1. 494 | AUGUST 2014 | VOLUME 10 www.nature.com/nrrheum
Department of
Rheumatology,
Klinikum Bad
Bramstedt University
Hospital of Schleswig-
Holstein, Oskar-
Alexanderstrasse 26,
24576 Bad Bramstedt,
Germany (E.C., F.M.).
Correspondence to:
E.C.
csernok@klinikumbb.de
Current and emerging techniques for ANCA
detection in vasculitis
Elena Csernok and Frank Moosig
Abstract | Detection of antineutrophil cytoplasmic antibodies (ANCAs) is a well-established diagnostic test used
to evaluate suspected necrotizing vasculitis of small blood vessels. Conditions associated with these antibodies,
collectively referred to as ANCA-associated vasculitides, include granulomatosis with polyangiitis (formerly known
as Wegener granulomatosis), microscopic polyangiitis, and eosinophilic granulomatosis with polyangiitis (formerly
known as Churg–Strauss syndrome). The diagnostic utility of ANCA testing depends on the type of assay
performed and on the clinical setting. Most laboratories worldwide use standard indirect immunofluorescence
tests (IFT) to screen for ANCA and then confirm positive IFT results with antigen-specific tests for proteinase 3
(PR3) and myeloperoxidase (MPO). Developments such as automated image analysis of immunofluorescence
patterns, so-called third-generation PR3-ANCA and MPO-ANCA ELISA, and multiplex technology have improved the
detection of ANCAs. However, challenges in routine clinical practice remain, including methodological aspects of
IFT performance, the diverse antigen-specific assays available, the diagnostic value of testing in clinical settings
and the prognostic value of serial ANCA monitoring in the prediction of disease relapse. This Review summarizes
the available data on ANCA testing, discusses the usefulness of the various ANCA assays and advises on the
clinical indications for the use of ANCA testing.
Csernok, E. Moosig, F. Nat. Rev. Rheumatol. 10, 494–501 (2014); published online 3 June 2014; doi:10.1038/nrrheum.2014.78
Introduction
Since the 1980s, the presence of antineutrophil cyto­
plasmic antibodies (ANCAs) has proven to be a valuable
serological marker that aids the diagnosis of small-vessel
vasculitis. The principle association with ANCAs origi­
nally defined the group of ANCA-associated vasculitides,
comprising granulomatosis with polyangiitis (GPA, for­
merly known as Wegener granulomatosis), microscopic
poly­angiitis (MPA) and eosinophilic granulomatosis
with polyangiitis (EGPA, formerly known as Churg–
Strauss syndrome), which have different frequencies of
ANCA-positivity.1–4
However, the spectrum of dis­eases
asso­ciated with ANCAs has since broadened to include a
range of other inflammatory and infectious dis­eases, call­
ing into question the diagnostic implications of ANCA
positivity (reviewed elsewhere5–7
). Furthermore, the appli­
cation of ANCA testing as a clinical tool is still con­sidered
controversial,8
with issues around methodo­logical aspects
(such as assay performance and testing strategies), when to
order ANCA testing (clinical ‘gating policy’), and the value
and therapeutic implications of sequential ANCA testing.
With respect to the last point, for instance, the reported
prevalence of ANCAs in GPA varies widely, from 50%
to 95% depending on disease stage, disease activity and
therapy at the time of sampling, and the ANCA detection
method used. Inconsistencies in ANCA detection fre­
quently cause incorrect applications and interpretations
in daily clinical practice.8
Despite these controversies, ANCA testing is widely
used by clinicians when considering a diagnosis of ANCA-
associated vasculitis (AAV). As indicated in the consensus
guidelines for ANCA testing and reporting document, and
according to the practice of most laboratories worldwide,
screening for vasculitis-associated ANCAs is done by
indirect immuno­fluorescence technique (IFT).9,10
Three
main patterns of fluorescence have been demonstrated
with IFT on e­thanol-fixed neutrophils (Figure 1). The
first, C‑ANCA, is a diffuse, cytoplasmic granular fluo­
rescence most prominent centrally, between the nuclear
lobes, and is noted overall in 90% of patients with active,
generalized GPA.11
The second, P‑ANCA, is a perinuclear
neutrophil staining pattern often with nuclear extension,
noted in those with MPA and EGPA. The third, so-called
atypical pattern, referred to as A‑ANCA, is rare and com­
bines both cytoplasmic and perinuclear or nuclear stain­
ing, with multiple antigen specificities; A‑ANCA can
occur in association with drug exposure, inflammatory
bowel disease or rheumatoid arthritis, most often in the
absence of vasculitis. The perinuclear or nuclear staining
pattern of P‑ANCA (or A‑ANCA) is actually an artefact
of ethanol fixation, as neutrophils show a cytoplasmic
staining pattern after formalin fixation. The consensus
guidelines require all sera to be examined by IFT on
et­hanol-fixed neutrophils.9,10
However, it is technically
difficult to differentiate P‑ANCA (or A‑ANCA) patterns
from the staining shown by antinuclear antibodies (ANA)
on this type of fixation. Several laboratories incorporate
a protocol in which formalin-fixed neutrophils are used,
Competing interests
The authors declare no competing interests.
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2. NATURE REVIEWS | RHEUMATOLOGY VOLUME 10 | AUGUST 2014 | 495
but the value of formalin fixation of neutrophils in differ­
entiating antibodies is controversial. Comparing the IFT
patterns of sera tested on ethanol-fixed and formalin-fixed
neutrophil preparations, our group demonstrated that for­
malin fixation does adequately differentiate between ANA
and P‑ANCA (or A‑ANCA) staining, and can been used
in the routine diagnostic laboratory.12
The use of differ­
ent fixation methods (ethanol and formalin) thus enables
differentiation between P‑ANCA (or A‑ANCA) and the
presence of ANA.
ThemajortargetantigensrecognizedbyANCAsinAAV
are proteinase 3 (PR3) and myeloperoxidase (MPO). PR3-
ANCA usually cause a C‑ANCA pattern and are mainly
associated with GPA. MPO-ANCA is associated with a
P‑ANCA pattern and is predominantly seen in patients
with MPA. Positive immuno­fluorescence results should
be followed by antigen-specific tests for PR3-ANCA and
MPO-ANCA.9
The tests most frequently used to identify
ANCAs with different specificities are ‘direct’ and ‘capture’
ELISA. The combination of both IFT and antigen-specific
assays (PR3-ANCA and MPO-ANCA) has been con­
firmed in several studies to be the best strategy for ANCA-
detection in vasculitis.11,13
In a specialized laboratory,
ANCA detection uses both methods (IFT and ELISA),
and contradictory results can be further analysed by other
methods, by using other cell substrates, and by investigat­
ing other target antigens (elastase, cathepsin G, bactericidal
permeability-­increasing protein and so on). It is important
to emphasize that the extensive ANCA-testing approach
used in these laboratories is rarely used in clinical practice,
where IFT with or without direct ELISA is routine.
A variety of different immunoassays for the detec­
tion of ANCAs have been developed in the past 10 years,
inclu­ding third-generation ELISA, dot blots and bead-
based multiplex assays, along with automated systems
for analy­sing ANCA fluorescence patterns. This Review
sum­marizes the most clinically relevant developments in
methodology and provides an overview on how ANCA
testing in vasculitis could be improved and simplified by
applying these techniques.
Automated analysis of ANCA patterns
Despite the emergence of alternative screening methods,
IFT remains the recommended method for ANCA screen­
ing in vasculitis. However, the reliability of this assay
depends on a number of factors, including the substrate,
conjugates and fixation methods used, the source of the
cells, storage conditions, and procedures for incubation
and washing.7
Furthermore, the assay is subjective, time
consuming, requires expertise and is labour intensive. To
address the limitations of conventional IFT, computer-
based image analysis of indirect immuno­fluorescence
(IIF) patterns has been applied to the analysis of ANCA
in neutrophil-based assays. Standardized, automated
analysis of IFT for ANCA detection provides several
advantages, including reduced consumption of samples
and reagents, shortened analysis time and less extensive
handling of samples. These systems, several of which are
commercially available, generally use digital acquisition
and computer-aided analysis of IIF images using pattern
recognition algorithms. These systems might distinguish
the presence or absence of ANCAs, or might also be able
to identify particular staining patterns.
One automated system used in ANCA detection was
first developed for ANA detection on human epithelial
type 2 (HEp2) cells. In contrast to HEp2 cells, neutro­
phils are characterized by nuclei of varying shapes, and
algorithms for the identification of staining patterns in
neutrophils are more complex than those for HEp2 cells.
For instance, signal assessment and pattern classifica­
tion cannot be performed inside or outside of the DAPI-
stained (that is, nuclear) area, as described for ANA
pattern detection. Furthermore, C‑ANCA and P‑ANCA
staining patterns are detected in the border regions of the
neutrophil nucleus, rendering the detection of ANCAs a
challenge for automation.14
Several studies have evaluated the usefulness of auto­
mated IIF systems for the objective interpretation of
ANCA patterns. In a 2003 study, Boomsma et al.15
first
Key points
■■ Screening for antineutrophil cytoplasmic antibodies (ANCAs) specific for
proteinase 3 (PR3) and myeloperoxidase (MPO) remains a basic tool in the
serological evaluation of systemic vasculitic disorders
■■ Automated analysis of ANCA immunofluorescence demonstrates good
agreement with conventional techniques when assessing patients with
suspected vasculitis and, in the future, might improve efficiency and
standardization in clinical laboratories
■■ Antigen-specific ANCA assays that use bridging molecules exhibit superior
performance compared with conventional assays and can be used for PR3-ANCA
and MPO-ANCA detection
■■ The new methods for ANCA detection and evaluation in ANCA-associated
vasculitis should be urgently evaluated in multicentre studies, in anticipation
of updating of standardization processes and a revision of existing strategies
a b
Figure 1 | ANCA IIF pattern differentiation by automated signal intensity analysis.
IIF images, taken automatically by AKLIDES®, can discriminate a | P‑ANCA-specific
and b | C‑ANCA-specific staining of neutrophils by use of mathematical algorithms
for pattern differentiation. Chromatin is stained by DAPI (blue) and specific ANCA
interactions are revealed by FITC (green)-labelled secondary anti-human IgG. DAPI
and FITC fluorescence intensity signals of respective images were combined and
illustrated in three dimensions (x, object length; y, object width; z, light intensity of
fluorescence signal). Abbreviations: ANCA, antineutrophil cytoplasmic antibody;
A‑ANCA, atypical ANCA staining pattern; C‑ANCA, cytoplasmic ANCA staining pattern;
DAPI, 4',6-diamidino‑2-phenylindole; FITC, fluorescein isothiocyanate; IIF, indirect
immunofluorescence; P‑ANCA, perinuclear ANCA staining pattern. Reproduced
from Knütter et al. Arthritis Res. Ther. 14, R271 (2012),17
which is published
under an open-access licence (http://creativecommons.org/licenses/by/2.0)
by BioMed Central Ltd.
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3. 496 | AUGUST 2014 | VOLUME 10 www.nature.com/nrrheum
compared a novel quantitative image analysis technique
with conventional IFT and various ELISAs for monitor­
ing ANCAs in a prospective cohort of 16 patients with
PR3-ANCA-positive GPA. With respect to the detection
of ANCA, the automated image analysis technique had
a lower diagnostic sensitivity (75%) than conventional
IFT (100%) or capture ELISA (100%). In determining
the ability of ANCA levels to predict disease relapse,
they found that increases in ANCA titres determined by
automated image analysis were followed by relapse in the
majority of patients with GPA (69%), a positive predic­
tive value somewhat better than that of conventional IFT
(56%) and comparable to that of direct ELISA (61–71%).
Furthermore, the quantification of ANCA titres by image
analysis was no different than by IFT, direct ELISA and
capture ELISA.15
However, the image analysis method
used in this study did not use different neutrophil-­
fixation methods, but rather used the recommended
method of ethanol fixation.
Three studies published in 2012 evaluated the
AKLIDES®(Medipan GmbH, Germany) automated
IIF analysis system,16–18
which is able to quantify fluo­
rescence intensity and interpret basic ANCA staining
patterns on combined ethanol-fixed and formalin-fixed
human neutrophils. Melegari et al.16
compared the diag­
nostic performance of AKLIDES®with that of both
manual interpretation of IIF by laboratory experts and
confirmatory tests (ANCA-specific ELISAs). The agree­
ment between traditional visual reading and AKLIDES®
of ANCA testing by IIF was 89.1%. However, this study
involved only a small number of samples (n = 46), lacked
any clinical information about the samples and reported
only positive–negative discrimination between ANCA
screening results.
Knütter et al.17
developed interpretation software for the
AKLIDES®system using pattern-recognition algorithms
that enabled positive–negative discrimination and clas­
sification of C‑ANCA and P‑ANCA. In a study of sera
from 342 patients with AAV and other rheumatic and
infectious diseases, and from healthy individuals, they
compared these algorithms and conventional fluorescence
microscopy in the analysis of ANCA patterns on ethanol-
fixed and formalin-fixed neutrophils (Figure 2). No sig­
nificant difference was found between the performance
of the visual and automated methods of ANCA detection
in patients with AAV and other rheumatic and infectious
diseases.17
The two methods showed strong agreement
in the positive–negative classification of samples and in
the interpretation of ANCA patterns on ethanol-fixed
and formalin-fixed neutrophils, confirming the useful­
ness of automated pattern-recognition algorithms for
the assessment of ANCA IIF patterns. The main differ­
ences between these two methods for the interpretation
of IIF patterns on ethanol-fixed neutrophils were found
for visual findings regarding the cytoplasmic and atypical
patterns. The automated reading defined 22 (25.0%) of 88
visually determined C‑ANCA patterns as negative results.
Furthermore, 10 (18.2%) of 55 A‑ANCA patterns deter­
mined visually were defined as nuclear patterns using the
automated pattern-­recognition algorithms.17
To further clarify the diagnostic value of the AKLIDES®
ANCA pattern-recognition system on et­hanol-fixed and
formalin-fixed neutrophils, Damoiseaux et al.18
used
sera from patients with AAV (n = 79) as well as distinct
cohorts of relevant control sera (n = 117). Five series of
control samples were used to examine the interference
with other autoantibodies: ANA-positive sera with dif­
ferent fluorescence patterns; antimitochondrial antibody-
positive samples; A‑ANCA positive sera; C‑ANCA with
BPI antigen specificities; and sera from healthy indi­
viduals. The data obtained in sera from patients with
PR3-ANCA-positive AAV revealed that the automated
system lacked sufficient sensitivity (74% compared with
92% for routine micro­scopy) on ethanol-fixed neutro­
phils, but the expected C‑ANCA pattern was very well
recognized on formalin-fixed neutrophils (95% and
97% recognized by the automated system and routine
microscopy, respectively). For MPO-ANCA positive
sera, the expected P‑ANCA pattern was best recognized
on ethanol-fixed neutrophils, whereas recognition of
C‑ANCA on fo­rmalin-fixed neutrophils was poor. Most
interference with ANCA pattern recognition occurred in
control sera with a homogeneous ANA pattern. In con­
trast to visual scoring, the AKLIDES®system could not
differentiate between P‑ANCA and the presence of ANA
in most of the samples.18
Furthermore, the sensitivity of
the AKLIDES®system for positive–negative discrimina­
tion was 94%, compared with 96% for visual scoring. This
study was not appropriate to calculate sensitivity or speci­
ficity for AAV, but at least showed that the sensitivity of
the automatic reading system might equal that of visual
scoring.18
Moreover, the immunofluorescence pattern of
ANCA predicts the ANCA-antigen specificity, and for
this purpose the reactivity on ethanol-fixed neutrophils
ethN
formN
Cytoplasmic Perinuclear Nuclear Atypical Atypical Atypical
Cytoplasmic Cytoplasmic Negative Cytoplasmic Negative Negative
10μm
Figure 2 | Characterization of neutrophils by automated pattern-recognition image
analysis. IIF images, taken automatically by AKLIDES®, of serum samples
demonstrate C‑ANCA, P‑ANCA and A‑ANCA patterns on ethanol-fixed and formalin-
fixed human neutrophils. Chromatin is stained by DAPI (blue) and specific ANCA
interactions are revealed by FITC (green)-labelled secondary anti-human IgG.
Abbreviations: ANCA, antineutrophil cytoplasmic antibody; A‑ANCA, atypical ANCA
staining pattern; C‑ANCA, cytoplasmic ANCA staining pattern; DAPI,
4’,6-diamidino‑2-phenylindole; ethN, ethanol-fixed neutrophils; FITC, fluorescein
isothiocyanate; formN, formalin-fixed neutrophils; IIF, indirect immunofluorescence;
P‑ANCA, perinuclear ANCA staining pattern. Reproduced from Knütter et al. Arthritis
Res. Ther. 14, R271 (2012),17
which is published under an open-access licence
(http://creativecommons.org/licenses/by/2.0) by BioMed Central Ltd.
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4. NATURE REVIEWS | RHEUMATOLOGY VOLUME 10 | AUGUST 2014 | 497
is the determining factor. In this respect, the sensitivity of
the automated system on ethanol-fixed neutrophils needs
to be optimized. Furthermore, data concerning the value
of image analysis for monitoring ANCA levels are lacking.
Altogether, studies to date show that automated reading
of IIF patterns is a promising technique for ANCA screen­
ing, but further improvement and evaluation in multi­
centre studies is required before it will be applicable in
routine clinical praxis.
Novel antigen-specific immunoassays
ELISA
Any new strategy for ANCA evaluation in vasculitis must
aim to identify the target antigens PR3 and MPO, because
the presence of PR3-ANCA or MPO-ANCA correlates
well with clinical, histological and genetic features.19–22
‘Direct’ ELISA, developed in the late 1990s, uses PR3 or
MPO antigens directly immobilized to the surface of the
ELISA plate (Figure 3); however, this assay shows con­
siderable variation in performance and often lacks sen­
sitivity.23
This so-called first-generation ANCA ELISA
has proven to be less sensitive than second-generation
ELISAs,23
which uses secondary antibodies specific to PR3
or MPO that have been attached to the plate to ‘capture’
ANCA (Figure 3). The capture ELISA technique reduces
the possible covering of epitopes by the plastic plate (as
can occur with direct ELISA) and demonstrates overall
superiority to direct ELISA in diagnostic performance.24–26
However, the capturing antibody might reduce the sensi­
tivity of capture ELISA by masking epitopes of the antigen
relevant to ANCA. Interestingly, PR3-ANCA detected by
capture ELISA correlates better with disease activity in
patients with AAV than PR3-ANCA detected by conven­
tional ELISA.27
As a consequence, many commercially
available capture ELISAs are in widespread clinical use.26,27
Third-generationELISA—‘anchor’ELISA—­immobilizes
antigen to the plastic plate via a bridging molecule
(Figure 3), preserving epitopes for binding; of note, almost
all of the available anchor assays are PR3-ANCA ELISA.
The anchor assay has demonstrated superiority to direct
ELISA and some capture ELISA assays.28
Third-generation
ANCA ELISA is now available (sometimes referred to by
commercial companies as ‘high-­sensitivity’ ELISA) and its
evaluation is ongoing28–33
(Table 1).
In a comparative study published in 2012, the diagnos­
tic performance of 11 commercially available PR3-ANCA
and MPO-ANCA ELISA assays—notably including direct,
capture and anchor ELISA systems—were ana­lysed for
their ability to distinguish sera from individuals with
AAV.32
All the assays evaluated were highly sensitive and
specific for GPA and MPA, and were able to differentiate
active AAV from other diseases (systemic lupus erythema­
tosus and rheumatoid arthritis). Maximum sensitivity for
PR3-ANCA in sera from patients with GPA was obtained
with the combination of IFT and either capture or anchor
ELISA, and for MPO-ANCA in sera from patients with
MPA with the combination of IFT and either capture or
direct ELISA (Table 1). In a second comparative study,
published in 2013, Radice et al.33
investigated the diag­
nostic performance of nine commercially available PR3-
ANCA kits for the diagnosis of GPA (Table 1). Two of the
assays were direct PR3-ANCA ELISA and the remaining
seven represented some of the tests developed in order
to improve the performance of the antigen-specific tests
(such as capture and anchor PR3-ANCA ELISA, chemi­
luminiscence immuno­assay, and fluorescence enzyme
immuno­assay).Thisstudyshowedthatthediagnosticvalue
generally seems to be improved with anchor and cap­ture
ELISA, in comparison with direct ELISA. Nota­bly, most of
the anchor and capture assays improved the positive pre­
dictive value for diagnosis of GPA, and most enabled better
discrimination between positive and nega­tive samples
owing to the reduction of borderline test results.33
Altogether, the antigen-specific anchor and capture
ANCA assays exhibited superior performance and could
be used for PR3-ANCA and MPO-ANCA detection. As a
consequence of the high sensitivity and specificity of these
assays, a revision of the current algorithm (Figure 4) for
ANCA detection is warranted. An alternative strategy has
been proposed that uses such novel sensitive tests as the
first-line screening assay, with confirmation of positive
results by IFT—this approach is practically an inversion of
the current sequence.34
However, although published data
support the applica­bility of these antigen-specific tests for
initial screening,13,34
further studies in large cohorts of
patients with inflammatory diseases are necessary before
the ANCA consensus statement should be revised. An
ongoing multi­centre international study is focused on this
goal. Over the next year, we expect to establish a more
efficient test algorithm that provides superior sensitivity
and specificity for the diagnosis of AAV.
Other immunoassays
Otherimmunoassaysforantigen-specificANCAdetection
include dot-blot assays and bead-based multiplex testing,
discussed briefly here, and other methods such as fluo­
rescence enzyme immunoassays and chemi­luminescence
technology that are beyond the scope of this Review.
Dot blots are a variant of ELISA that use an antigen-
coated nitrocellulose strip. This assay is not quantitative,
but it is especially suitable for examination of a single
serum in case of emergency because it is a single-strip
preparation.35
A biochip technology (EUROPLUS®,
EUROIMMUNAG,Germany)hasalsobeendevelopedfor
Specific
antibodies
in patient
serum
PR3
Enzyme-conjugated
anti-human IgG
Bridging
molecule
Monoclonal
antibody
Direct ELISA Anchor ELISACapture ELISA
Figure 3 | Overview of ELISA procedures for ANCA detection. Using the example
of antigen-specific tests for PR3, direct ELISA immobilizes the antigen to the solid
phase, whereas in capture and anchor ELISA the antigen is bound to a bridging
molecule (in the case of capture ELISA, an antibody) attached to the solid phase.
Abbreviations: ANCA, antineutrophil cytoplasmic antibodies; PR3, proteinase 3.
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5. 498 | AUGUST 2014 | VOLUME 10 www.nature.com/nrrheum
rapid ANCA testing.35
In this assay, IFT is com­bined with a
dot-blot test for PR3-ANCA and MPO-ANCA. However,
the results obtained are only qualitative and should be
confirm­ed and quantified by other ANCA assays.
Bead-based multiplex assay is a variant of the solid-
phase assay in which capture antigens are bound to
colour-coded beads in suspension that are then ana­
lysed using flow cytometry. The main advantage of this
method is the ability to simultaneously detect multiple
autoantibodies relevant to vasculitis (ANCAs and anti-
glomerular basement membrane [GBM] antibodies) in a
small serum sample. The potential disadvantages are the
same as with direct ELISA; that is, the antigen-binding
potential of the PR3 and MPO and its potential loss after
the coating process. A study by Trevisin and colleagues36
in sera from individuals with active and treated vasculitis
and inflammatory bowel disease found a flow cyto­metric
immunoassay for PR3-ANCA and MPO-ANCA had a
specificity of 88%, compared with 96% and 94% for IFT
and ELISA, respectively. The flow cytometric assay was
almost as sensi­tive as IFT, and more sensitive than, but just
as specific as, most of the 12 commercial ELISAs tested in
detecting ANCAs in sera from patients with vasculitis.36
However, the utility of this method for initial screening of
patients with suspected vasculitis needs to be evaluated in
prospective studies.
Clinical usefulness of ANCA testing
At present, ANCA testing is widely used by clinicians
when AAV is suspected. Accurate identification of all
patients with active AAV and the avoidance of mis­diag­
nosis are best achieved by use of IFT combined with an
antigen-specific assay.11
Using this strategy, PR3-ANCA
or MPO-ANCA are detectable in nearly all patients with
active, generalized GPA or MPA, but only in approxi­
mately 60% of patients with limited (“initial phase”)
dis­ease.32,37
In cases of emergency, such as the pu­lmonary-
renal syndrome (the differential diagnosis of which should
include testing for anti-GBM antibodies, which are
speci­fic for Goodpasture syndrome), several rapid screen­
ing assays for ANCA (dot blots, biochip technology) are
available.35,38
If the results are clearly posi­tive, for example,
high-titre ANCA on IFT and proof of a defined speci­ficity,
immunosuppressive therapy should be started imme­
diately. However, patients with ANCA should receive
a careful workup because the presence of ANCA can
indicate conditions other than vasculitis, such as infec­
tions or exposure to drugs, which might be worsened
Table 1 | Comparison of methods for testing for PR3-ANCA and MPO-ANCA in ANCA-associated vasculitis
Patient population
(n) vs comparison
group (n)
Method Sensitivity
(%)
Specificity
(%)
PPV
(%)
NPV
(%)
AUC/ROC Comments
GPA (86) vs
non-vasculitic
disease (450)28
IFT
Direct PR3-ANCA ELISA
Capture ELISA
Anchor ELISA
92
60
72
96
99
99
99.3
98.5
Nd Nd 0.96 (0.94–0.98)
0.80 (0.76–0.83)
0.86 (0.82–0.89)
0.96 (0.94–0.98)
Histological
diagnosis
Retrospective
study
GPA (232) vs
inflammatory
diseases (661)29
IFT
Anchor ELISA
77.9
80.4
90.9
97.4
73
88
93
93
Nd Histological
diagnosis
Prospective
study
GPA (59*) vs
inflammatory and
infectious diseases
(585)30
Hn–hr PR3-ANCA ELISA
Capture ELISA
Direct (hn) PR3-ANCA
94
66
64
99
(predefined)
Nd Nd Nd Histological
diagnosis
Retrospective
study
GPA (34) vs SLE
(65)31
Direct PR3-ANCA
Anchor ELISA
97.1 98.4 Nd Nd 0.999
(0.947–1.00)
Clinical
diagnosis
Retrospective
study
GPA (40) vs RA
or SLE (20)32
IFT
Direct PR3-ANCA (n = 5 kits)
Capture ELISA (n = 2 kits)
Anchor ELISA (n = 4 kits)
62.5
45–55
60–62.5
60–62.5
95–100 Nd Nd Nd Histological
diagnosis
Retrospective
study
MPA (40) vs RA
or SLE (20)32
IFT
Direct MPO-ANCA ELISA
(n = 8 kits)
Capture ELISA (n = 2 kits)
Anchor ELISA (n = 1 kit)
82.5
62.5–85
80
75
95–100 Nd Nd Nd Histological
diagnosis
Retrospective
study
GPA (55) vs
suspected
vasculitis (175)33
IFT
Direct PR3-ANCA ELISA
(n = 2 kits)
Capture ELISA (n = 2 kits)
Anchor ELISA (n = 3 kits)
Other assays (n = 2)
69.1
61.8–72.7
70.9–72.7
61.8–72.7
72.7–74.5
100
95.4–96.4
95.9–99.5
98.5–99.0
95.9–97.9
Nd Nd Nd
0.856–0.879
0.862–0.878
0.833–0.881
0.878–0.902
Clinical
diagnosis
Retrospective
study
*47 of 59 patients in the GPA group had a cytoplasmic ANCA pattern on IFT. Abbreviations: ANCA, antineutrophil cytoplasmic antibody; AUC, area under the
curve; GPA, granulomatosis with polyangiitis; hn, human native; hr, human recombinant; IFT, indirect immunofluorescence technique; MPA, microscopic
polyangiitis; MPO, myeloperoxidase; Nd, not determined; NPV, negative predictive value; PPV, positive predictive value; PR3, proteinase 3; RA, rheumatoid
arthritis; ROC, receiver operating characteristics; SLE, systemic lupus erythematosus.
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6. NATURE REVIEWS | RHEUMATOLOGY VOLUME 10 | AUGUST 2014 | 499
by immunosuppression. Thus, rapid ANCA tests might
be helpful as an adjunct to urgent therapeutic decisions,
but cannot supplant the critical appraisal of all findings
including patient history, clinical assessment and imaging
as well as other laboratory tests. Histological confirmation
of vasculitis is still the gold-standard and should be sought
in every patient.
The use of ANCA to monitor disease activity and guide
treatment decisions in AAV is controversial (reviewed
else­where7
). A meta-analysis published in 2012 con­cluded
that serial measurements of ANCA in patients in remis­
sion has limited value, as neither an increase in ANCA
titre nor the persistence of ANCA during remission were
highly predictive of relapse.39
Furthermore, the correlation
of ANCA levels with disease activity is reportedly assay-
dependent, with the PR3-ANCA capture ELISA showing
some potential as a surrogate marker of disease activity in
patients with GPA.27
At present, an increase in ANCA titre
during f­ollow-up of patients with AAV, in the absence of
signs of relapse, should prompt intensification of moni­
toring by measures such as urinalysis. The utility of novel
antigen-specific immunoassays—anchor ELISA—for
monitoring disease activity and guiding t­reatment of
patients with AAV requires longitudinal studies.
Whereas PR3-ANCA and MPO-ANCA are highly
specific for AAV, they have very little diagnostic value for
nonvasculitic conditions. ANCA testing performed in
a population with a low pretest probability of vasculitis
is expected to yield a large number of ANCA-positive
results.13,40
Overestimating the diagnostic relevance of a
positive ANCA test could misdirect clinicians and delay
appropriate treatment. The diagnostic accuracy of ANCA
testing should be improved by an increased pretest prob­
ability of vasculitis. The use of a ‘gating policy’ that adheres
to clinical guidelines for limiting ANCA testing on the
basis of clinical criteria9,10
makes ANCA screening more
relevant clinically, as demonstrated in several studies.41–43
Investigating the effect of such a policy at a single regional
centre, Arnold et al.43
followed up on patients for whom
ANCA testing was deemed inappropriate and found that
no diagnoses of AAV were delayed or missed.
Commercial ELISA screening can be used to detect
ANCAswithspecificitiesotherthanPR3andMPO,suchas
elastase, cathepsin G, bactericidal permeability-­increasing
protein, lactoferrin and others. However, these specific
ANCAs have no diagnostic value for patients with AAV.
Conclusions
The development of image analysis technology and novel
antigen-specific immunoassays for detection of ANCAs
is an important contribution to the diagnostic repertoire
in the assessment of AAV. The evolution of ANCA test­
ing needs to incorporate automated, multiplexed and
increas­ingly sensitive technologies that can correctly
identify ANCAs in a routine clinical setting. Automated
approaches to the performance and interpretation of
ANCA IIF offer the opportunity to improve standardi­
zation and to address other shortcomings of manual
analysis such as its labour intensiveness and its main
di­sadvantage: subjectivity.
This automation is also expected to have a substantial
effect on diagnostic testing by requiring less sample and
reagent, analysis time and sample handling. Image analy­
sis technology has proven to be an interesting tool for
ANCA screening, but we expect to see further standardi­
zation and improvement of this technique over the next
few years before its routine application in clinical practice
will be feasible.
Within specialized labs conducting clinical studies
(as opposed to labs consulted in the course of clinical
prac­tice), the use of automated ANCA IIF analysis is
expected to advance quickly and is already considered as
an alternative to conventional manual IFT. Nonetheless,
continuous efforts are necessary to ensure this method
over­comes all the disadvantages of the conventional
manual approach. One important task for the near future
is to assess the reliability of these automated assays in
multicentre studies.
In the development of advanced PR3-ANCA and
MPO-ANCA assays, the binding of antigen onto the
solid phase by means of bridging molecules improves
the accessibility of the antigen-relevant epitope to the
ANCA. Consequently, the sensitivity of capture as well
as anchor ELISAs seems to be superior to that of conven­
tional ELISA (which directly immobilizes antigen) and
to IFT. However, despite several comparative studies, it
is still unclear which immunoassays for the detection of
PR3-ANCA and MPO-ANCA provide the highest clinical
+
A-ANCA patternP-ANCA patternC-ANCA pattern
MPO-ANCA ELISA
PR3-ANCA ELISA
MPO-ANCA ELISA
P-ANCA
(MPO-ANCA)
Other
ANCA
ELISA
MPO-ANCA
ELISA
Other ANCA
ELISA
Cytoplasmic
staining
on both
ethanol-fixed
and formalin-fixed
cells
Mixed cytoplasmic
and perinuclear/
nuclear staining on
ethanol-fixed cells
Cytoplasmic
staining on
formalin-fixed cells
Cytoplasmic
staining on
formalin-fixed cells
Nuclear
staining on
formalin-fixed cells
Perinuclear/nuclear staining
on ethanol-fixed cells
IFT on ethanol-fixed and formalin-fixed neutrophils
MPO-ANCA ELISA
PR3-ANCA ELISA
Other ANCA ELISA
IFT on HEp2PR3-ANCA ELISA
ANA A-ANCA
C-ANCA
(PR3-ANCA)
– + – +– + –
Figure 4 | ANCA testing algorithm routinely used in the authors’ laboratory. The
testing strategy for ANCA in patients with suspected vasculitis includes initial
screening by IFT on ethanol-fixed and formalin-fixed neutrophils, and confirmation
of positive results with ELISA. This algorithm conforms to international consensus
guidelines for screening on ethanol-fixed neutrophils,9,10
but our laboratory uses
additional tests on formalin-fixed neutrophils and HEp2 to better discriminate
between P‑ANCA (or A‑ANCA) and ANA. Abbreviations: ANA, antinuclear antibodies;
ANCA, antineutrophil cytoplasmic antibody; A‑ANCA, atypical ANCA staining pattern;
C‑ANCA, cytoplasmic ANCA staining pattern; HEp2, human epithelial type 2 cells;
IFT, indirect immunofluorescence test; MPO, myeloperoxidase; P‑ANCA, perinuclear
ANCA staining pattern; PR3, proteinase 3.
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7. 500 | AUGUST 2014 | VOLUME 10 www.nature.com/nrrheum
accuracy. Furthermore, the clinical utility of measuring
PR3-ANCA and MPO-ANCA for monitoring disease
activity remains a topic for further investigation.
The availability of highly sensitive PR3-ANCA and
MPO-ANCA assays has raised questions about the two-
stage diagnostic strategy currently recommended for
ANCA detection.9,10
An alternative algorithm13,34
might
use such high-sensitivity solid-phase assays as the initial
step, with confirmation of positive results by IFT. More­
over, it has been postulated that the need to perform IIF–
based assays might be eliminated altogether.7
How­ever,
at present, the evidence that IIF can be replaced by PR3-
ANCA and MPO-ANCA assays is insufficient, and future
studies are needed to establish whether the al­ternative
ANCA-detection algorithm is feasible.
Altogether, these studies enable us to draw some
conclusions. First, IFT performs consistently well but
seems to be slightly less sensitive than anchor ELISA,
which can also show a higher positive predictive value.
Anchor ELISA, however, awaits further evaluation for its
utility in monitoring treated and relapsing disease and
with regard to its reproducibility in nonspecialist labo­
ratories. The current practice of screening for ANCAs by
IFT and confirming IFT-positive sera in antigen-specific
immuno­assays is reasonable for now, but improvements
in the methods for ANCA detection and the development
of novel detection technologies could affect the validity of
the current international recommendations, lead to
updating of the standardization process and a revision
of existing ANCA diagnostic strategies. Clearly, though,
the development of any new testing strategy for ANCAs
in vasculitis must identify the ANCA target antigens, as
PR3-ANCA and MPO-ANCA serotype correlate well
with the disease expression.
Review criteria
A search for original articles published between
January 2000 and October 2013 was performed in
MEDLINE without language restrictions. Search terms
used, in various combinations, included: “vasculitis”,
“antineutrophil cytoplasmic antibodies”, “proteinase
3‑ANCA”, “myeloperoxidase-ANCA”, “methods”,
“ELISA”, “indirect immunofluorescence”, “automation”
and “diagnostic utility”. All articles identified were
full-text papers. The reference list was last updated
February 2014.
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Author contributions
E.C. researched data for and wrote the article,
F.M. made a substantial contribution to discussion
of content, and both authors reviewed/edited the
manuscript before submission.
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