
Contributor Disclosures
Donald B Bloch, MD Nothing to disclose Robert H Shmerling, MD Consultant/Advisory Board:
Knowyourmeds [Advise on matters relating to the Company’s business, technology and products (medication
information)]. Monica Ramirez Curtis, MD, MPH Nothing to disclose
Contributor disclosures are reviewed for conflicts of interest by the editorial group. When found, these are
addressed by vetting through a multi-level review process, and through requirements for references to be
provided to support the content. Appropriately referenced content is required of all authors and must conform
to UpToDate standards of evidence.
Conflict of interest policy

Clinical Significance of Antinuclear Antibody Staining Patterns and Associated Autoantibodies - UpToDate

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Clinical Significance of Antinuclear Antibody Staining Patterns and Associated Autoantibodies - UpToDate

Uploaded by

Copyright:

Available Formats

24/10/2018 Clinical significance of antinuclear antibody staining patterns and associated autoantibodies - UpToDate

Official reprint from UpToDate®

Author: Donald B Bloch, MD

ANA STAINING PATTERNS INVOLVING THE NUCLEUS

● Targets of antibodies – Histone proteins, double-stranded (ds)DNA, single-stranded DNA.

● Targets of antibodies – Sm, U1 ribonuclear protein (RNP).

Dense fine speckled pattern

● Targets of autoantibodies – Components of the PM/Scl complex (PM-Scl-75, PM-Scl-100), U3RNP

[8]. (See "Clinical manifestations of dermatomyositis and polymyositis in adults", section on

● Targets of antibodies – Centromere proteins A, B, and C.

● Disease association – Anticentromere antibodies are present in approximately 30 percent of patients

PML-Sp100 nuclear body

● Target of antibodies – Speckled-100 kD (Sp100), promyelocytic leukemia protein (PML).

● Target of antibodies – p80-coilin.

Nuclear envelope pattern

● Targets of antibodies – Nuclear lamin proteins, lamin-associated proteins.

Nuclear pore complex pattern

● Targets of antibodies – Glycoprotein-210 kD (Gp210), nucleoporin-62 kD (Nup62).

CELL CYCLE-ASSOCIATED STAINING PATTERNS

● Target of antibodies – Delta chain of DNA polymerase.

● Target of antibodies – CENP-F.

NuMA1 staining pattern

● Target of antibodies – NuMA1.

NuMA2 staining pattern

CYTOPLASMIC STAINING PATTERNS

● Targets of antibodies – E2 component of pyruvate dehydrogenase complex (also known as

Cytoplasmic fine speckled

● Disease association – Antibodies directed against aminoacyl-tRNA synthetases are present in 25 to 35

● Targets of antibodies – Actin and actin-associated proteins, cytokeratin, tropomyosin, vimentin.

Rods and rings

some, but not all, commercial HEp-2 cell slides [52].

● Targets of antibodies – Inosine monophosphate dehydrogenase 2 (IMPDH2) and cytidine triphosphate

Use of UpToDate is subject to the Subscription and License Agreement.

Topic 1823 Version 15.0

Conflict of interest policy

You might also like