Auris Nasus Larynx 44 (2017) 735–741

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Auris Nasus Larynx

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A novel strategy with combined assays for detection of anti-

neutrophil cytoplasmic antibody (ANCA) in clinically ANCA-
negative granulomatosis with polyangiitis patients
Kaori Tateyama a, Satoru Kodama a, Kan Kishibe b, Yasuaki Harabuchi b,
Masashi Suzuki a,*
a
Department of Otolaryngology, Head and Neck Surgery, Oita University Faculty of Medicine, Oita, Japan
b
Department of Otolaryngology, Head and Neck Surgery, Asahikawa Medical University, Hokkaido, Japan

A R T I C L E I N F O A B S T R A C T

Article history: Objective: Granulomatosis with polyangiitis (GPA) that is localized to the upper airway presents a
Received 11 October 2016 diagnostic challenge because of a tendency towards anti-neutrophil cytoplasmic antibody (ANCA)-
Accepted 1 March 2017 negativity. The purpose of this study was to investigate whether positivity of ANCA detection might
Available online 27 June 2017
be elicited with combined use of enzyme-linked immunosorbent assay (ELISA) kits.
Methods: Twenty-nine serum samples obtained from GPA patients were used in this study. In
Keywords:
addition to routine biochemical investigation for ANCA, tests for detecting PR3-, MPO-ANCAs,
Anti-neutrophil cytoplasmic antibody
(ANCA)
and minor ANCAs were performed with commercially available ELISA kits. Cytoplasmic (C)-
ANCA-associated vasculitis ANCA and perinuclear (P)-ANCA were evaluated using the indirect immunofluorescence (IIF)
Granulomatosis with polyangiitis technique.
ANCA detection Results: Twelve patients were positive for PR3- or MPO-ANCA in the clinical laboratory test, and
Minor ANCA 17 patients were negative for both ANCAs. Of the 17 ANCA-negative patients, four were positive
for PR3- or MPO-ANCA, and three were positive for minor ANCA according to results obtained
from six different ELISA kits. These findings indicated that performing detection tests with six
different ELISA kits might improve the positivity of ANCA and might contribute to establishing the
diagnosis of ANCA-associated vasculitis. Together with results from IIF, the samples of eight
patients with clinically ANCA-negative results (8/17, 47.1%) were converted to ANCA-positive
results, and the ANCA detection rate was significantly improved from 12/29 (41.4%) to 20/29
(69.0%, p = 0.03).
Conclusions: Additional detection techniques should be used to confirm the results of clinically
ANCA-negative samples, particularly when vasculitis is suspected. Minor ANCAs should also be
evaluated with detection tests when PR3- and MPO-ANCA are negative.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction

Anti-neutrophil cytoplasmic antibody (ANCA)-associated

* Corresponding author at: Department of Otolaryngology, Head and Neck
Surgery, Oita University, Faculty of Medicine, Idaigaoka, Hazama-cho, Yufu,
Oita 879-5593, Japan. Fax: +81 97 549 0762.
E-mail address: suzukim@oita-u.ac.jp (M. Suzuki).
vasculitis (AAV) is a necrotizing vasculitis that is characterized
by the presence of few or no immune deposits. AAV
predominantly affects small vessels, and is associated with
ANCA specific for myeloperoxidase (MPO)-ANCA or

http://dx.doi.org/10.1016/j.anl.2017.03.002
0385-8146/© 2017 Elsevier B.V. All rights reserved.
736 K. Tateyama et al. / Auris Nasus Larynx 44 (2017) 735–741
proteinase 3 (PR3)-ANCA [1]. AAV includes three major
diseases, namely granulomatosis with polyangiitis (GPA),
microscopic polyangiitis (MPA), and eosinophilic granuloma-
tosis with polyangiitis (EGPA). Otorhinolaryngologists might
encounter limited manifestations of GPA, particularly when the
disease is localized to the upper airway, which is not easy to
diagnose at the early stages of the disease. The positivity of
MPO-ANCA and PR3-ANCA in Japanese GPA patients
is reported to be 17–54%, and 46–73%, respectively
[2–4]. Approximately half of the localized GPA cases show
negative results on serologic test for ANCA [5], although they
exhibit clinical manifestations identical to those seen in ANCA-
positive GPA.
Currently in clinical practice, diagnosis is usually based on
presence of ANCA and histological examination of the affected
organ. ANCA is valuable for the diagnosis of AAV, although a
negative result should not necessarily rule out a diagnosis of the
disease. In patients without ANCA positivity, the following
three possibilities are considered. First, the patients might have
ANCA that could not be detected with current methods.
Second, the ANCA might be of as yet undiscovered specificity.
Third, pathogenic mechanisms that do not involve ANCA may
be involved [1]. In routine clinical practice, only single enzyme
immunoassays (EIA) are used to detect ANCA; however, there
are various commercially available EIA such as enzyme-linked
immunosorbent assay (ELISA) and chemiluminescence en-
zyme immunoassay (CLEIA) for detecting ANCA with varying
degrees of performance efficacy and cut off values. Thus, the
clinician should be aware of the different performance
characteristics of various ELISA kits [6]. In the present study,
we assumed that applying different detection methods to cases
that were otherwise diagnosed as ANCA negative in the clinical
test would improve the positivity rate of ANCA. In addition,
some cases might include ANCAs that do not involve PR3- or
MPO-ANCA. Currently, many target antigens for ANCA have
been identified, including elastase, bactericidal/permeability
increasing protein (BPI), cathepsin G, lactoferrin, lysozyme,
and azurocidin [7]. These are known as minor ANCAs that are
observed less frequently than MPO- and PR3-ANCAs. We also
assume that the minor ANCA are involved in the pathology of
both PR3- and MPO-ANCA-negative cases.
The purpose of this study was to investigate whether the
positivity of ANCA might improve with combined use of
ELISA kits. We also investigated the presence of minor ANCAs
in the serum of ANCA-negative cases.
2. Materials and methods
2.1. Patients and serum samples
Fifteen GPA patients treated in Oita university Hospital and
14 GPA patients treated in Asahikawa medical university
Hospital from 2000 to 2014 were included in the present study.
All experiments were approved by the Institutional Review
Board of each university hospital. Diagnosis of AAV was
determined according to the European Medicines Agency
(EMEA) algorithm [8,9]. In the EMEA algorithm, surrogate
markers for GPA in head and neck lesions are defined as
follows: (1) bloody nasal discharge and crusting for > 1 month,
or nasal ulceration; (2) chronic sinusitis, otitis media, or
mastoiditis for > 3 months; (3) retro-orbital mass or
inflammation (pseudotumor); (4) subglottic stenosis; and (5)
saddle nose deformity/destructive sinonasal disease. The
following information was collected from the medical records:
sex, age at initial visit, initial and cumulative disease
manifestation, and ANCA positivity by clinical examination.
As a routine clinical practice in Japan, serum MPO- and PR3-
ANCA are measured by one of three EIA methods, namely,
ELISA, CLEIA, or fluorescence enzyme immunoassay (FEIA),
all of which have been authorized by the Ministry of Health and
Welfare of Japan. For detection of ANCA by CLEIA, we used
STACIA (Mitsubishi Chemical Medience Corporation, Tokyo,
and MBL Co., Ltd, Nagoya, Japan) with MEBLux (MBL Co.,
Ltd, Tokyo Nagoya, Japan). For ELISA, Nipro Nephroscholar
MPO-ANC II (Nipro, Osaka, Japan) was approved for use in
2000 and was widely used until 2012, after which the MPO-
STACIA MEBLux test was authorized and has since been
widely available [10]. For FEIA, Elia PR3s-ANCA, MPOs-
ANCA (Phadia GmbH, Freiburg, Germany) has been used since
2011.
Blood samples were obtained with informed consent from all
patients. After centrifugation, serum samples were divided into

aliquots and stored at 80 C. Seven samples were obtained
from patients during the inactive phase of the follow-up period,
and 22 samples were obtained from patients in the active phase
with an untreated status.
2.2. Detection of PR3- and MPO-ANCA
In addition to the routine clinical examination, test for
ANCA reactivity in patient’s serum was performed in our
laboratory using commercially available ELISA kits. For
detection of PR3- and MPO-ANCA by direct ELISA, Wieslab
PR3-ANCA and MPO-ANCA ELISA kits (Wieslab AB,
Malmo, Sweden) were used according to the respective
manufacturer’s instructions. Briefly, the 96 wells of a microtiter
plate were coated with purified proteinase 3 or myeloperox-
idase. Each sample was diluted 1:100 with diluent (990 ml
diluent + 10 ml serum) and incubated for 60 min with specific
antibodies in diluted serum. Subsequently, the wells were
washed three times to remove unbound antibodies and other
non-specific components. Alkaline phosphatase-labeled anti-
bodies conjugated to human IgG were then added to the wells
and incubated for 30 min. After a wash step, detection of
specific antibodies was achieved by adding a substrate solution
to the plate. In this set-up, the amount of bound antibodies
correlates with the color intensity and is measured in terms of
absorbance (optical density). The absorbance is then compared
against a calibration curve, and the results are presented in IU/
ml, adapted to the AF-CDC international standard for PR3. For
this method, the cut-off value was 6 IU/ml for direct PR3, and
8 IU/ml for direct MPO.
For detection of PR3-ANCA and MPO-ANCA by capture
ELISA, the Wieslab capture PR3-ANCA and capture MPO-
ANCA ELISA kits (Wieslab AB) were used according to the
respective manufacturer’s instructions. The 96 wells of a
 K. Tateyama et al. / Auris Nasus Larynx 44 (2017) 735–741
microtiter plate were coated with purified anti-PR3 monoclonal
antibody and proteinase-3 or anti-MPO monoclonal antibody
and myeloperoxidase, respectively. The detection procedures
are the same as those for direct ELISA. For this method, the cut-
off value was 7 IU/ml for capture PR3, and 7 IU/ml for capture
MPO.
For detection of PR3-ANCA by anchor ELISA, anti-PR3 hs
ELISA kit (Nunc, GmbH & Co. KG, Wiesbaden, Germany)
was used according to the manufacturer’s instructions. The
96 wells of microtiter plates were coated with highly purified
PR3. Human sera were diluted at 1:200 with sample buffer and
the coated microtiter plates were incubated with 100 mL of
serum per well (in duplicate) for 30 min at room temperature.
After washing, peroxidase-conjugated anti-human IgG was
added and the microtiter plates were incubated for an additional
15 min. After washing, a tetramethyl-benzidine-containing
substrate solution was added to each well and incubated for
another 15 min. The enzyme reaction was stopped by adding
100 mL hydrochloric acid. The plates were analyzed at 450 nm
using a microtiter plate reader. The cut-off value was 10 IU/ml.
2.3. Detection of minor ANCA
In addition to major antigens PR3-ANCA and MPO-ANCA,
the Wieslab ANCA panel kit (Wieslab AB) was used for
qualitative analysis of antibodies to minor ANCA antigens as
well (including azurocidin, BPI, cathepsin G, elastase,
lactoferrin, and lysozyme). Briefly, the wells of the microtiter
strips were coated with purified ANCA antigens and incubated
with specific antibodies in diluted serum. The wells were then
washed to remove unbound antibodies and the other
components. In a second incubation, alkaline phosphatase-
labeled antibodies conjugated to human IgG were added and
allowed to bind to the antibodies in the wells. After a wash step,
specific antibodies were detected by incubation with a substrate
solution. The amount of bound antibodies correlates to the color
intensity and is measured in terms of absorbance (optic density
[OD]). The plates were analyzed at 405 nm using a microtiter
plate reader. OD ratios were calculated as follows: OD of
patient sample for the antigen/OD of patient sample blank. The
cut-off value was 4.
2.4. Indirect immunofluorescence assay
The indirect immunofluorescence (IIF) assay for the
detection of cytoplasmic (C)- and perinuclear (P)-ANCA
was performed by SRL Inc. (Tokyo, Japan). IIF was performed
on commercially available slides of ethanol and formalin-fixed
normal human neutrophils. The MBL Fluoro ANCA test Code
Nos. 4710 and 4720 (MBL) are equivalent to the Binding Site
ANCA ethanol kit Code FK016 and ANCA formalin kit Code
FK017 (Binding Site), respectively. Slides were examined by
fluorescence microscopy for ANCA staining patterns (C-
ANCA and P-ANCA). When peri-nuclear or nuclear immuno-
fluorescence was detected on ethanol-fixed granulocytes, the
IIF was repeated using formalin-fixed neutrophil preparations.
Samples were interpreted as P-ANCA positive if they displayed
cytoplasmic staining on formalin-fixed slides [11].
737
2.5. Statistical analyses
Two-group comparisons were performed using the Mann–
Whitney U test, the chi-square test, or Fisher’s exact test. All
statistical analyses were performed using JMP 11 (SAS Institute
Inc., Cary, NC). Statistical significance was set at p < 0.05.
3. Results
3.1. Patient characteristics
A total of 29 patients were diagnosed with AAV. According
to the EMEA, 18 patients were diagnosed with GPA (Table 1).
Eight patients fulfilled the American College of Rheumatology
(ACR) criteria, one patient showed Chapel Hill Consensus
Conference (CHCC)-defined GPA histology, and nine patients
were positive for ANCA in the presence of GPA surrogate
markers. Eleven patients (37.9%) had clinical features of GPA
surrogate markers without ANCA positivity and histology
compatible with small-vessel vasculitis. Of 11 unclassifiable
patients, 10 patients had symptoms localized to head and neck,
and one patient had pulmonary involvement with head and neck
lesions. These patients were diagnosed with GPA by clinical
features alone (i.e., intractable otitis media or sinusitis resistant
to antibiotics, no malignant tumor or inflammatory disease, and
response to treatment with corticosteroids). Characteristics of
ANCA-positive and ANCA-negative patients according to the
results from clinical tests are shown in Table 2. Seventeen
patients initially presented with nasal symptoms (58.6%), and
17 presented with aural symptoms (58.6%); among them, five
patients presented with both ear and nose symptoms. Thirteen
patients (44.8%) had other organ system involvement,
including 11 patients with lung involvement (37.9%), and
two patients with kidney involvement (3.4%). Eighteen patients
(62.1%) had ENT symptoms only. One patient developed
subglottic stenosis during the clinical course and required
tracheostomy. One patient had exophthalmos due to an orbital
mass. Two patients with aural symptoms developed hypertro-
phic pachymeningitis, and three had facial palsy. In serum tests
during the clinical course, PR3-ANCA was positively detected
in seven patients (24.1%), and MPO-ANCA was positive in five
patients (17.2%). Thus, 12 patients (41.4%) were positive for
ANCA by clinical laboratory tests, and 17 patients (58.6%)
Table 1
Diagnosis of GPA according to EMEA algolism.
ANCA positive ANCA negative
(n = 12) (n = 17)
ACR criteria 3 (25.0%) 4 (23.5%)
GPA histology 0 1 (5.9%)
MPA histology, GPA 0 0
surrogate markers
ANCA positive, GPA 9 (75.0%) 0
surrogate markers
Uncalssifiable 0 11 (64.7%)
EMEA, Europian medical agency; ACR, American College of Rheumatology;
ANCA, antineutrophil cytoplasmic antibody; GPA, granulomatosis with
polyangiitis; MPA, microscopic polyangiitis.
 738 K. Tateyama et al. / Auris Nasus Larynx 44 (2017) 735–741

Table 2
Characteristics of clinically ANCA positive and ANCA negative patients.

ANCA positive ANCA negative P value

Number of patients 12 17
Gender (female rate, %) 9 (75.0%) 13 (76.5%) 0.93
Median age at onset (range), 71 (36–81) 63 (37–83) 0.72
years
ANCA status
PR3-ANCA positive (%) 7 (58.3%) 0
MPO-ANCA positive (%) 5 (41.7%) 0
ANCA negative (%) 0 17
Organ involvement
Ear (%) 8 (66.7%) 9 (52.9%) 0.46
Nose (%) 6 (50.0%) 11 (64.7%) 0.43
Kidney (%) 2 (16.7%) 1 (5.9%) 0.55
Lung (%) 2 (16.7%) 3 (17.7%) 1
Disease activity Fig. 1. Detection of ANCA with combined methods.
Active 9 (75.0%) 13 (76.5%) 1 Detection of ANCA and its improvement with combined methods. Twelve
Inactive 3 (25.0%) 4 (23.5%) 1 patients were positive for PR3-ANCA or MPO-ANCA by clinical laboratory
test, and 17 patients were negative for both ANCA. Of the 17 ANCA-negative
ANCA, antineutrophil cytoplasmic antibody; MPO, myeloperoxidase; PR3, patients, four were shown as positive for PR3- or MPO-ANCA, and three were
proteinase-3. shown as positive for minor ANCA in the experiments using 6 different ELISA
kits. Adding the result of IIF assay, the ANCA detection rate significantly
improved from 12/29 (41.4%) to 20/29 (69.0%, p = 0.03).
ANCA, antineutrophil cytoplasmic antibody; MPO, myeloperoxidase; PR3,
were negative for both ANCA during the whole follow-up proteinase-3; ELISA, enzyme-linked immunosorbent assay; IIF, indirect
period (Fig. 1). immunofluorescence.

3.2. Direct ELISA, capture ELISA, and anchor ELISA
Patient’s sera were also examined in our laboratory with five
commercially available ELISA kits, including direct ELISA for
PR3-ANCA and MPO-ANCA, capture ELISA for PR3-ANCA
and MPO-ANCA, and anchor ELISA for PR3-ANCA
(Tables 3 and 4). Serum samples that tested positive by at
least one of the five ELISA kits were considered as positive for
ANCA. According to these results, a total 14 patients (48.3%)
were positive, and of the 12 ANCA-positive patients, 10 patients
were positive by these ELISA kits. Two serum samples from
ANCA-positive patients were shown as negative for ANCA
discrepantly. On the other hand, four samples (23.5%) from
17 ANCA-negative patients were shown as positive by the
ELISA tests (Fig. 1). This result indicates that using ELISA kits
might improve the positivity of ANCA in serum samples, and
might therefore contribute to better diagnosis of AAV.
3.3. Minor ANCA
Minor ANCAs were also examined with a commercially
available ELISA kit. Interestingly, three patients were positive
for minor ANCA, including two for BPI and one for elastase
(Table 4). Although no clinically ANCA-positive patients were
shown as positive for minor ANCA, three of the 17 ANCA-
negative patients (17.6%) were shown as positive for minor
ANCA (Fig. 1). All of these patients had been shown as
negative for PR3-ANCA and MPO-ANCA by clinical and

Table 3
Q9 ANCA positive patients in the clinical test.

Case Age Gender E/L/K Legion in head Disease Pathology Clinical Direct Capture Anchor IIF Minor
and neck activity test PR3/MPO PR3/MPO PR3/MPO PR3 ANCA
1 71 F ELK Ear, nose Inactive + +/ (CLEIA) / +/ +
2 56 F E Nose, larynx Inactive +/ (CLEIA) +/ +/ +
3 36 M E Ear, HP Inactive +/ (ELISA) +/ +/ + + (C)
4 81 F ELK Ear, nose Active +/ (ELISA) +/ +/ + No data
5 72 M E Nose Active +/ (ELISA) +/ +/ + + (C)
6 55 M E Nose, FP Active +/ (FEIA) / +/ +
7 60 F EL Nose Active + +/ (CLEIA) +/ +/ + + (P)
8 72 F EL Ear Active /+ (CLEIA) / / + (P)
9 72 F EL Ear Active /+ (CLEIA) / /
10 73 F E Ear, FP, HP Active /+ (CLEIA) / /+ + (P)
11 70 F EL Ear Active /+ (CLEIA) /+ /+ + (P)
12 49 F EL Ear Active /+ (CLEIA) /+ /+ + (P)
 K. Tateyama et al. / Auris Nasus Larynx 44 (2017) 735–741 739

Table 4
ANCA negative patients in the clinical test.

Case Age Gender E/L/K Legion in head Disease Pathology Clinical test Direct Capture Anchor IIF Minor
and neck activity PR3/MPO PR3/MPO PR3/MPO PR3 ANCA
13 64 F E Ear, HP Inactive / (CLEIA) / /
14 79 F ELK Ear, nose Inactive + / (FEIA) / / + (BPI)
15 83 F E Nose Inactive / (CLEIA) / /
16 63 F EL Nose Inactive + / (ELISA) / +/ +
17 39 F EL Ear Active / (CLEIA) / /
18 50 M E Nose Active / (CLEIA) / / +
(Elastase)
19 76 F EL Ear, nose Active + / (CLEIA) /+ /+
20 78 F E Nose Active + / (ELISA) / /
21 37 F E Nose Active / (ELISA) / /
22 67 F E Ear, HP Active + / (ELISA) /+ /
23 56 F EL Nose, larynx Active / (CLEIA) / / No data
24 59 M E Ear, nose, FP Active / (ELISA) / / No data
25 38 M E Nose Active / (CLEIA) / /
26 76 M E Ear Active / (CLEIA) / / + (BPI)
27 58 F E Ear Active / (CLEIA) / /
28 49 F E Ear Active / (CLEIA) / / + (P)
29 64 F E Nose Active / (CLEIA) /+ / + (P)
E/L/K, ear nose throat/lung/kidney; HP, hypertrophic pachymeningitis; FP, facial palsy; ELISA, enzyme-linked immunosorbent assay; CLEIA, chemiluminescence
enzyme immunoassay; FEIA, fluorescence enzyme immunoassay; (C), C-ANCA; (P), P-ANCA; BPI, bactericidal/permeability increasing protein;
ANCA, antineutrophil cytoplasmic antibody; MPO, myeloperoxidase; PR3, proteinase-3.

laboratory examination. Overall, seven of the 17 clinically
ANCA-negative patients (41.2%) were re-classified as ANCA-
positive (including for minor ANCA) based on results of the six
ELISA kits.
3.4. IIF assay
The IIF assay was performed by SRL Inc. (Tokyo, Japan).
Nine patients (31.0%) were positive by IIF. Of 12 ANCA-
positive patients, seven patients were positive by IIF, and five
patients were negative (Table 3). Two of the 17 ANCA-
negative patients were shown as positive by IIF, of which one
was positive by ELISA and one was negative by ELISA
(Table 4).
In addition, eight patients of the 17 clinically ANCA-
negative patients (47.1%) were re-classified as ANCA-positive
based on results from the current study. Thus, ANCA detection
rate significantly improved from 12/29 (41.4%) to 20/29
(69.0%) using combined ANCA detection methods (Fig. 1;
p = 0.03).
4. Discussion
Occasionally, otorhinolaryngological involvement may be
the first and only sign of AAV. Initial symptoms of GPA in 80–
95% of cases are observed in the head and neck region, most
commonly as sinusitis, saddle nose deformity, septal perfora-
tion, subglottic stenosis, and otitis media [12,13]. Owing to
ANCA negativity, otolaryngologists often encounter difficulty
in diagnosing AAV that is limited to the upper airway. A
potential delay in diagnosis may lead to progression of a disease
that is initially limited to head and neck to a systemic disease.
Additionally, otitis media is occasionally accompanied by facial
palsy and hypertrophic pachymeningitis, both of which
demonstrate an unfavorable clinical course that can impair
hearing and increase mortality [14,15]. Therefore, positive
detection of ANCA is important for diagnosis and management
of the disease. In our clinical cases, 29 GPA patients were
treated and analyzed. Of these, 12 patients were positive on
routine clinical examination; however, 17 patients were
negative for ANCA. Although serological testing for ANCA
is essential for diagnosing AAV, negative results could not rule
out the possibility of the disease. ANCA-negative AAV is
implied if the patient otherwise fulfills the definition for AAV
but has negative results on serologic testing for ANCA. Patients
with ANCA-negative AAV might have ANCA that could not be
detected with current methods. Alternatively, the disease might
be of as yet undiscovered specificity, or it may have
pathological mechanisms that do not involve ANCA [1]. The
most important finding from the present study is the
improvement of ANCA-positivity by the use of combination
methods involving clinical and laboratory tests. In serum
samples from 17 ANCA-negative patients, seven samples tested
positive with commercially available ELISA kits. Together
with results from IIF, eight ANCA-negative patients were re-
classified as ANCA-positive. Overall, the ANCA detection rate
was significantly improved by using combined ANCA
detection methods (p = 0.03).
Over the last decade, a variety of different methods have
been developed for the detection of ANCA. The direct,
noncompetitive ELISA test used to be the first-generation
method of choice. The second-generation (capture ELISA) or
third-generation (anchor ELISA) tests are now the more highly
sensitive and specific methods for detection of ANCA
[16–19]. Recently, the availability of CLEIA has further
improved the detection efficacy of ANCA in the clinical
laboratory; however, it is still not possible to diagnose and treat
all patients with AAV in current clinical practice since
740 K. Tateyama et al. / Auris Nasus Larynx 44 (2017) 735–741
approximately half of the patients are often ANCA-negative. In
addition, clinicians should take into account the differences
between ELISA systems. For detection of PR3-ANCA, both the
sensitivity and specificity are increased when the capture
ELISA is compared with the direct ELISA. However, for MPO-
ANCA, capture ELISA seems to be more specific but not more
sensitive for ANCA detection in AAV [17,18]. In the present
study, reproducibility of data was confirmed in clinically
ANCA-positive sera by using new generation ELISA kits (i.e.,
capture and anchor ELISA). However, the improvement in the
detection of ANCA was not remarkable in clinically ANCA-
negative sera when using the currently available generations of
ELISA techniques. This result suggested that other target
antigens for ANCA (besides PR3 and MPO) might be present.
Another important result was our demonstration of the
presence of minor ANCAs by ELISA. Thus, we detected BPI-
ANCA positivity in two patients and elastase-ANCA-positivity
in one patient, suggesting that negativity for PR3-ANCA and
MPO-ANCA may be due to presence of ANCAs other than
these two major ANCAs.
Minor ANCAs are antibodies against constituents of the
neutrophil granules. These antibodies are predicted to
upregulate neutrophil activity, which could contribute to tissue
damage. Antibodies to minor antigens, such as BPI-ANCA and
elastase-ANCA, have been reported in cases of PR3- and MPO-
ANCA-negative systemic vasculitis whose levels parallel
disease activity [20–22]. Moreover, minor ANCAs may exhibit
a different IIF pattern. Samples that show atypical fluorescence
patterns or in which C-or P-ANCA are positive despite a
negative result for PR3- or MPO-ANCA should be tested for the
presence of minor-ANCA [23]. Therefore, testing for anti-
bodies to minor antigens is recommended under IIF reactivity in
the absence of PR3-ANCA and MPO-ANCA negativity
[24]. Based on our results in this study, the algorithm for
ANCA detection is shown in Fig. 2. For patients with a negative
Fig. 2. Algorithm for ANCA detection.
With routine clinical examinations, IIF should be performed for confirmation of
the results. PR3- and MPO-ANCA negative serum or an atypical pattern of
ANCA should be tested for minor ANCA.
IIF, indirect immunofluorescence; ANCA, antineutrophil cytoplasmic
antibody; C, cytoplasmic-ANCA; P, perinuclear-ANCA; MPO,
myeloperoxidase; PR3, proteinase-3.
result for ANCA in the routine clinical examination, other EIA
methods, such as capture ELISA or anchor ELISA, should be
performed. IIF is recommended for confirmation of an ELISA
result [24]. When PR3 or MPO are negative with other ELISA
kits, final testing for minor ANCAs should be performed.
Although detection of minor ANCA is not commonly
performed in clinical practice and is considered to have less
diagnostic value for patients with AAV [23], our findings from
the present study suggest that some AAV patients may test
positive for minor ANCAs. Therefore, testing for minor
ANCAs may be useful in the diagnosis of AAV in PR3-ANCA
and MPO-ANCA-negative patients.
The diagnosis of GPA relies on the combination of clinical
findings, ANCA status and histological result of the affected
organ. Histological findings are often non-specific inflamma-
tion in head and neck limited GPA. For such a patients, positive
result of ANCA may strongly support the diagnosis. When the
result of ANCA was determined by combined use of other EIA
methods or IIF, the diagnosis of AAV would be more reliable.
Although ANCA is a specific marker for AAV, false positive
result for ANCA is observed in drug exposure or inflammatory
bowel disease [25,26]. In recent years, the use of CLEIA for
detection of ANCA has gained increasing attention because of
its high sensitivity and specificity; however, CLEIA yielded a
larger number of false-positive results than ELISA [10]. A
false-positive ANCA test could lead to misdiagnosis. Diagnosis
of GPA should be made in combination with clinical
manifestations, histological findings, and careful exclusion of
other diseases.
In conclusion, clinically ANCA-negative samples should be
confirmed using other EIA detection techniques for antibodies
against particular target antigens, mainly PR3 and MPO,
particularly when vasculitis is clinically suspected. Minor
ANCAs should also be tested when PR3- and MPO-ANCA are
negative. Moreover, development of novel detection technolo-
gies is essential for improvements in the methods for ANCA
detection.
Acknowledgments
This work is supported by Grant-in-Aid from the Ministry of
Education, Science and Culture of Japan (16K20262).
We thank Mrs. A. Iwamoto for assistance with the ELISA.
This study was performed as a work of the working group for
a nationwide survey of OMAAV organized by the Japan
Otological Society.
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