Nephrol Dial Transplant (2015) 30: i8–i13
doi: 10.1093/ndt/gfv070
NDT Perspectives
A historical essay on detection of anti-neutrophil
cytoplasmic antibodies
Niels Rasmussen1, Allan Wiik1 and David R. Jayne2
1
Department of Autoimmune Serology, Statens Seruminstitut, Copenhagen, Denmark and 2Medicine, Addenbrooke’s Hospital, Cambridge, UK
Correspondence and offprint requests to: Niels Rasmussen; E-mail: [email protected]
included to screen for autoantibodies in the protocols for the
Cattegat Study Group of Wegener’s Granulomatosis (CSGWG)
in 1981. In 1982, an Australian group published a short communication, describing similar anti-neutrophil cytoplasm autoantibodies in eight patients with pauci-immune segmental
necrotizing glomerulonephritis [2]. With the subsequent paper
from the CSGWG [3] and the ensuing paper by the DutchCSGWG collaboration [4] more specifically relating the neutrophil
cytoplasmic staining autoantibodies, then called anticytoplasmic antibodies (ACPAs), to Wegener’s granulomatosis/
granulomatosis with polyangiitis (Wegener’s) (GPA) in 1985,
testing for these autoantibodies, termed cytoplasmic staining
anti-neutrophil cytoplasmic antibodies (C-ANCAs) in 1989,
has become an important diagnostic tool for GPA. C-ANCAs
in GPA are produced as a polyclonal response to an as yet unidentified agent or disease process and may belong to any of the
immunoglobulin classes, IgG, IgM and IgA, but predominantly
IgG in the active disease phases [2, 3]. C-ANCAs are typically
present during the initial active disease phase, disappearing
during remission (Figure 1) and reappearing at relapse [2–4].
ANCA can be detected not only using neutrophils and monocytes
as substrate but also using the leukemic HL-60 cell line [5, 6].
It was originally speculated that Ross River virus could have a
causal role for the disease [2], but no particular infectious agent
has ever been shown to cause GPA. There is no cross-reactivity
with neutrophil constituents from other species like rodents [2] or
more specifically rats, mice, rabbits, cows, horses, cats or hens [5].
A B S T R AC T
In this essay we describe a number of the known and not so
known experiences of the early anti-neutrophil cytoplasmic
antibodies (ANCAs) days, explaining why and how we reached
consensus on the standard indirect immunofluorescence (IIF)
techniques, the naming of the two principal C- and P-ANCA
patterns, why we chose to use IIF as the standard technique,
how the solid phase assays have developed and where we stand
today, the use of ANCA for diagnosis and the importance of
using several techniques for that purpose, how ANCA titres are
related to disease activity and the clinical impact of this, and
finally the implications of ANCA being a natural, polyclonal
antibody response against various epitopes in relation to diagnostics and disease patterns.
Keywords: ANCA, autoantibodies, biomarkers, crescentic
glomerulonephritis, vasculitis
T H E F I R S T O B S E R VAT I O N S
The indirect immunofluorescence (IIF) technique for detection
of autoantibodies to neutrophil granulocytes was initially used to
identify neutrophil-specific antibodies in patients with rheumatoid arthritis and Felty’s syndrome [1]. Some of these antibodies
stained the nuclei and perinuclear region of neutrophils and
monocytes and were therefore designated granulocyte-specific
anti-nuclear antibodies (GS-ANA), but early attempts at demonstrating their antigenic targets were unsuccessful.
By use of this technique, antibodies staining cytoplasmic
granules of neutrophils (anti-PMN-cytoplasm autoantibodies)
in a female patient with crescentic glomerulonephritis were
incidentally found in 1973, and this technique was therefore
© The Author 2015. Published by Oxford University Press
on behalf of ERA-EDTA. All rights reserved.
T H E I I F A S S AY S F O R A N C A
Several IIF techniques have been used for the detection of
ANCA, differing in source of neutrophils, purification, application on slides and fixation method. The most prominent
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difference turned out to be the agent used for fixing the neutrophils. Using formalin would keep all substances fixed in their
native position in the neutrophils, whereas ethanol would allow
for some substances to be dissolved and redistributed in or
outside the neutrophils. When using ethanol, myeloperoxidase
(MPO) dissolves from the primary granules and attaches to the
cell nucleus yielding a perinuclear staining pattern [7] like GSANA, whereas proteinase3 (PR3) does not and therefore yield
a cytoplasmic staining pattern. At the first international workshop on ANCA in 1988, it was agreed to use this method with
washed human leucocytes as substrate smeared onto glass slides
and fixed with ethanol as a standard procedure for IIF detection
of ANCA, as this method had already been fine-tuned for that
purpose [8]. The nomenclature for these staining patterns,
C-ANCA for the cytoplasmic pattern and P-ANCA for the perinuclear pattern (Figure 2), was agreed on at the second International ANCA Workshop in 1989.
THE ANCA ANTIGENS
MPO was the first ANCA antigen to be identified and reported
in 1988 [7] as one of the ANCA antigens yielding a P-ANCA
T H E E A R LY S O L I D P H A S E A S S AY S F O R
DETECTION OF ANCA
F I G U R E 1 : The first description of the disappearance of C-ANCA
(anti-PMN-aab) titre in a GPA patient brought in complete remission
on treatment, presented at the First International Academic Conference in Immunology and Immunopathology as applied to Otology
and Rhinology, 11–12 April 1984 in Utrecht, the Netherlands [3].
The first radioimmunoassay for ANCA detection using neutrophil cytoplasm as antigen was described in 1987 [17], the first
enzyme-linked immunosorbent assay (ELISA) using affinity
purified antigen for C-ANCA detection was described in 1988
[18] and the first commercially available kit for C-ANCA detection using extract from the primary/azurophil/alpha granules of
neutrophils was described in 1990 [19]. At the same time,
several other solid phase assays were developed, including direct
ELISAs and capture ELISAs (using a mouse-monoclonal catching antibody to present the antigen). Gradually, ELISAs became
more specific using purified MPO or PR3 as antigens.
F I G U R E 2 : The IIF staining patterns for C-ANCA (A) and P-ANCA (B) using ethanol fixed neutrophil granulocytes.
History of ANCA
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NDT PERSPECTIVES
IIF pattern. Another P-ANCA antigen was identified as
human neutrophil elastase (HNE) [9] and soon after, reports
from several groups identified the C-ANCA pattern antigen in
GPA as a 29 kDa serine protease, PR3 [10–14] in 1989 and
1990. These antigens were all located to the primary/azurophilic granules in the cytoplasm of the neutrophils, along with cathepsin-G, bactericidal/permeability increasing protein (BPI),
HNE and azurocidin. Lactoferrin is located in the secondary/
specific granules, and—like alkaline phosphatase located in the
tertiary/secretory granules and plasma membrane—both have
been implicated as P-ANCA antigens, too.
Only PR3-ANCA and MPO-ANCA are important in relation
to the small vessel vasculitides. Whereas PR3-ANCA/C-ANCA
was identified from the very beginning as a marker for GPA,
MPO-ANCA soon turned out to be a marker for microscopic
polyangiitis (MPA). The importance of PR3-ANCA and MPOANCA as disease markers has recently been emphasized in a
genomewide association study of ANCA associated vasculitis,
as the most marked associations were differentially related to
PR3-ANCA and MPO-ANCA [15]. MPO-ANCA, however, is
also found in 30–38% of patients with eosinophilic granulomatosis with polyangiitis (Churg-Strauss syndrome), primarily
in patients with vasculitis symptoms [16], and as discussed
below, PR3-ANCA and MPO-ANCA may also be detected in
other diseases, although the sub-specificities of these ANCAs
may differ from those closely related to GPA and MPA.
T H E E C / B C R S TA N D A R D I Z AT I O N O F S O L I D
P H A S E A S S AY S
At the second International ANCA Workshop in 1989, it became
obvious that the emerging solid phase methodologies created
for detecting and quantifying PR3-ANCA and MPO-ANCA
gave rise to different results and thus necessitated efforts to
standardize ANCA determination [20]. With a grant from the
EU/EC in 1990 from the EC/BCR programme, 14 European
laboratories participated, using three selected preparations of
native human PR3 and one preparation of MPO [21–23] for
their assays. These results were compared to results in the IIF.
The IIF tests were comparable and after corrections of the
solid phase methods, the coefficient of variation was reduced
to 20%, concluding that the assays could be used for standardized PR3- and MPO-ANCA testing.
NDT PERSPECTIVES
ANCA DETECTION FOR DIAGNOSTIC
PURPOSES
However, it is not only necessary to standardize the ANCA
assays but also necessary to secure that the clinical indications
for ANCA testing are relevant in order to obtain a pre-test probability high enough to justify the test and in order to evaluate
the sensitivity and specificity. The Lancet paper from 1985
showed a diagnostic sensitivity of 93% for active GPA [4]. Here,
the sera were selected to make sure that the patients in each
group were typical patients. In contrast, the sera for the EC/
BCR diagnostic study [23] were unselected sera obtained from
consecutive series of patients retrospectively and prospectively
in relation to a specific date. This included referred patients on
treatment and patients with a suspected but not histopathology
proven disease. The overall sensitivity for all PR3/C-ANCA
assays was only 64% for GPA. This illustrates that a gating
policy for requesting an ANCA test is necessary, as the diagnostic sensitivity and specificity depend entirely on the GPA population examined and the inflammatory disease control
population used for selecting a suitably high specificity. This
has complicated comparisons between reports on diagnostic
sensitivity and specificity of the many different methodologies
described since the end of the 1980s.
THE DECISION TO USE IIF AS THE PRIMARY
TEST FOR ANCA
After the EC/BCR standardization, it became relevant to
evaluate how, when and which assays should be used for clinical diagnostic use. At an international consensus meeting in
Australia [24], it was decided to use the IIF test as the primary
diagnostic screening test, based on the extensive experience
with the diagnostic potential of the HEp2-cell assay for ANA
screening and classification [25]. In accordance with the EC/
BCR study [23], a positive IIF test should always be followed
by a specific MPO- or PR3-ANCA test, and ideally all three
should be used in each case. This cluster of results would potentially sort out those P-ANCA-/GS-ANA-positive sera that
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were relevant for small vessel vasculitis from those that could
appear in a number of other connective tissue diseases.
ANCA DETECTION IN DRUG-INDUCED
VA S C U L I T I S , I N T E S T I N A L B O W E L D I S E A S E
AND CYSTIC FIBROSIS
P-ANCA patterns and atypical (A-ANCA) patterns have been
observed in a variety of other diseases than the systemic small
vessel vasculitides. It is well-known that certain drugs given
over prolonged periods of time can give rise to development of
ANCA-positive vasculitis or lupus-like disease. In 1991, it was
demonstrated that MPO-ANCA and NHE-ANCA were associated with hydralazine-induced glomerulonephritis [26], and
later ANCA against lactoferrin, ANA [27] and ANCA against
cathepsin-G was demonstrated in propylthiouracil-induced
vasculitis, producing a picture of a multiple autoantibody response in these diseases.
A special form of drug-induced disease with necrotizing inflammation is cocaine-induced midline destructive lesions,
which is a differential diagnosis to localized GPA in the
midface. This condition is highly correlated to HNE-ANCA
with a consistent P-ANCA pattern, even though PR3-ANCA
is also observed in about half of the cases [28].
The inflammatory bowel diseases, ulcerative colitis (UC)
and Crohn’s disease have been reported to be associated with
a P-ANCA or A-ANCA pattern since the mid-1990s, but determination of the antigen specificity of these ANCA patterns
has been difficult and has caused a number of conflicting
results. Just lately, using a chemiluminescence immunoassay,
the A-ANCA pattern has been correlated to PR3-ANCA in
31% of UC patients [29] and to 38% of Primary Sclerosing
Cholangitis patients [30]. The finding of PR3-ANCA yielding an A-ANCA pattern, however, is discussed but not
explained.
In cystic fibrosis, the presence of ANCA against BPI is correlated to the severity of pulmonary involvement with vasculitis and is also associated with the load of chronic infection
with Psudomonas aeruginosa (PA). These BPI-ANCAs, reflecting inflammation of the airways, are not only of IgG class
but frequently also of IgA class. Operations to remove infected
and diseased tissue in the form of a lung transplant or extensive image guided sinus surgery significantly reduced the IgG
as well as the IgA BPI-ANCA levels [31].
Whether the decrease of the infectious burden and/or the
removal of inflamed tissue is the reason for the decrease in BPI
levels remains to be shown. The correlation between the load
of chronic infection with PA and the presence of BPI-ANCA,
however, suggests that the infection somehow triggers ANCA
production. The presence of ANCA has been reported in a
number of infectious conditions including bacterial endocarditis and HIV. Recently, it has been shown that bacterial DNA
motifs may trigger production of PR3-ANCA as well as MPOANCA in vitro [32]. As new disease activity in GPA is frequently preceded by a ‘flu-like condition’, a transient infection
may trigger or contribute to disease activity as well as ANCA
production.
N. Rasmussen et al.
PR3-ANCA AND RELAPSE
C A N R E S U LT S O F S O L I D P H A S E A S S AY S F O R
PR3-ANCA BE COMPARED?
Although the many solid phase assays for PR3-ANCA have
good performances for diagnostic sensitivity and specificity,
they have turned out to have different performance patterns,
i.e. highly variable correlations of PR3-ANCA values between
assays [36, 37]. This can be illustrated by applying the dendrogram method on the changes of PR3-ANCA values from entry
to relapse for each patient for each of the nine commercial
solid phase assays used in the RELANCA study [33]. The dendrogram method applied on the changes from active disease at
entry to active disease at relapse revealed that some assays had
similar performance, whereas other assays were completely
discordant (Figure 3).
As PR3-ANCA are normal human, polyclonal autoantibodies [38], ANCA from different persons must be expected to
behave differently in different assays depending on the binding
profiles of these assays in relation to distribution of IgG subclasses [39], specificity for the epitopes exposed over time [40]
and in relation to disease activity [41], binding to free PR3 complexed to alpha-1-antitrypsin in serum [42] and binding to
reverse PR3-ANCA [43].
This means that PR3-ANCA values from one assay cannot
necessarily be compared with values from another assay and
that values from discordant assays cannot be standardized. The
marketing of a PR3-ANCA standard from CDC can therefore
only be used to standardize each single assay and secure
History of ANCA
F I G U R E 3 : Dendrogram illustrating the concordance/discordance
between the performance of nine solid phase assays with the level of
the horizontal bars illustrating the concordance of the connected
assays, where the level of 1 (height) is good concordance and levels >2
are discordance.
constant performance of that assay when new batches of
antigen are introduced.
The dendrogram results support previous findings that combining results from several PR3-ANCA assays may increase the
diagnostic potential of PR3-ANCA detection [44]. A screening
of PR3-ANCA assays using the dendrogram method can identify each assay as belonging to a certain cluster of assays, which
could be helpful to guide the local clinical laboratory to pick out
a relevant panel of assays from the assays available in that
region, in order to optimize sensitivity and specificity.
EPITOPE SPECIFICITY OF ANCA
In 1995, it was demonstrated that at least four different epitopes
on PR3 were relevant for PR3-ANCA binding [45]. Since then,
several groups have been working on the implications of
PR3-ANCA epitope specificity. The major problem is that the
relevant PR3 epitopes are conformational, complicating the
possibilities to use linear epitopes for meaningful diagnostic
purposes and pathogenic studies. Production of chimeric
human/mouse PR3 recombinant proteins made way for
epitope-mapping of anti-PR3 antibodies in 2004 [46] and in
2010, using a refined construct of their original recombinant
proteins, the presence of four major surface epitopes with
functional importance in relation to enzymatic activity and
disease activity was described [41]. A design of specific PR3epitope-ANCA assays was therefore suggested to be of potential clinical interest.
In contrast to PR3, some meaningful epitopes on MPO are
linear. In 1998, it was reported that five recombinant fragments of human MPO identified with rabbit antibodies could
sub-classify MPO-ANCA disease in Japan [47]. These findings
have been further confirmed in 2007 [48].
In 2006, the technique of producing chimeric human/
mouse constructs was translated to MPO, but the results suggested that further refinements using shorter fragments of the
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NDT PERSPECTIVES
The RELANCA study [33] planned in 1995 as a part of the first
series of randomized controlled trials by the European Vasculitis Study Group (EUVAS) using nine PR3-ANCA commercial
solid phase assays in monthly serum samples from the
NORAM trial, confirmed the original observations of reappearance of C-ANCA at relapse [2–4], but as already concluded in
1989, a rising titre/value was sensitive but not specific for the
development of a relapse [34]. The correlation between a rise in
titre and relapse is of interest from a pathogenic point of view,
but from a clinical point of view, the relevant window for
observing a rapid increase due to clinical relapse may be a few
weeks. Decrease and increase due to change in therapy develop
more slowly. Detecting a relapse therefore requires a sampling
frequency of a few weeks, which is almost never the case in the
clinical situation. This probably explains why increases in PR3ANCA levels were not found to correlate to relapses in the
WGET study, using three monthly sampling [35]. Nevertheless,
an increase in PR3-ANCA titres may reflect a relapse. It is therefore recommended that increasing titres are used as an alert
signal for a possible relapse, but that therapeutic action is taken
only if a clinical relapse can be confirmed.
So far, MPO-ANCA titre increases have not been linked to
a similar risk of relapse, but recent studies of MPO-epitopespecific ANCA indicate that certain epitope specificities are
linked to relapse as discussed below.
epitopes had to be performed [49]. This was described in
2013, using a highly sensitive epitope excision/mass spectrometry approach, reporting on 5 linear as well as 20 conformational epitopes relevant for MPO-ANCA, some being related
to disease activity [50].
These findings make it relevant to consider not only the
implications for setting up specific functional PR3- and MPOANCA assays, but also the implications for further research in
the possible pathogenic role of ANCA. Depending on the
functional aspects of the individual epitope-specific autoantibodies, these might differ in capacity to exert the pathogenic
functions suggested to be effective according to in vitro and
in vivo experiments.
NDT PERSPECTIVES
AC K N O W L E D G E M E N T S
The EUVAS organization that grew out from the group of colleagues assembled at the first International ANCA Workshop
in Copenhagen in 1988. Without the numerous contributions
from this mass of talent, none of the selected results mentioned in this review could have been achieved. MSc. Severin
Olesen Larsen, Statens Serum Institute, has contributed with
the statistical handling of data from the EUVAS Serum Bank
using the dendrogram method.
C O N F L I C T O F I N T E R E S T S TAT E M E N T
None declared.
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Received for publication: 18.12.2014; Accepted in revised form: 24.2.2014
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