REVIEWS

Understanding and interpreting

antinuclear antibody tests in systemic
rheumatic diseases
Xavier Bossuyt   1 ✉, Ellen De Langhe2,3, Maria Orietta Borghi   4,5 and Pier Luigi Meroni   4
Abstract | Antinuclear antibodies (ANAs) are valuable laboratory markers to screen for and
support the diagnosis of various rheumatic diseases (known as ANA-​associated rheumatic
diseases). The importance of ANA testing has been reinforced by the inclusion of ANA positivity
as an entry criterion in the 2019 systemic lupus erythematosus classification criteria. In addition,
specific ANAs (such as antibodies to Sm, double-​stranded DNA (dsDNA), SSA/Ro60, U1RNP,
topoisomerase I, centromere protein B (CENPB), RNA polymerase III and Jo1) are included in
classification criteria for other rheumatic diseases. A number of techniques are available for
detecting antibodies to a selection of clinically relevant antigens (such as indirect immunofluo­
rescence and solid phase assays). In this Review, we discuss the advantages and limitations of
these techniques, as well as the clinical relevance of the differences between the techniques,
to provide guidance in understanding and interpreting ANA test results. Such understanding
not only necessitates insight into the sensitivity and specificity of each assay, but also into the
importance of the disease context and antibody level. We also highlight the value of titre-​specific
information (such as likelihood ratios).

The presence of antinuclear antibodies (ANAs) is associ­ the main advantage of these assays being their high
1
Department of Microbiology,
ated with various systemic rheumatic diseases, including reproducibility and specificity.
Immunology and
Transplantation, KU Leuven
systemic lupus erythematosus (SLE), systemic sclerosis Notably, ANA positivity (ANA with a titre ≥1:80 by
and Department of (SSc), primary Sjögren syndrome, mixed connective HEp‐2 IIF or an equivalent positive test by SPA at least
Laboratory Medicine, tissue disease (MCTD) and idiopathic inflammatory once) was confirmed as a new entry criterion for the new
University Hospitals Leuven, myopathies (IIMs, such as polymyositis and dermato­ 2019 SLE classification criteria3. In these criteria, specific
Leuven, Belgium.
myositis)1. These diseases are collectively referred to antibodies against double-​stranded DNA (dsDNA) and
2
Department of Rheumatology,
as ANA-​associated rheumatic diseases, and several Sm were included as immunological domains for SLE
University Hospitals Leuven,
Leuven, Belgium.
autoantibodies that are specific to each disease have classification3. Although ANAs are useful for screening
3
Laboratory of Tissue
been identified. for SSc, primary Sjögren syndrome, MCTD and IIM,
Homeostasis and Disease, The gold standard approach to screening for ANAs ANA positivity is not included in the classification cri­
Department of Development in rheumatic diseases is detection via indirect immuno­ teria for these diseases. However, positivity for some
and Regeneration, Skeletal fluorescence (IIF) assays using human epithelial type 2 specific autoantibodies is included in these criteria:
Biology and Engineering
cells (HEp-2 cells) (referred to here as HEp-2 IIF assays)2. antibodies to SSA for primary Sjögren syndrome, anti­
Research Center, KU Leuven,
Leuven, Belgium.
In the case of a positive ANA result, follow-​up testing bodies to centromere protein B (CENPB), topoisomer­
4
Istituto Auxologico Italiano,
is performed to identify the specific autoanti­b odies ase 1 (also known as Scl70) and RNA polymerase III for
IRCCS; Immunorheumatology present. However, HEp-2 IIF is laborious, necessitates SSc, antibodies to U1RNP for MCTD and antibodies to
Research Laboratory, skilled operators and has a high inter-​observer vari­ Jo1 (histidyl tRNA synthetase) for IIM4–7. Antibodies
Milan, Italy. ability and low specificity 1. ANAs can be present in to U1RNP at high levels are suggestive of MCTD.
5
Department of Clinical patients with various other diseases, including non- The inclusion of ANA as a new entry criterion in
Sciences & Community rheumatic diseases (for example, autoimmune liver the 2019 classification criteria for SLE, along with the
Health, University of Milan,
Milan, Italy.
diseases), and can also be present in healthy indivi­ emergence of new techniques and the improvement of
✉e-​mail: xavier.bossuyt@ duals1. Therefore, researchers have developed alter­ previous techniques, has raised new questions regarding
uzleuven.be native (automated) solid phase immunoassays (SPAs) ANA testing in the clinical setting. Should HEp-2 IIF
https://doi.org/10.1038/ to screen for autoantibodies that are specifically remain the ‘gold standard’ for ANA detection? What is
s41584-020-00522-​w associated with ANA-associated rheumatic diseases, the best approach to interpreting a positive HEp-2 IIF

Nature Reviews | Rheumatology

Reviews
Key points
• Clinicians should be aware of the type of assay used for antinuclear antibody
detection and the advantages and disadvantages of using immunofluorescence (IIF)
assays and solid phase immunoassays (SPAs) for screening.
• IIF assays can vary in performance between different laboratories, whereas the
performance of fully automated assays is more consistent.
• The performance characteristics of IIF assays and SPAs are disease-​dependent;
IIF assays are more sensitive than SPA for screening for systemic sclerosis Solid phase assays
(and systemic lupus erythematosus) but not Sjögren syndrome.
• For both IIF assays and SPAs, no single cut-​off has both good sensitivity and good
specificity; combining IIF with SPA has the highest clinical value.
• A dichotomous interpretation of test results overlooks important information at the
antibody level; this limitation of antinuclear antibody testing can be overcome by
reporting test result-​specific likelihood ratios.
• The differences among the assays are not only important considerations when
screening and diagnosing patients, but also when using these assays for classifying
patients (for example, for clinical trial enrolment).
test result, and what is the relevance of the HEp-2 IIF
titre? What titre should be used as the cut-​off point?
What is the clinical performance of the new screening
SPAs, and how do these assays compare with HEp-2
IIF? What are the optimal testing strategies, and what
is the value in using a combination of HEp-2 IIF and
SPA testing in the diagnostic workflow? All these issues
justify a reassessment of ANA testing. In this Review,
we attempt to answer the above-​mentioned questions
and argue that information gets lost when a single
cut-​off value is applied when interpreting test results
(dicho­tomous interpretation). Different techniques
have varying performance characteristics and pro­
vide distinct types of information. Physicians habit­
ually interpret test results in a dichotomous way and
often are unaware of the intrinsic differences between
the assays and their clinical relevance. This Review is
aimed at improving the understanding of the clinical
performance of the avail­able techniques to empower
clinicians to better comprehend and interpret the
results in the context of systemic ANA-​associated
rheumatic diseases.
Available ANA assays
Indirect immunofluorescence
Traditionally, ANAs are detected by IIF on rodent tissue
sections or on HEp‐2 cells. With HEp-2 IIF, the auto­
antibodies that attach to the HEp-2 cell substrate are
visualized with a fluorescence microscope after stain­
ing with fluorescein-​labelled anti-​immunoglobulin
antibodies (Fig. 1a). In addition to recording the highest
antibody titre that provides a positive test result, the pat­
tern produced by the autoantibodies can provide further
clinically useful information as certain patterns are asso­
ciated with particular autoantibodies. The main ANA
staining patterns are homogeneous, speckled, nucleolar
Specificity and centromere. The International Consensus on ANA
The ability of a test to correctly Patterns working group have made efforts to harmo­
exclude individuals who do not nize the HEp-2 pattern nomenclature8. Table 1 gives an
have the disease; the specificity
is calculated as the fraction of
overview of the patterns associated with disease-​related
individuals without the disease autoantibodies. Examples of some of the most prevalent
who test negative. HEp-2 IIF patterns are shown in Supplementary Fig. 1.
As ANA testing by HEp-2 IIF reveals reactivity not only
to nuclear antigens but also to cytoplasmic and mitotic
antigens, the term anti-​cell antibodies is increasingly
being used by researchers9; however, this term is yet to
disperse to the clinic. Cytoplasmic reactivity is clinically
relevant and should be considered and reported as a
positive ANA test result.
Alternative, automated screening SPAs are gradually
being introduced into clinical laboratories. In these
assays, the solid phase (for example, a plate, bead or
membrane) is coated with a mixture of relevant (puri­
fied or recombinant) specific autoantigens (that in
some assays can be complemented with a cell extract).
Autoantibodies present in a patient sample attach to
the autoantigen and are bound by a detection antibody
linked to an enzyme that generates a colorimetric reac­
tion (such as with enzyme-​linked immunosorbent assays
(ELISAs)) or a fluorescent reaction (such as with fluoro­
metric enzyme-​linked immunoassays (FEIAs)) (Fig. 1b).
Alternatively, the detection antibody can be linked to a
chemiluminescent compound (such as with chemilumi­
nescence immunoassays (CIAs)) (Fig. 1b). Supplementary
Table 1 gives an overview of the blend of autoantigens
included in such screening assays. Following a positive
screening SPA test result, the specific reactivity of the
autoantibody should be determined in follow-​up assays,
in which the solid phase is coated with a single autoan­
tigen, unless a multiplexed assay was used for screen­
ing and the autoantibody specificity has already been
identified.
Various multiplexed SPAs are available. For example,
in the addressable laser bead immunoassay (ALBIA),
each autoantigen is coupled to a bead with a distinctive
intrinsic fluorescent signal. This approach enables the
simultaneous, multiplexed measurement of autoanti­
bodies to various autoantigens using flow cytometry
(Fig. 1b). Assays that use alternative technologies (such
as particle-​based multianalyte technology (PMAT))
(Fig. 1b) are also under development10.
Finally, line or dot immunoassays also enable multi­
plexed detection of autoantibodies to various auto­
antigens; these antigens are typically immobilized as
a line or dot on a nitrocellulose membrane (Fig. 1b).
In general, such assays are used to identify the specific
autoantibodies present in patients who have had a pos­
itive screening result (for example, positivity by HEp-2
IIF) or in patients who are highly suspected of having
an ANA-​associated rheumatic disease (for example,
for detecting IIM-​specific or SSc-​specific antibodies in
patients suspected of having IIM or SSc, respectively).
How well do the assays perform?
Performance characteristics: HEp-2 IIF
The interpretation of an HEp-2 IIF test result is depen­
dent on the antibody titre and pattern. In this section,
we discuss the importance of considering both the
antibody titre and pattern, as well as additional aspects
such as the use of automated HEp-2 IIF, the clinical rele­
vance of HEp-2 IIF negativity and variation in HEp-2
IIF testing.
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Sensitivity
The ability of a test to correctly
detect patients with a disease;
the sensitivity of a test is
calculated as the fraction of
patients with the disease who
test positive.
Likelihood ratio
The ratio of the probability
of a particular test result for
a patient with a particular
disease and the probability
of the same test result for an
individual without the disease.
Nature Reviews | Rheumatology
ANA titre and the value of titre-​specific information.
The international recommendations for the assessment
of antibodies to cellular antigens, which were formu­
lated to support ANA screening during the first level
of diagnosis for systemic autoimmune-​rheumatic dis­
eases, define the HEp-2 IIF cut-​off value as the titre that
corresponds to the 95th percentile of local age-​matched
and gender-​matched healthy individuals11. In an inter­
national study on healthy individuals, this 95th percen­
tile corresponded to a titre of 1:160 (ref.12). However, the
international group of experts who created these recom­
mendations recognized that, at a 1:160 dilution, the
sensitivity of HEp-2 IIF for ANA-​associated rheumatic
diseases is not perfect and that a negative result at such
a dilution does not necessarily exclude disease11.
At variance with the suggested HEp-2 IIF cut-​off
titre of 1:160 in the diagnostic workflow for SLE (and
other systemic autoimmune rheumatic disease) men­
tioned above11, the steering committee for the 2019 SLE
classification criteria concluded that a titre of 1:80 has
sufficiently high sensitivity (97.8%) to be considered as
an entry criterion3,13.This conclusion was based on a lit­
erature review and meta-​regression analysis of the per­
formance of HEp-2 IIF antibody testing for classifying
SLE13. A cut-​off titre of 1:80 was chosen to optimize the
sensitivity of HEp-2 IIF for SLE classification; however,
it should be noted that the specificity associated with the
1:80 cut-​off titre is low (74.7%)13.
Typically, a single cut-​off value is used to interpret
test results. However, using a single HEp-2 IIF cut-​off
value with a dichotomous interpretation (positive or
negative) overlooks the fact that the chance (likelihood)
of disease increases with increasing antibody level (titre).
A dichotomous approach does not convey informa­
tion inherent to the antibody level (or the specific test
result). For ANA detection by HEp-2 IIF, each test result
(antibody titre) has a specific likelihood ratio of disease
(known as the titre-​specific likelihood ratio). The con­
cept of likelihood ratios is explained in detail in Box 1.
Box 2 explains how the likelihood ratio can be used to
estimate the post-​test probability or positive predictive
value (PPV). Estimates of the titre-​specific likelihood
ratios of HEp-2 IIF suggest that the likelihood ratio
increases with increasing antibody level14. For example,
the clinical and diagnostic value of a titre of 1:80 (like­
lihood ratio of 0.5) differs from that of a titre of 1:640
(likelihood ratio of 19). A 1:640 titre has a likelihood
ratio of 19, meaning that samples from patients with SLE
are 19 times more likely to give a positive test result at
this titre than control samples, whereas a 1:80 titre has
a likelihood ratio of 0.5, meaning that control samples
are twice as likely to give a positive test result at this titre
than samples from patients with SLE. Such titre-​specific
information is more valuable than a dichotomous inter­
pretation that uses a single cut-​off point. Applying a
single cut-​off point ignores the clinical information
inherent in the antibody level (titre). Titre-​specific like­
lihood ratios (and PPVs) have also been estimated for
ANA-​associated rheumatic diseases15,16.
Taken together, titre-​specific likelihood ratios hold
more clinical value than using a single cut-​off value
and, therefore, enable a more accurate interpretation of
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HEp-2 IIF test results. The high sensitivity associated
with a HEp-2 IIF cut-​off of 1:80 ANA enables such a
cut-​off to be used as an entry criterion for SLE classifi­
cation. However, one should be careful when interpret­
ing a 1:80 result during disease diagnosis, given the low
likelihood ratio associated with such a result. In patients
with such low ANA test result, the diagnostic workflow
and eventually the diagnosis will have to rely on clinical
manifestations or characteristics to avoid false diagnoses
and unnecessary treatments.
Staining pattern. The antibody staining patterns with
HEp-2 IIF assays provide useful information that can
inform the disease diagnosis8,17,18. The classical nuclear
patterns are speckled, homogeneous, nucleolar and
centromere. For each of these patterns, the PPV for
ANA-​associated rheumatic diseases increases with
increasing antibody titre18. Of these patterns, the cen­
tromere pattern has the highest PPV for ANA-​associated
rheumatic diseases and is highly associated with SSc18.
Except for the centromere pattern, which is specific for
anti-​CENPB antibodies, the HEp-2 IIF pattern does not
enable the identification of the specificity of the anti­
body, even though associations between the antibody
pattern and specificity have been described (Table 1).
For example, anti-​double-​stranded DNA antibodies
are associated with a homogeneous pattern, anti-​SSA/
Ro60 antibodies are associated with a fine speckled pat­
tern, anti-​U1RNP and anti-​Sm antibodies are associated
with a large/coarse speckled pattern and anti-​PM/Scl
and anti-​fibrillarin (U3 RNP) antibodies are associated
with the nucleolar pattern. Some cytoplasmic patterns
(such as a (dense) fine speckled pattern) are associated
with the presence of antisynthetase anti­bodies (such
as antibodies towards the amino-​acyl transfer RNA
synthetases Jo1, PL7, PL12, OJ, EJ, KS, Ha or Zo), anti-​
signal recognition peptide (SRP) or anti-​ribosomal P
antibodies. Of note, some of the cytoplasmic antibod­
ies (for example, the antisynthetase antibodies) can be
detected at a low titre. In any case, a positive HEp-2
IIF test should be confirmed with an antigen-​specific
immunoassay. A nuclear dense fine speckled pattern
on HEp-2 cells can also occur at high titres for sam­
ples from healthy individuals17, owing to the presence
of anti-​DFS70 antibodies. Hence, confirming the anti­
body specificity of this pattern with a specific assay is
important, as determining the antibody specificity using
microscopy can be difficult. Anti-​DFS70 antibodies are
present in 1–8% of healthy individuals, mainly in young
females19, but are also present in a wide range of diseases
(reviewed elsewhere20). The physiological importance of
these antibodies is not well understood. The presence
of anti-​DFS70 antibodies might explain the finding of
a high-​titre ANA test result for some individuals who
have no evidence of an ANA-​associated rheumatic dis­
ease. Some researchers have proposed that the presence
of monospecific anti-​DFS70 antibodies (that is, anti-​
DFS70 in the absence of a disease-​associated antibody)
can be used as a biomarker to exclude individuals with­
out an ANA-​associated rheumatic disease20,21. However,
other researchers have argued that the absence of
disease-​associated ANAs and clinical symptoms should
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a Indirect immunofluorescence
Antibody labelling Antibody detection

Fluorescent tag

Detection antibody
Autoantibody
(patient sample)
Autoantigen Microscopic evaluation
in HEp-2 cell (by indirect
immunofluorescence)

Glass
slide

Antibody titre Antibody staining pattern

(for example, in the nucleus and cytoplasm
Autoantigens 1:80) (for example, fine speckled)

HEp-2 cell

b Solid-phase assays
Antibody labelling Antibody detection

Solid phase assay Substrate or

Detection oxidizing agent
Signal
antibody
• Colorimetric
Autoantibody • Fluorescent
(patient sample) Autoantigen • Chemiluminescent

Enzyme (ELISA, FEIA

Dot or line blot (multiplexed assay) and blotting) or
chemiluminescent
compound (CIA)
Nitrocellulose
membrane

Different autoantigens

ALBIA and PMAT (multiplexed assays)

Detection by flow
cytometry (for
ALBIA) or by camera
Antibody label (for PMAT)
Detection
antibody Detection by a laser
(for ALBIA) or a
Autoantibody 1 Autoantibody 2 light-emitting diode
(patient (patient (for PMAT)
sample) sample)
Bead or particle
detection by a
different laser
(for ALBIA) or
light-emitting diode
(for PMAT)

Different beads (for ALBIA) or particles (for PMAT)

with a unique intrinsic fluorescent signal (ALBIA) or
signature (PMAT), each coupled to a specific antigen

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◀ Fig. 1 | overview of methods used for aNa detection. a | In immunofluorescence (IIF)
assays, patient samples are typically incubated on a slide covered with a monolayer of
human epithelial type 2 (HEp-2) cells. Any antinuclear antibodies (ANAs) from the patient
that bind to autoantigens in the HEp-2 cells are detected by fluorophore-​labelled anti-
immunoglobulin antibodies (detection antibodies), which are then visualized using a
fluorescence microscope. The test report includes the antibody titre and the antibody
staining pattern. b | In standard solid phase assays, a solid phase surface (for example,
polystyrene or nitrocellulose) is coated with (a mixture of) autoantigens. Any ANAs
present in the patient sample that attach to the autoantigens are then bound by
detection antibodies that provide a readout of the ANA concentration. This readout
can be generated by linking the detection antibody with an enzyme that produces either
a colorimetric signal (known as enzyme-​linked immunosorbent assays (ELISAs)) or a
fluorescent signal (known as fluorometric enzyme-​linked immunoassays (FEIAs)) in the
presence of a particular substrate. Alternatively, the detection antibody can be linked
to a compound that emits a chemiluminescent signal in the presence of a particular
solution (known as chemiluminescence immunoassays (CIAs)). In a line or dot blot
(multiplex) immunoassay, the autoantigens are usually immobilized as a line or dot on
a nitrocellulose membrane. The autoantibodies bound to the nitrocellulose membrane
are then detected with anti-​immunoglobulin antibodies linked to an enzyme (similarly
producing a colorimetric signal). In the addressable laser bead immunoassay (ALBIA) and
particle-​based multi-​analyte technology (PMAT) assay (which are further examples of
multiplexed assays), different autoantigens are coupled to different beads (for ALBIA)
or particles (for PMAT), each with a distinctive intrinsic fluorescent signal (for ALBIA) or
a unique signature (PMAT). Following incubation of the patient sample with the beads
or particles, any bound autoantibodies present in the sample are then sandwiched by
fluorophore-​labelled detection antibodies. The samples are subsequently analysed
by a flow cytometry-​based system using two lasers (for ALBIA) or a camera-​based system
using two light-​emitting diodes (for PMAT), which enables the simultaneous detection
of multiple ANAs with different specificities. Other assay formats might become
available in the future.
contribute to the exclusion of an ANA-associated rheu­
matic disease, rather than the presence of anti-DFS70
antibodies22.
Automated HEp-2 IIF assays. Over the past decade,
HEp-2 IIF systems have been developed that enable
automated image acquisition. Such automated HEp-2
IIF systems generate quantitative fluorescence intensity
data23,24, which the manufacturers propose could be used to
estimate the end-​point titres25. However, in addition
to the antibody titre, the antibody pattern might also
affect the fluorescence intensity25. With some commercial
systems, the likelihood ratio for ANA-​associated rheum­
atic diseases increases with increasing fluorescence light
intensity23,26,27, and light intensity unit-​specific likelihood
ratios have been calculated28. These data suggest that
the higher the fluorescence intensity as measured by
an automated HEp-2 IIF system, the higher the chance
(likelihood) of an ANA-​associated rheumatic disease. In
addition to staining intensity, some automated systems
are able to identify some of the major ANA patterns.
However, it is important that an experienced technician
reviews the antibody staining pattern as not all patterns
are recognized by automated HEp-2 IIF systems and as
some automated systems might fail to accurately assign
the pattern.
Relevance of negative HEp-2 IIF test result. An impor­
tant aspect to consider for ANA testing is that an HEp-2
IIF might miss the presence of certain antibodies, such as
antibodies to SSA/Ro60, Ro52 (also known as TRIM21),
ribosomal P, Jo1 and other amino-​acyl transfer RNA
synthetases, SRP, HMGCR, NXP1 or MDA5, because of
Nature Reviews | Rheumatology
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the low abundance of the antigen and/or because of the
fixation method29–34. If the patient is suspected to have a
disease associated with one of the aforementioned anti­
bodies, a negative HEp-2 IIF should not elicit hesitation,
but should rather trigger the clinician to request an
alternative diagnostic assay (such as a SPA).
Assay variability. Different HEp-2 IIF kits are avail­
able that might yield varying results; two studies have
shown that variation among HEp-2 IIF kits can influ­
ence ANA detection in patients with established SLE35,36.
In one of the studies, some patients with established
SLE had a negative ANA test result, and the frequency
of ANA negativity (which varied from 0.6% to 27.6%)
varied depending on the assay kit used35,36. Conversely,
in a correspondence to one of these studies, an Italian
study reported good sensitivity of manual and various
automated HEp-2 IIF methods37. Nevertheless, based on
these findings, the researchers questioned whether ANA
positivity should be employed to determine eligibility for
clinical trials35,36.
External quality assessment schemes (using reference
samples) have revealed that the reported HEp-2 IIF titre
can vary between different laboratories38,39, which was
put down to variations in methodology (such as the sub­
strate, fixative, conjugate and optics used) and subjective
evaluation of the positivity38,39. Such variation also occurs
for automated ANA HEp-2 IIF40. The use of a secondary
reference material could reduce the between-​laboratory
variation39, but such reference materials are not used as
standard in clinical practice.
Summary and future perspectives. The performance
characteristics (such as the sensitivity, specificity, likeli­
hood ratio and PPV) of HEp-2 IIF are usually reported
for a single cut-​off. However, such a dichotomous
approach overlooks the fact that the likelihood of dis­
ease increases with increasing antibody level. Therefore,
when interpreting the results of a HEp-2 IIF in clinical
practice, clinicians should consider the antibody titre
(including the relevant titre-​specific likelihood ratio), as
well as the fluorescence pattern, to avoid loss of essential
additional information.
Performance characteristics: SPA
Different SPAs, including ELISA, FEIAs and CIAs, are
increasingly being introduced in clinical laboratories
to screen for ANA-​associated rheumatic diseases. Line
blots and dot blots are typically not used to screen for
ANA-​associated rheumatic diseases and are instead usu­
ally used for the identification of specific autoantibodies.
By comparison, the high degree of automation of ALBIA
and PMAT makes these multiplexed assays more apt for
screening. In this section, we discuss the performance
characteristics of SPAs in screening for ANA-​associated
rheumatic diseases (as a group and for individual dis­
eases), how the screening SPAs differ from each other
and whether screening SPAs can replace HEp-2 IIF.
Supplementary Table 2 gives an overview of the studies
published after 2010 that have reported data on the
performance characteristics of SPA and HEp-2 IIF for
ANA-​associated rheumatic diseases14,15,24,41–48.
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Table 1 | autoantigens and hep-2 IIF patterns of aNas associated with various rheumatic diseases
antibody specificity associated disease hep-2 IIF nuclear staining hep-2 IIF cytoplasmic staining
Broad expression in ANA-​associated diseases
ssDNA No specific disease Frequently negative None
Histone Drug-​induced lupus, SLE, JIA and Homogeneous None
various other diseases
SSA/Ro60a Sjögren syndrome and SLE Fine speckled; can be missed None
Ro52 (TRIM21) Sjögren syndrome, SLE, IIM, SSc and Frequently negative None
various other diseases
U1RNP (70 K, A and C)a SLE and MCTD Large/coarse speckled None
cN1A Inclusion body myositis, Sjögren Undefined Undefined
syndrome and SLE
Systemic lupus erythematosus
Double-​stranded DNAa SLE Homogeneous None
Nucleosome or chromatin SLE Homogeneous None
Sm a
SLE Large/coarse speckled None
Ribosomal P SLE None Dense fine speckled; can be missed
PCNA SLE PCNA-​like (pleomorphic speckled) None
Systemic sclerosis
Topoisomerase Ia (Scl70) SSc Topo I-​like None
CENPBa SSc Centromere None
RNA polymerase III a
SSc Large/coarse speckled None
Fibrillarin SSc Clumpy nucleolar; perichromosomal None
staining in mitotic HEp-2 cells
Th/To SSc Homogeneous nucleolar None
Nor90 SSc Punctate nucleolar; staining in the nucleolar None
organizer regions during metaphase
U11/U12 RNP SSc Large/coarse speckled None
BICD2 SSc Nuclear None
Sjögren syndrome
SSB/La Sjögren syndrome Fine speckled None
Overlap syndromes
RuvBL1/2 SSc overlap with IIM Fine speckled None
PM/Scl SSc overlap with IIM Homogeneous nucleolar and weak fine None
speckled
Ku Overlap syndromes Fine speckled None
Idiopathic inflammatory myopathies
Jo1a Antisynthetase syndrome None Fine speckled; can be missed
PL7 , PL12, OJ, EJ, KS, Ha Antisynthetase syndrome None (Dense) fine speckled; can be
or Zo missed
Mi2 Dermatomyositis Fine speckled None
MDA5 Dermatomyositis None Fine speckled in a subset of cells;
can be missed
TIF1γ Dermatomyositis Fine speckled; can be missed None
NXP2 Dermatomyositis Multiple nuclear dots; can be missed None
SAE Dermatomyositis Fine speckled None
SRP Necrotizing myositis None Fine speckled; can be missed
HMGCR Necrotizing myositis None Staining (fine speckled) in only a
few cells; can be missed
Other
DFS70 Rare in ANA-​associated rheumatic Dense, fine speckled None
diseases
Immunofluorescence (IIF) assays with human epithelial type 2 cells (HEp-2 cells, HEp-2 IIF). Several of these autoantibodies are covered by numerous solid phase
assays from different brands. ANA, antinuclear antibody; IIM, inflammatory myopathies; JIA, juvenile idiopathic arthritis; MCTD, mixed connective tissue disease;
SLE, systemic lupus erythematosus; SSc, systemic sclerosis. aIncluded in classification criteria.

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Box 1 | likelihood ratios
The likelihood ratio is the fraction of patients with the disease who have a particular
test result divided by the fraction of individuals without the disease who also have this test extract had a high sensitivity (90%) (at least as high as
result. The positive likelihood ratio (that is, the likelihood ratio of a positive test result)
and the negative likelihood ratio (that is, the likelihood ratio of a negative test result) can
be calculated from the sensitivity and specificity of the test. For example, a test that
has a sensitivity of 80% (that is, 80% of the patients with the disease test positive) and porates only a mixture of separate antigens (thus no cel­
a specificity of 90% (that is, 10% of individuals without the disease test positive) has a
positive likelihood ratio of 8 (that is, 0.8 divided by 0.1). Hence, for this particular test, specificity (90.4%) than HEp-2 IIF48. Thus, it is important
a positive test result is 8 times more likely in a patient with the disease than in an
individual without the disease. Using the same example, the negative likelihood ratio is
the fraction of patients with the disease who test negative (that is, 20 out of 100 patients)
divided by the fraction of individuals without the disease who test negative (that is,
90 out of 100 individuals), which is 0.222 (that is, 0.2 divided by 0.9). Hence, for this
example, a negative test result is 4.5 times less likely in a patient with the disease than HEp-2 IIF is more sensitive than FEIA, CIA and ALBIA
in an individual without the disease. A high positive likelihood ratio (ideally >10) is best
for confirming a disease diagnosis, whereas a low negative likelihood ratio (ideally <0.1)
is best for excluding the disease.
In the example mentioned above, a single cut-​off point is used and the result is either
positive or negative. For many tests, however, more information is available than just For IIM, the sensitivity of FEIA, CIA, ALBIA and
a positive or negative result. For example, antinuclear antibody tests provide an antibody
titre; for each titre, a titre-​specific likelihood ratio can be calculated. If an antinuclear
antibody (ANA) titre of 1:1,280 is positive in 20% of patients with an ANA-​associated
rheumatic disease and only in 1% of individuals without a disease, then the likelihood
ratio associated with a titre of 1:1,280 would be 20 (that is, the fraction of patients with
the disease and with this specific test result (0.2) divided by the fraction of individuals
without disease and with this specific test result (0.01)). Similarly, if an ANA titre of 1:320 is
positive in 40% of patients with disease and in 4% of individuals without disease, then the
likelihood ratio associated with an ANA titre of 1:320 would be 10 (0.4 divided by 0.04).
For quantitative (continuous scale) results, test result interval-​specific likelihood
ratios can be estimated by dividing the fraction of patients with disease and with a test
result within the specified interval by the fraction of individuals without the disease
with a test result in the same interval 69. The test result-​specific likelihood ratio also
corresponds to the slope of the tangent on the receiver operating characteristics curve
at the point that corresponds to the specific test result69,70.
Determining the likelihood ratio is complex and requires sufficient well-​characterized
patients and controls and, therefore, it is not realistic to expect each individual labora-
tory to determine the likelihood ratios of the tests they are performing. Data for titre result-
specific and test result-​specific likelihood ratios can be obtained from the literature14–16,28
or can be deduced from published large meta-​regression data13. We encourage laboratory
immunologists, clinicians and in vitro diagnostic companies to cooperate (in multi-centre,
multi-​national initiatives) to establish test result-​specific likelihood ratios.
SPA screening for ANA-​associated rheumatic diseases.
Data from various studies have suggested that FEIA,
CIA and ALBIA are less sensitive but more specific for
screening for ANA-​associated rheumatic diseases than
HEp-2 IIF14,15,24,43–48 (Supplementary Table 2). In a meta-​
analysis of fully paired studies, the sensitivity of FEIA,
CIA and HEp-2 IIF (1:80 cut-​off) for ANA-​associated
rheumatic diseases was calculated to be 78.5%, 85.9% and
89.1–89.2%, respectively, whereas the specificity was cal­
culated to be 93.6%, 86.1% and 70.9–72.4%, respectively49.
In one study that directly compared the performance of
FEIA, CIA and HEp-2 IIF24, the overall performance, as
assessed by the area under receiver operator character­
istics curve, was similar for all three assays24. Thus, the
differences in performance between the assays largely
relate to the way the cut-​off points are chosen (by the
manufacturer). When the cut-​off was defined as the value
that corresponded to a specificity of ~95%, then the sen­
sitivities of HEp-2 IIF, FEIA and CIA for ANA-​associated
rheumatic diseases were comparable (79%, 82% and 78%
for HEp-2 IIF, FEIA and CIA, respectively)24. In terms
Nature Reviews | Rheumatology
Reviews
of ELISAs, one study that compared different ELISAs
found that the performance was dependent on the type of
ELISA used: ELISAs that incorporate a HEp-2 or HeLA
HEp-2 IIF (87.4%)) but a low specificity (45–60%) (lower
than HEp-2 IIF (72.3%)), whereas an ELISA that incor­
lular extract) had a lower sensitivity (76%) and higher
to know the type of ELISA used for ANA screening to
adequately interpret the test results.
SPA screening for individual ANA-​associated rheu-
matic diseases. Data from several studies suggest that
for screening for SSc and SLE but that these latter SPAs
are more sensitive than HEp-2 IIF for screening for
Sjögren syndrome14,15,41–47 (Supplementary Table 2).
HEp-2 IIF (depending on the study) is relatively low
(36–89.3% for studies that included >10 patients with
IIM)14,15,24,44,46–48 (Supplementary Table 2). These findings
could be explained by the fact that the screening SPAs
do not include all known IIM-specific antibodies and that
not all IIM-specific antibodies are adequately detected
by HEp-2 IIF (such as cytoplasmic Jo1). A syste­matic
review and meta-analysis that compared HEp-2 IIF
with FEIA reported a sensitivity of 94% and 79%, respec­
tively, for SSc, of 87% and 81%, respectively, for SLE,
of 63% and 50%, respectively, for IIM and of 80% and
88%, respectively, for Sjögren syndrome50. These data
should be viewed with caution given the small number
of patients included in the study50. The higher sensiti­
vity of HEp-2 IIF compared with SPA for SSc could be
because not all SSc-​specific antibodies are included in
the SPA screening assays.
Test result-​specific likelihood ratios. Researchers have
estimated test result-​specific likelihood ratios for ANA-​
associated rheumatic diseases for automated FEIA and
CIA28. Such information could help to define thresholds
for each assay that correspond to predefined likelihood
ratios28. By using such an approach, the interpretation of
ANA test results can be aligned across different assays
from different manufacturers.
Assay variability. Similar to the variation reported for
different HEp-2 IIF kits, different screening SPA kits
might also give varying results. For example, a meta-​
analysis found high variability in test performance
charac­teristics across studies for ELISAs and HEp-2 IIF
but not for FEIA49. The high variability of ELISA and low
variability of FEIA could be explained by the fact that
assays with diverse configurations (setups) are available
from different manufacturers for ELISA, whereas only
one fully automated assay (from a single manufacturer)
is available for FEIA.
Summary and future perspective. Taken together, the
performance characteristics of screening SPAs are both
assay-​dependent and disease-​dependent. Clinicians
should consider these aspects when interpreting the
Reviews
Box 2 | Clinical test indices: predictive values, probabilities and odds
Positive and negative predictive values
The positive predictive value (PPV) is the proportion of positive test results that are
true-positive results in a reference group out of all positive results, and provides a
measure of the probability of disease in an individual with a positive test result.
number of true‐positives
PPV =
(number of true‐positives + number of false‐positives)
The PPV is dependent on the prevalence of the disease, and the sensitivity and
specificity of a test. If, out of 100 individuals, 10 individuals have the disease of interest
(that is, a prevalence of 10%) and a particular test has a sensitivity of 80% and specificity
of 90%, then among these 100 individuals, 17 will have a positive test result (8 out of
10 individuals with the disease (true-​positives) and 9 out of 90 individuals without the
disease (false-​positives)). For this example, the PPV is 0.47 (that is, 8 divided by 17).
The negative predictive value (NPV) is the probability of not having the disease in a
patient with a negative test result.
Post-​test and pre-​test probabilities
The post-​test probability also provides a measure of the probability of disease in a
patient with a positive test result, and is similar to the PPV (and the two terms are
sometimes referred to synonymously), except that the post-​test probability refers to IIF test result together with a negative FEIA test result
a probability for a particular individual, rather than that established from a reference
group. The pre-​test probability is a measure of the probability of the target disease for a
particular individual before the test is performed, whereas the prevalence is established
from a reference group.
Post-​test and pre-​test odds
The post-​test and pre-​test odds are estimates of the odds that a patient has a target
disorder before and after the test result are known. The relationship between odds and
probability are summarized by the following equations:
probability
Odds =
(1 − probability)
odds
Probability =
(1+ odds)
Relation to the likelihood ratio
The post-​test odds of a disease can be estimated on the basis of the pre-​test odds and
the likelihood ratio of a test using the following equations:
Post‐test odds = pre‐test odds × likelihood ratio
Similarly, the post-​test probability (or positive predictive value, assuming that the two
are equal) can be estimated on the basis of the pre-​test probability (prevalence) and the
likelihood ratio, using a combination of the equations listed above.
ANA test results provided. Different commercial assays
have different compositions of autoantigens, and the
clinician should be aware of which autoantigens are
included in the SPA used. None of the assays is overall
superior to the other assays or to HEp-2 IIF, but rather
each assay has different strengths. Test result-​specific
likelihood ratios could help to improve and harmonize
the interpretation of test results.
Some relevant autoantibodies probably remain
uncharacterized at this time. Thus, even SPA screen­
ing assays that include a complete array of known
autoantibodies could fail to detect an uncharacterized
disease-​related autoantibody. HEp-2 IIF can potentially
pick up hitherto unidentified antibodies, which can be
clinically helpful and useful for research.
Value of combining ANA assay data
Data from HEp-2 IIF and SPAs can provide comple­
mentary information, and combining data from both
assay types could have diagnostic value. For example,
a systematic review of studies reporting on the diag­
nostic test accuracy of FEIA and/or HEp-2 IIF for
ANA-​associated rheumatic diseases showed that a
double-​positive test result (that is, positivity by both
assays) had a higher likelihood ratio for ANA-​associated
rheumatic diseases than a positive test result from just
one of the assays (26.2 for a double-​positive test result
versus 14.4 for positivity by FEIA alone or 5.1 for posi­
tivity by HEp-2 IIF alone) and a negative test result for
both assays had a lower likelihood ratio than a nega­
tive test result from a single assay (0.15 versus 0.21 for
negativity by HEp-2 IIF alone or 0.33 for negativity by
FEIA alone)50. Thus, combining results from a FEIA
and a HEp-2 IIF is more powerful than performing
either assay alone, as concordant positive or concordant
negative results can better rule in or rule out disease,
respectively. For discordant test results, a positive FEIA
test result together with a negative HEp-2 IIF test result
had a higher likelihood ratio than a positive HEp-2
(likelihood ratio 2.4 versus 1.4)50. Thus, samples that are
positive by FEIA but negative by HEp-2 IIF are more
likely to be from a patient with an ANA-​associated rheu­
matic disease than samples that are negative by FEIA but
positive by HEp-2 IIF. One study performed a similar
analysis of the performance of HEp-2 IIF with or with­
out CIA and found a similar outcome: double positi­vity
by both HEp-2 IIF and CIA had a higher likelihood
ratio (12.2) than positivity by HEp-2 IIF alone (2.4) or
by CIA alone (7.3)24. Finally, according to the results of
one study16, for each HEp-2 IIF antibody titre, the PPV
for ANA-​associated rheumatic diseases increases when
combined with a positive FEIA test result and decreases
when combined with a negative FEIA test result. This
result further supports the value of combining HEp-2
IIF with FEIA.
Combining information obtained from HEp-2 IIF
assays and FEIAs or CIAs has the added benefit that
either assay might detect antibodies that are undetected
by the other assay. HEp-2 IIF can detect some antibodies
at high titres in diagnostic samples of ANA-​associated
rheumatic diseases that are undetected by SPA14,51. Such
high-​titre antibodies are associated with a high speci­
ficity for ANA-​associated rheumatic diseases. Eight
percent of patients with an ANA-​associated rheumatic
disease have a negative test result by FEIA but a highly
positive test result by HEp-2 IIF (that is a higher titre
than the 95% specificity cut-​off value)51. By contrast,
patients with a weakly positive or negative test result by
HEp-2 IIF and a positive FEIA test result have a higher
likelihood ratio for ANA-​associated rheumatic diseases
(for example, Sjögren syndrome) than patients with a
weakly positive or negative test result by HEp-2 IIF and
a negative FEIA test result51. Double positivity for HEp-2
IIF and FEIA (which occurs in 71% of all patients with
an ANA-​associated rheumatic disease) has the highest
likelihood ratio for ANA-​associated rheumatic diseases51.
Taken together, considering information from both
the HEp-2 IIF and the FEIA or CIA can be beneficial
when interpreting ANA test results: the likelihood ratio
for ANA-​associated rheumatic diseases increases with
double positivity, and both types of assays can detect
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antibodies that are undetected or only weakly positive
by the other assay type51.
How to best interpret results
Which cut-​off to use?
Current cut-​off values. Applying a single cut-​off and
interpreting ANA test results in a dichotomous manner
provides an oversimplified picture and misses out on
information that is inherent in the antibody level. For
example, HEp-2 IIF is generally considered a sensitive
but unspecific assay. However, if the cut-​off value for
HEp-2 IIF is adapted, this assay can become highly spe­
cific. The treating physician should ensure that they are
aware of these relevant differences.
Value of titre-​specific information. We propose that
studies assessing the performance of ANA assays should
include test result-​specific likelihood ratios (for example,
titre-​specific or light intensity unit-​specific likelihood
ratios for HEp-2 IIF assays). Similarly, we encourage
assay developers to include test result-​specific likelihood
ratios so that this information is available to clinicians.
Reporting test result-​specific likelihood ratios can not
only improve clinical interpretation but can also help to
align test result interpretation between different assays
and methods28. Antibody levels that are below the anti­
body level that corresponds to a likelihood ratio of 0.1
are useful to exclude disease, whereas antibody levels
that exceed the antibody level that corresponds to a
likelihood ratio of 10 are useful to confirm the disease.
Antibody levels that correspond to a likelihood ratio of
1 indicate that the test result will be helpful in neither
excluding nor confirming the disease. The likelihood
ratio can also be used to estimate the post-​test probability
of disease based on the pre-​test probability (Box 2).
Value of the HEp-2 IIF pattern
In addition to the antibody titre, the antibody pattern
on HEp-2 IIF has further diagnostic value and should
also be considered during the diagnostic workflow.
A centromere pattern is highly associated with SSc5,8.
Clinicians should check whether the specific antibody
identified fits with the pattern observed on HEp-2 IIF.
Proposed testing approaches
The optimal test approach to ANA screening is disease-
dependent (Table 2)52. For SLE, an ANA titre of ≥1:80 on
HEp‐2 cells or an equivalent positive test at least once
is an entry criterion for the new disease classification
criteria3. Thus, unlike previous criteria, these classifi­
cation criteria enable the use of SPAs as alternatives to
HEp-2 IIF; however, the criteria did not define what
counts as ‘an equivalent positive test’. Logically, equi­
valence should be determined on the basis of the assay
sensitivity. Given that the ANA status can depend on the
assay used35,36, the optimal strategy might be to combine
the use of HEp-2 IIF and SPAs24,51–57. Such a strategy had
added diagnostic value55. Researchers have called atten­
tion to the fact that the ANA status of patients with SLE
might depend on the therapy, the stage of disease and/or
the natural history of the disease, the aspects of which
deserve further more detailed investigations55,56.
Nature Reviews | Rheumatology
Reviews
For Sjögren syndrome, FEIA and CIA are highly sen­
sitive and specific for disease and can be directly per­
formed (without the need for HEp-2 IIF). For SSc, HEp-2
IIF is a good screening assay, given its high sensitivity.
The centromere pattern is highly associated with SSc5,8,
and the nucleolar pattern is also associated with dis­
ease, albeit to a lesser extent. Other SSc-​associated anti­
bodies include antibodies to fibrillarin, PM/Scl, Th/To
or Nor90, which all produce a nucleolar pattern; how­
ever, none of the existing screening SPAs (FEIA and CIA)
is able to detect all these antibodies (see Supplementary
Table 1). Multiplexed line blot or dot blot assays that
detect all of the currently known SSc-​specific antibodies
exist, but the sensitivity and specificity for SSc of these
assays has not been fully documented.
For IIMs, both HEp-2 IIF and SPA screening assays
(FEIA and CIA) might not detect some IIM-​specific
antibodies. As the number of characterized target anti­
gens of IIM-​specific antibodies is continuing to grow,
more specific (multiplexed) testing for IIM-​specific
antibodies (for example, using dedicated dot blots or
line blots) is recommended for patients for whom there
is a high level of clinical suspicion of IIM58. The clin­
ical performance (such as the sensitivity, specificity
and likelihood ratio) of commercial line or dot blots
might differ59. For some line or dot multiplexed assays,
a weakly positive result might have a low specificity60,61.
Concordance between the HEp-2 IIF pattern and results
of the specific antibody testing can enhance the speci­
ficity of the testing overall62,63. Not all IIM-​specific anti­
bodies are included in the SPA screening assays. For
example, anti-​RuvBL1/2 antibodies, which typically give
a speckled nuclear pattern on HEp-2 IIF and are associ­
ated with SSc-​overlap with IIM64, are not detectable by
commercial assays. These autoantibodies can be reliably
detected by immunoprecipitation.
An important consideration is that other diseases
(including non-​rheumatic diseases) have ANA testing as
part of their diagnostic or classification criteria, includ­
ing autoimmune hepatitis65,66, primary biliary cholangitis
(PBC)67 and juvenile idiopathic arthritis68. For autoim­
mune hepatitis and juvenile idiopathic arthritis, ANA
by HEp-2 IIF remains the only option for ANA testing
as the target autoantigens are largely unknown and not
included in SPAs. In PBC, the ANA target autoanti­
gens include sp100 and gp210, which give a multiple
nuclear dot pattern and a nuclear envelope pattern,
respectively67. HEp-2 IIF can also reveal the presence of
anti-​mitochondrial antibodies in PBC, which produce a
reticular cytoplasmic staining pattern.
Conclusions
Overall, a variety of different methods are currently
available for ANA detection in the framework of
ANA-​associated rheumatic diseases. HEp-2 IIF assays
have been used by researchers and clinicians for several
decades and rely on the end point titration and visual
reading of the antibody patterns. However, the HEp-2
IIF has the disadvantage of variability between different
laboratories and a low specificity at low-​antibody titres.
Alternative SPAs that measure disease-​specific anti­
bodies have been introduced. Compared with HEp-2 IIF
Reviews

Table 2 | approach to aNa detection in aNa-​associated rheumatic diseases

Disease overall approach Staining pattern by hep-2 IIF Specificity of aNas
detected on SPas
Systemic lupus IIF has a high sensitivity, but low specificity. Combining IIF Nuclear homogeneous dsDNA
erythematosus with SPA adds value
Nucleosome or chromatin
Nuclear fine speckled (can be missed) SSA/Ro60
Nuclear large/coarse speckled Sm
U1RNP
Cytoplasmic dense fine speckled Ribosomal P
(can be missed)
PCNA-​like (pleomorphic speckled) PCNA
Sjögren IIF might miss some anti-​SSA/Ro60 antibodies. SPAs for Nuclear fine speckled SSA/Ro60
syndrome SSA/Ro60 (and SSB/La) have a good sensitivity and can
be directly performed without the need for a HEp-2 IIF SSB/La
screening test
Systemic sclerosis HEp-2 IIF is a good screening assay and has a high Centromere CENPB
sensitivity. SPAs can detect the majority of SSc-​specific
antibodies, but not all. Topo I-​like Topoisomerase I (Scl70)

It is important to check whether the specific antibody Nuclear speckled RNA polymerase III
identified by SPA fits with the IIF pattern U11/U12 RNP
Ku (overlap)
RuvBL1/2 (overlap IIM)a
Nucleolar (clumpy, homogeneous or Fibrillarin
punctate)
Th/To
PM/Scl (overlap)
Nor90
Poorly defined (speckled) BICD2
Idiopathic HEp-2 IIF has a limited sensitivity. Employ SPAs in cases Cytoplasmic (dense) fine speckled Various amino-​acyl transfer
inflammatory where there is a high level of clinical suspicion. Cautious (can be missed) RNA synthetases: Jo1, PL7 ,
myopathies interpretation of weakly positive results on SPAs is PL12, OJ, EJ, KS, Ha or Zo
necessary owing to the possibility of weakly false-​positive
results with some assays. The available assays do not SRP
cover the full antibody spectrum. It is important to check Cytoplasmic (fine speckled, in a HMGCR
whether the specific antibody identified by SPA fits with subset of cells) (can be missed)
IIF pattern MDA5
Nuclear, fine speckled Mi2
SAE
TIF1γ
Ku (overlap)
RuvBL1/2 (overlap with SSc)a
Nuclear multiple dots (can be missed) NXP2
Mixed connective HEp-2 IIF is a good screening assay. High levels of antibodies Nuclear large/coarse speckled U1RNP
tissue disease to U1RNP (detected by SPA) is suggestive of MCTD
ANA, antinuclear antibody; IIF, immunofluorescence; MCTD, mixed connective tissue disease; PCNA, proliferating cell nuclear antigen;SPA, solid phase assay;
SRP, signal recognition peptide; SSc, systemic sclerosis. aDetected by immunoprecipitation (a technique for autoantibody detection based on immunoprecipitation
of the autoantigen from a radiolabelled cell extract). The technique is used in only a few (research) laboratories.

assays, the performance of fully automated systems such
as FEIA (and probably CIA) is more consistent between
laboratories49,50. In terms of clinical performance, neither
the HEp-2 IIF nor the SPAs (such as FEIA or CIA) are
superior to each other. A test result with a high sensitiv­
ity, such as when using a HEp-2 IIF with a 1:80 cut-​off
titre, is useful for excluding the presence of disease,
whereas a test result with a high specificity (for exam­
ple, when using a FEIA or HEp-2 IIF with a high cut-​off
or threshold) is useful for confirming the presence of
disease. Clinicians should be aware of the clinically
relevant differences in performance between HEp-2
IIF assays and various SPAs. The differences among the
assays should also be kept in mind when the tests are
used as an entry criterion for classification, in which
case a test with a high sensitivity for detecting disease
is needed.
The performance of HEp-2 IIF assays and SPAs
(including FEIA and CIA) is disease-​dependent. SPA is
superior to HEp-2 IIF for Sjögren syndrome, and HEp-2
IIF is more sensitive than SPAs for SSc and SLE. For IIM,
the use of neither a HEp-2 IIF nor a SPA is optimal.

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 Reviews

For patients for whom there is a high level of clinical
suspicion of IIM, the presence of IIM-​specific anti­
bodies should be determined by use of specific assays
(for example, line or dot blot assay). Thus, the optimal
test strategy depends on the disease of interest. Neither
HEp-2 IIF assays nor SPAs (that is, FEIAs or CIAs) alone
can identify all patients with an ANA-​associated rheu­
matic disease, and using a combination of data from
both types of assays provides the most clinically valuable
information. Double positivity for both types of assays
has the highest likelihood ratio for disease. HEp-2 IIF
might detect relevant antibodies that are undetected
by SPAs, whereas SPAs can detect antibodies that are
undetected or weakly positive by HEp-2 IIF (such as
anti-​SSA/Ro or anti-​Jo1 antibodies). No single cut-​off
value for HEp-2 IIF assays or SPAs (including FEIA and
CIAs) is associated with both good sensitivity and good
specificity. Ideally, a screening assay should be followed
by an antibody-​specific assay. However, each laboratory
should define the test strategy that best addresses the
local situation.
Finally, and notably, interpreting ANA test results
in a dichotomous manner loses important informa­
tion, which can be overcome by instead considering
test result-​specific likelihood ratios. We propose that
test result-​specific likelihood ratios should be reported
whenever possible. Such an approach should not only
enable better interpretation of test results but should
also help to harmonize test results across different assays
from different manufacturers. This approach, however,
will require a change in the mindset among clinicians,
laboratory professionals and the diagnostic industry.
Published online xx xx xxxx

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Author contributions
X.B. researched data for the article and wrote the article.
P.L.M. and M.O.B. made substantial contributions to dis­
cussion of the content. E.D.L., M.O.B. and P.L.M. reviewed
and edited the manuscript before submission
Competing interests
X.B. has been a consultant for Inova Diagnostics and
Thermo-​F isher and has received lecture fees from Inova
Diagnostics, Menarini and Thermo Fisher. P.L.M. has been a
consultant for Inova Diagnostics and Thermo-​Fisher and has
received lecture fees from Inova Diagnostics, and Thermo
Fisher. E.D.L. and M.O.B. declare no competing interests.
Peer review information
Nature Reviews Rheumatology thanks L. Andrade,
J. Damoiseaux and the other, anonymous, reviewer(s) for
their contribution to the peer review of this work.
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Supplementary information
Supplementary information is available for this paper at
https://doi.org/10.1038/s41584-020-00522-​w.
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