INOVA NEWS

Antineutrophil Cytoplasmic Antibodies:

Historical perspectives and new advances

No. 8
IN THIS ISSUE

p2 An introduction to ANCA-associated

First described in 1982, antineutrophil cytoplasmatic antibodies (ANCA) represent

vasculitis | Antonella Radice, PhD

a diagnostically important group of autoantibodies. Indirect immunofluorescence

p5 Diagnostic methods for ANCA: past and present |

(IIF) on ethanol fixed human neutrophils was not only the first method described for
the detection of ANCA, it is still proclaimed as the method of choice for the first line
screening of sera from patients suspected to suffer from ANCA associated vasculitis
(AAV). The labor-intensive methodology inherent to IIF prompted new technological
advancements such as automated digital image analysis. Such technology allows for
standardization, reduction of hands-on time, and facilitation of case review.
While low sensitivity and specificity of early enzyme immunoassays (ELISAs) caused
much controversy, recent advances, such as the development of second and third
generation capture ELISAs offered significant improvements in diagnostic accuracy.
Ongoing innovations in the field of antigen specific solid phase immunoassays have
continuously improved the analytical and clinical performance characteristics of these
assays, supporting both diagnosis and follow-up. The recently developed chemiluminescent immunoassays (CIA) offer results in 30 minutes while allowing for high
sensitivity and specificity. These advances have helped to facilitate new opportunities
in the formerly unresolved associations of ANCA, such as the specific detection of
PR3-ANCA in ulcerative colitis.
This issue of the INOVA Newsletter covers historical perspectives from thirty years ago
to the current advances available today.
I hope you will enjoy reading!

Johannes Schulte-Pelkum, PhD

Director of Research and Development
INOVA Diagnostics, Inc.

Johannes Schulte-Pelkum, PhD

p9 ANCA testing in emergency setting |

Lucile Musset, PhD; Makoto Miyara, MD, PhD

p12 A new look at ANCA with NOVA VIEW |

Gabriella Lakos, MD, PhD; Carol Buchner, MT (ASCP)

p16 Anti-PR3 antibodies in ulcerative colitis |

Michael Mahler, PhD; Gary L. Norman, PhD;
Marcos Lopez-Hoyos, MD, PhD

An introduction to
ANCA-associated
vasculitis

a.)

b.)

Antonella Radice, PhD

Laboratory Manager - Immunology,
Autoimmunity and Microbiology
San Carlo Borromeo Hospital
Milan, Italy

Discovery of ANCA
Antibodies to neutrophil cytoplasmic components
(ANCA) were first described in 1982 by Davies et al. in
some patients with necrotizing glomerulonephritis
(FNGN) and symptoms of systemic vasculitis.1 In 1985
van der Woude et al. reported the strong association of
ANCA producing a diffuse granular cytoplasmic staining pattern (cANCA) on ethanol-fixed neutrophils and
granulomatosis with polyangiitis (formerly Wegeners
granulomatosis (WG)); a few years later, ANCA, producing a perinuclear fluoroscopic pattern (pANCA) on the
same cellular substrate were described in patients with
idiopathic necrotizing crescentic glomerulonephritis
(iNCGN) and microscopic polyangiitis (MPA).1 (Figure 1a.
and Figure 1b.)
The method available at that time to detect the cANCA
and pANCA pattern was the indirect immunofluorescence test (IIF) on normal human ethanol-fixed leukocytes.2 Over 30 years since its first use, IIF remains the
desired method to screen samples in patients suspected of vasculitis.

Figure 1a.) cANCA is largely due to the presence of autoantibodies targeting the serine protease proteinase-3 (PR3-ANCA).
Figure 1b.) pANCA is caused by antibodies directed against many
antigens, among which myeloperoxidase (MPO-ANCA) is the
most frequent in primary systemic vasculitis.3

Function of target antigens-PR3 and MPO

The target antigens of ANCA are located in the
primary granules of neutrophils and have antibacterial properties. PR3 and MPO are recognized in most
ANCA-positive small vessell vasculitidies. PR3 is a
weak cationic protein consisting of 228 amino acid
residues (MW 29-30 kD), belonging to the trypsin family
of serine proteases. PR3 is synthesized as a preproenzmye and subsequently processed in four steps
into the mature form. It is stored in the azurophilic
granules of neutrophils, but can also be found within
the membrane of secretory vesicles, and also expressed
at the plasma membrane. PR3 is physiologically inhibited by 1-antitrypsin.4 PR3 disintegrates tissue to allow
the passage of neutrophils into an inflammatory focus
and is also involved in neutrophil maturation. MPO is
a 150 kD heterdimer peroxidase enzyme abundantly
expressed in neutrophils. The enzyme is characterized
by a powerful bactericidal activity, whose peroxidase
activity is physiologically inhibited by ceruloplasmin.5
The identification and purification of PR3 and MPO
antigens resulted in development of several immunoassays for the quantitative detection of antibodies specific for
PR3 and MPO; the conventional enzyme-linked immunosorbent assays (ELISAs) and, more recently, methods
based on different fully automated technologies.3

2 |INOVA NEWS No. 8

Pathogenesis of ANCA disease

ANCA classification

Numerous in vitro studies demonstrate that both

MPO-ANCA and PR3-ANCA are capable of activating neutrophils and monocytes through Fab2 and Fc
engagement, which initiates several signal transduction pathways (Figure 2).6 Activation of these leukocytes results in adhesion to endothelial cells, causing
endothelial damage. Studies in animal models provide
conclusive evidence that anti-MPO antibodies induce
necrotizing and crescentic glomerulonephritis and
systemic small vessel vasculitis. Anti-MPO antibodies in
the absence of functional T cells are capable of causing
glomerulonephritis and vasculitis, and the induction
of this disease is dependent on neutrophils.6 This can
be exacerbated by a variety of cytokines, is dependent
on activation of the alternative pathway of complement, and is abrogated by inhibition of the alternative
pathway and by anti-C5 receptor antibodies. 6 These
studies provide a basis for exploring novel therapeutic
strategies.

ANCA are the serological hallmarks of idiopathic systemic

vasculitis, and the term ANCA-associated vasculitis (AAV)
has been used to collectively name those primary small
vessel vasculitic syndromes in which circulating ANCA are
commonly found (microscopic polyangiitis and its renal
limited form, granulomatosis with polyangiitis, ChurgStrauss syndrome).7 This approach, adopted by the
Chapel Hill International Consensus Conference (CHCC)
and by the European Vasculitis Study Group (EUVAS), is
supported by the striking clinical and histological similarities between the AAV, the widespread use of ANCA as
a diagnostic marker, and the growing evidence of their
pathogenetic potential.8

Neutrophil

Infection

ANCA

Release of
proinflammatory
cytokines

Names of the common forms of vasculitis have been

recently revised so that the eponyms such as WG and
CSS have been changed with granulomatosis with
polyangiitis (GPA) and eosinophilic granulomatosis
with polyangiitis (EGPA), respectively.9 The new disease
nomenclature system adopted by the 2012 CHCC
for defining small vessel vasculitis is listed in Figure 3.

Priming

Chemotaxis

Giant cell arteritis

Takayasu arteritis
Aorta
Polyarteritis nodosa
Kawasaki disease

Large- to
medium-sized
artery

The Chapel Hill Consensus Conference

nomenclature defines 10 primary
vasculitides based on vessel size.2

Small-sized
artery

C5a, chemokines

Henoch-Schonlein purpura
Cryoglobulinemic vasculitis

Arteriole
Capilary

Anti-GBM

Leukocytoclastic
vasculitis

Microscopic
polyangitis

Granulomatosis
with polyangitis
Churg-Strauss

ANCA
associated
vasculitis

Venule

In situ ICX
formation
+
AP Complement
activation

Vein

Figure 3. The Chapel Hill Consensus nomenclature defines 10

primary vasculitides based mainly on vessel size
O2

ANCA antigen
CD11b
Endothelial adhesion
molecules
Cytokine recptor

O2

Endothelium

Fc receptor
Cytokine/chemokine
C5a receptor
C5a

Figure 2. Pathogenesis of ANCA-associated vasculitis

After the standardization of the methods for ANCA

detection and the evaluation of their clinical application
was agreed upon, a document produced by an international consensus of experts was published, with suggestions for the correct ANCA testing and reporting.10

Adapted from Falk, R and Jennette, J. 2010 J Am Soc Nephrol

TESTING FOR ANTINUCLEAR ANTIBODIES |

An introduction to ANCA-associated vasculitis...Continued

ANCA testing
The International Consensus Statement on Testing
and Reporting of ANCA was created to enhance the
diagnostic usefulness of ANCA testing in patients
suspected of having vasculitis. Testing for ANCA in the
presence of certain clinical indications (Table 1) yields
a high positive predictive value. Guidelines stipulate
testing samples with IIF on ethanol fixed leukocytes (or
purified neutrophils) and PR3/MPO by solid phase assay
such as ELISA (Table 2). Testing all IIF positive samples
by ELISA produces sensitivities of 73% and 67% for GPA
and MPA, respectively, and a diagnostic specificity of
99%.9

IN CONCLUSION
In conclusion:

ANCA are the serological hallmarks of idiopathic systemic vasculitis, and the term AAV has been
used to collectively name those primarly small
vessel vasculitic syndromes in which circulating
ANCA are commonly found.
Names of the common forms of vasculitis have
been recently revised so that the eponyms such
as Wegeners granulamatosis and Churg Strauss
syndrome have been changed with granulomatosis with polyangiitis (GPA) and eosinophilic granulomatosis with polyangiitis (EGPA), respectively.

Table 1. Indications for ANCA testing

Clinical indication for ANCA testing

The International Consensus Statement on

Testing and Reporting of ANCA recommends
screening by ANCA by IIF and to confirm any
positivity by both PR3 and MPO ELISA.

Glomerulonephritis, especially rapidly progressive

glomerulonephritis
Pulmonary hemorrage
Cutaneous vasculitis
Multiple lung nodules
Chronic destructive disease of the upper airways
Long standing sinusitis or otitis
Subglottic tracheal stenosis
Peripheral neuropathy
Retro-orbital mass
Other possible indications for ANCA testing
Pulmonary fibrosis, with systemic features
Episcleritis, uveitis, retinal vasculitis, with systemic features

Table 2. Summary of the international consensus statement on

testing of ANCA
Minimum requirements
Perform IIF on all sera; 10% of ANCA-positive sera are
detected only by IIF
Samples with IIF positivity should immediately be tested for
both PR3 and MPO ELISA
Optimal requirements
IIF titration should be performed for sera positive only by IIF
The inclusion of the most recent positive serum in the IIF or
ELISA studies may be useful in demonstrating a change in
antibody level

4 |INOVA NEWS No. 8

References
1.

Radice A, Sinico RA Antineutrophil cytoplasmic antibodies (ANCA).

Autoimmunity 2005; 38(1):93-103.
2. Wiik A. Delineation of a standard procedure for indirect
immunofluorescence detection of ANCA. APMIS 1989; 6:1213.
3. Radice A, Bianchi L, Sinico RA. Anti-neutrophil cytoplasmic
autoantibodies: methodological aspects and clinical significance in
systemic vasculitis.Autoimmunity Rew 2013; 12(4):487-495
4. Radice A, Sabadini E, Sinico RA. Antineutrophil cytoplasmic
autoantibodies with specificity for proteinase 3. In: Y. Shoenfeld, M.E.
Gershwin, P.L. Meroni (Eds), Autoantibodies 2007 (2nd ed), Elsevier B.V;
14:105110.
5. Kallenberg CGM. Antineutrophil cytoplasmic autoantibodies with
specificity for myeloperoxidase. In: Y. Shoenfeld, M.E. Gershwin, P.L.
Meroni (Eds), Autoantibodies 2007 (2nd ed), Elsevier B.V; 14:95-103.
6. Falk, R, Jennette JC. ANCA disease: where is this field heading? J Am Soc
Nephrol 2010; 5:745-752.
7. Seo P, Stone JH.The Antineutrophil Cytoplasmic Antibody-Associated
Vasculitides. Am J Med 2004; 117:39-50.
8. Jennette JC, Falk RJ, Hu P, Xiao H. Pathogenesis of antineutrophil
cytoplasmic autoantibody-associated small-vessel vasculitis. Annu Rev
Pathol. 2013 Jan 24;8:139-60.
9. 2012 Revised International Chapel Hill Consensus Conference
Nomenclature of Vasculitides. Jennette JC, Falk RJ, Bacon PA , Basu N ,
Cid MC, Ferrario MC et al. Arthritis & Rheum 2013; 65(1):1-11.
10. Savige J, Gillis D, Benson E, Davies D, Esnault V, Falk RJ et al. International
Consensus Statement on Testing and Reporting of Antineutrophil
Cytoplasmic Antibodies (ANCA). Am J Clin Pathol 1999; 111:507513.

Diagnostic
methods for
ANCA: past and
present
Johannes Schulte-Pelkum, PhD
Director of Research and Development - Immunopathology
INOVA Diagnostics
San Diego, CA

Introduction
Antineutrophil cytoplasmic antibodies (ANCA) are one
of the most common causes of small vessel vasculitis
and are diagnostic hallmarks of microscopic polyangiitis (MPA), Wegeners Granulomatosis1,more recently
referred to as granulomatosis with polyangiitis (GPA)2
and Churg Strauss syndrome, now called eosinophilic
granulomatosis with polyangiitis (EGPA).
ANCA were first reported in 1982 by Davies et al.3 and
investigated to a greater extent in 1985 by van der
Woude et al.4 Myeloperoxidase (MPO) and proteinase 3 (PR3) were soon discovered as major antigenic
targets for ANCA in neutrophils. After these antigens
were described,5, 6 continual developments were made
to improve associated diagnostic tests. The importance of serological testing cannot be underestimated
in patients suffering from vasculitis. These individuals
must be swiftly diagnosed and proper therapy must be
administered to avoid renal failure.7, 8
The initial screening method for these autoantibodies is,
according to the International Consensus Statement on
Testing and Reporting of ANCA, IIF on fixed neutrophils of
human origin. The result of an initial IIF screening is verified
by ELISA.9 In the past, second and third generation tests
often did not reach the sensitivity and specificity of IIF on
ethanol or acetone fixed neutrophils,10, 11 but new methods
such as lateral flow and multiplex assays are now available
with increased sensitivity and specificity.12, 13 The major
contribution for the improved performance was the result
of different immobilization strageties culminating in better
exposure of relevant epitopes.

The requirement of sample batching for ELISA has now

driven innovation to random access test systems with
reduction in assay times. Systems combining random
access and chemiluminiscent immunoassay (CIA)
technology have been developed and offer
drastically reduced assay times of just 30 minutes.14

First generation ELISA Tests

After the target proteins for pANCA and cANCA were
described,5, 6 the first ELISA was developed using
purified native PR3 and MPO antigens of various origin.
These assays used simple direct coating methods and
the purity of the antigens often was not very high.
Most of all the tests lacked comparability of results and
did not show strong correlation to IIF methods.10 This
lack of sensitivity was the result of different epitopes
on the PR3 antigen being blocked by the direct
binding process. The lack of standardization led to the
International Consensus Statement on Testing and
Reporting of ANCA by a combination of ELISA and IIF
test methods.9

Second and third generation tests

Second generation ANCA tests utilize a capture ligand,
generally consisting of a monoclonal antibody directed towards a specific epitope. This method allows the
protein to retain its three dimensional structure15 while
providing significantly higher sensitivity compared
to first generation tests. Protein modification such as
biotinylation led to third generation ELISAs, also aimed
to prevent protein distortion. Spacer molecules are
attached to PR3 antigen16 and the complex binds to the
surface of the ELISA plate by streptavidin coupling. This
method further increased the sensitivity and specificity
and showed improved correlation with IIF.17

Other ELISA methods

An alternative approach to achieve sensitivity comparable to IIF is a direct coated ELISA using native purified
PR3 in mixture with a recombinant human PR3 antigen.
This method purportedly offers significantly higher
sensitivity compared to first generation direct coated
ELISA tests and second generation capture ELISA. A
promising performance analysis of this method was
published by Damoiseaux et al. in 2009.18 However,
a recent study presented at the 16th international Vasculitis & ANCA Workshop in Paris did not show
a significantly higher diagnostic performance of this
approach.19
TESTING FOR ANTINUCLEAR ANTIBODIES |

Diagnostic methods for ANCA: past and present...Continued

Lateral flow and multiplex assays
Historically, lateral flow assays (LFA) were regarded as fast,
but lacking sensitivity compared to ELISA tests.20-22 The
development of liquid antigens for LFA has significantly
improved their diagnostic performance. The sensitivity of
LFA is comparable to that of third generation ELISA tests
with a result time of 20 minutes.12,13 Since LFA is based
on a universal IgG with the antigens added in solution,
this test system can accommodate multiple analytes. Presenting the antigen in the liquid phase prevents
potential epitope masking, as seen in some solid
phase assays.
Multiplex technology allows for the detection of multiple antibodies in a single assay run. The antigen antibody
reaction is visualized using fluorescent dyes and is read
by two lasers, one identifying the bead and the second
exciting the fluorophore coupled to the conjugate. This
technology allows detection of up to 100 different analytes in only one reaction. For the diagnosis of ANCA, tests
are available which simultaneously measure antibodies
against PR3 and MPO. The sensitivity for PR3 is comparable to first generation solid phase ELISA, when a cut off
setting optimized for 100% specificity is used.23

ed and washed repeatedly. The isoluminol conjugate

is oxidized when sodium hydroxide and peroxide
solutions (Triggers) are added to the cuvette, and the
flash of light produced from this reaction is measured as
relative light units (RLUs) by the BIO-FLASH optical system.
The RLUs are proportional to the amount of isoluminol conjugate that is bound to the human IgG, which
is in turn proportional to the amount of autoantibodies bound to the antigen on the beads. Based on the
results of running two calibrators, an instrument specific Working Curve is created, which is used to calculate
chemiluminescent units (CUs) for each sample.
a.)

b.)

BIO-FLASH- Rapid Response

Chemiluminescent Analyzer
The QUANTA Flash PR3 and MPO assays (INOVA
Diagnostics San Diego, CA) are novel CIAs performed
on the BIO-FLASH instrument using native purified
antigens from human neutrophils (Figure 1). The CIAs
are designed for the fully automated BIO-FLASH instrument, containing a luminometer as signal detector,
as well as all the hardware and liquid handling accessories necessary to perform the assay. Native PR3
purified from human neutrophils is covalently bound
to paramagnetic particles. Native MPO purified from
human neutrophils is bound to paramagnetic particles
via an anchor molecule to improve immunoreactivity.
Patient serum is prediluted by the BIO-FLASH instrument with sample buffer in a small disposable plastic
cuvette. Small amounts of the diluted patient serum,
the beads, and the assay buffer are all combined into
a second cuvette, mixed, and then incubated for 9.5
minutes at 37 C. The magnetized beads are sedimented
and washed several times followed by addition of isoluminol conjugated antibody, and again incubated for 9.5
minutes at 37 C. The magnetized beads are sediment6 |INOVA NEWS No. 8

Figure 1. a.) Principle of the novel chemiluminescent immunoassays.14 Paramagnetic beads are coupled with native PR3 or MPO.
The beads are then incubated with diluted patient samples. After
9.5 min incubation unbound antibodies are removed by washing.
Anti-human IgG isoluminol conjugate (Tracer) is added and
binds immobilized antibodies. After another 9.5 min incubation
unbound Tracer is removed by washing. b.) Trigger 1 and Trigger
2 are injected and emerging light is measured. After injection of
Trigger 1 and Trigger 2, the luminescence is measured as relative
luminescence units (RLU).

Table 1. Agreement between QUANTA Lite ELISA and QUANTA Flash CIA
QUANTA Lite PR3

Kappa = 0.92
QUANTA Flash
PR3 CIA

Percent agreement (95% confidence)

Positive

Negative

Total

Positive

31

3*

34

Pos agreement 100% (88.8-100.0%)

Negative

37

37

Neg agreement 92.5% (79.6-98.4%)

Total

31

40

71

Total agreement 95.8% (88.1-99.1%)

*all samples were from GPA patients

QUANTA Lite MPO

Kappa = 0.83
QUANTA Flash
MPO CIA

Percent agreement (95% confidence)

Positive

Negative

Total

Positive

29

6*

35

Pos agreement 100% (88.1-100.0%)

Negative

36

36

Neg agreement 85.7% (71.5-94.6%)

Total

29

42

71

Total agreement 91.5% (92.5-96.8%)

*3 samples from MPA and 3 from GPA patients

Table 2. Agreement between QUANTA Flash and IIF

cANCA

Kappa = 0.83
QUANTA Flash
PR3 CIA

Percent agreement (95% confidence)

Positive

Negative

Total

Positive

28

6*

34

Pos agreement 100% (87.7-100.0%)

Negative

37

37

Neg agreement 86.6% (72.1-94.7%)

Total

28

43

71

Total agreement 91.5% (82.5-96.8%)

*5 samples from GPA and 1 from MPA patient

pANCA

Kappa = 0.80
QUANTA Flash
MPO CIA

Percent agreement (95% confidence)

Positive

Negative

Total

Positive

30

5*

35

Pos agreement 93.8% (79.2-99.2%)

Negative

2#

34

36

Neg agreement 87.2% (72.6-95.7%)

Total

32

39

71

Total agreement 90.1% (80.7-95.9%)

*3 samples from MPA and 2 from GPA patients #1 sample from MPA and 1 from GPA patients

Comparison of QUANTA Lite ELISA and

QUANTA Flash CIA
Seventy one samples (41 GPA and 30 MPA, N=71) were
used to compare the PR3 and MPO ANCA QUANTA
Flash CIA with QUANTA Lite PR3 and MPO ELISA.14
The positive agreement of the PR3 CIA compared to
ELISA was 100%, the negative agreement 92.5%, and
the overall agreement 95.8% (Table 1).14 The positive
percent agreement of the MPO CIA compared to the
ELISA was 100%, the negative agreement 85.7%, and
the overall agreement 91.5% (Table 1).14

Comparison of QUANTA Flash CIA with IIF

The results of the QUANTA Flash PR3 and MPO CIA were
compared to IIF results. MPO-ANCA was analyzed vs.
the presence of a pANCA pattern and PR3-ANCA vs. the
presence of cANCA. In the entire cohort, 28 samples
showed a cANCA pattern in IIF, and 34 were positive
by CIA.14 For MPO-ANCA, 32 samples showed a pANCA
pattern, and 35 were positive by CIA. The agreement with
MPO-ANCA was 90.1% for CIA (Table 2).14 For PR3-ANCA
the agreement was 91.5% for CIA (Table 2).14

TESTING FOR ANTINUCLEAR ANTIBODIES |

Diagnostic methods for ANCA: past and present...Continued

IN CONCLUSION
In conclusion:

Many algorithms have been proposed as

the best possible way to detect ANCA. The
IIF screening on fixed neutrophils followed
by verification of positive results remains
the recommended method according to the
International Consensus Statement on ANCA
Testing and Reporting.
ELISA methods often are burdened with batching and relatively long result times. Accurate,
rapid diagnosis followed by appropriate
therapy is paramount to halting the deleterious
effects of ANCA vasculitic disease. Many clinicians now desire assays which allow quick turnaround time along with diagnostic accuracy.
QUANTA Flash PR3 and MPO CIAs are sensitive and specific assays for rapid detection (30
min) of PR3 and MPO ANCA, which fulfills the
compelling need for a rapid detection system.
Moreover, these CIAs allow quantitative detection of antibodies with a broad assay range.

7.

8.

9.

10.
11.

12.

13.

14.

15.

16.
17.

18.

Reference List
1.
2.
3.
4.

5.

6.

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Granulomatosis with polyangiitis (Wegeners): an alternative name for
Wegeners granulomatosis. Arthritis Rheum 2011, 63: 863-864.
Davies DJ, Moran JE, Niall JF, Ryan GB: Segmental necrotising
glomerulonephritis with antineutrophil antibody: possible arbovirus
aetiology? Br Med J (Clin Res Ed) 1982, 285: 606.
van der Woude FJ, Rasmussen N, Lobatto S, Wiik A, Permin H, van Es
LA et al.: Autoantibodies against neutrophils and monocytes: tool for
diagnosis and marker of disease activity in Wegeners granulomatosis.
Lancet 1985, 1: 425-429.
Falk RJ, Jennette JC: Anti-neutrophil cytoplasmic autoantibodies with
specificity for myeloperoxidase in patients with systemic vasculitis and
idiopathic necrotizing and crescentic glomerulonephritis. N Engl J Med
1988, 318: 1651-1657.
Niles JL, McCluskey RT, Ahmad MF, Arnaout MA: Wegeners
granulomatosis autoantigen is a novel neutrophil serine proteinase.
Blood 1989, 74: 1888-1893.

8 |INOVA NEWS No. 8

19.

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Conrad K, Schler W, Hiepe F, Fritzler M: Myeloperoxidase Antibodies.

In Autoantibodies in Systemic Autoimmune Diseases- A Diagnostic
Reference. 2 edition. Edited by Conrad K, Schler W, Hiepe F, Fritzler M.
Pabst; 2007:111-113.
Conrad K, Schler W, Hiepe F, Fritzler M: Proteinase 3 Antibodies.
In Autoantibodies in Systemic Autoimmune Diseases- A Diagnostic
Reference. 2 edition. Edited by Conrad K, Schler W, Hiepe F, Fritzler M.
Pabst; 2007:147-149.
Savige J, Gillis D, Benson E, Davies D, Esnault V, Falk RJ et al.:
International Consensus Statement on Testing and Reporting of
Antineutrophil Cytoplasmic Antibodies (ANCA). Am J Clin Pathol 1999,
111: 507-513.
Wang G, Csernok E, De GK, Gross WL: Comparison of eight commercial
kits for quantitation of antineutrophil cytoplasmic antibodies (ANCA). J
Immunol Methods 1997, 208: 203-211.
Csernok E, Ahlquist D, Ullrich S, Gross WL: A critical evaluation of
commercial immunoassays for antineutrophil cytoplasmic antibodies
directed against proteinase 3 and myeloperoxidase in Wegeners
granulomatosis and microscopic polyangiitis. Rheumatology (Oxford)
2002, 41: 1313-1317.
Schulte-Pelkum J, Offermann N, Fooke M: New highly sensitive
and specific Lateral Flow Tests for the detection of Proteinase 3,
Myeloperoxidase and Glomerular Basement Membrane antibodies. 8th
International Congress on Autoimmunity May 9-13th 2012, Granada,
Spain 2012, Poster: 234.
Lucassen R, Schulte-Pelkum J, Petschinka M, Fooke M: New sensitive
and reliable Lateral flow assay for the detection of proteinase 3 and
myeloperoxidase antibodies. Report on the 10th Dresden Symposium
on Autoantibodies 2011, Poster: 694-695.
Mahler M, Radice A, Yang W, Bentow C, Seaman A, Bianchi L et al.:
Development and performance evaluation of novel chemiluminescence
assays for detection of anti-PR3 and anti-MPO antibodies. Clin Chim
Acta 2012, 413: 719-726.
Csernok E, Holle J, Hellmich B, Willem J, Tervaert C, Kallenberg CG et al.:
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174-180.
Avidin-Biotin Chemistry: A Handbook. 2008.
Roggenbuck D, Buettner T, Hoffmann L, Schmechta H, Reinhold
D, Conrad K: High-sensitivity detection of autoantibodies
against proteinase-3 by a novel third-generation enzyme-linked
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Damoiseaux J, Dahnrich C, Rosemann A, Probst C, Komorowski L,
Stegeman CA et al.: A novel enzyme-linked immunosorbent assay using
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Radice A, Bianchi L, Gliona S, Trezzi B, Maggiore U, Sinico RA:
Comparison of PR3 specific ANCA Assays performance for diagnosis of
Granulomatosis with polyangiitis (GPA). 16th International Vasculitis
and ANCA Workshop 2013, -Poster.
Posthuma-Trumpie GA, Korf J, van AA: Lateral flow (immuno)assay: its
strengths, weaknesses, opportunities and threats. A literature survey.
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Bonenberger J, Doumanas M: Overcoming sensitivity limitations
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Gordon J, Michel G: Analytical sensitivity limits for lateral flow
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Damoiseaux J, Vaessen M, Knapen Y, Csernok E, Stegeman CA, Van
PP et al.: Evaluation of the FIDIS vasculitis multiplex immunoassay
for diagnosis and follow-up of ANCA-associated vasculitis and
Goodpastures disease. Ann N Y Acad Sci 2007, 1109: 454-463.

ANCA testing
in emergency
setting
Lucile Musset, PhD
Director Laboratory of Immunochemistry
Pitie Salpetriere Hospital
Paris, France
Makoto Miyara, MD, PhD
Sr. Lecturer
Pitie Salpetriere Hospital
Paris, France

Introduction
Antineutrophil cytoplasmic antibodies (ANCA) are
a heterogeneous group of autoantibodies whose
antigenic targets include proteinase-3 (PR3) and myeloperoxydase (MPO).1 Detection of these autoantibodies
is useful for the diagnosis and follow-up of small vessel
vasculitis such as granulomatosis with polyangiitis
(GPA), microscopic polyangiitis (MPA), and eosinophilic
granulomatosis with polyangiitis (EGPA).2 These
autoantibodies and their respective prevalence are
reported in Table 1.3
PR3 and MPO autoantibodies can be detected using
various techniques and different multi step strategies.4 The first step is typically a screening test using
an indirect immunofluorescence (IIF) assay on human
neutrophils which have been fixed with ethanol.
Autoantibodies against antigens other than PR3 and
MPO can cause positive results with ANCA testing by
IIF and the sensitivity of IIF is not perfect. Therefore
confirmation and identification of PR3 and MPO ANCA
antibodies is very important and can be obtained using
different solid phase technologies such as immunodot,

Table 1. Prevalence of PR3 and MPO-ANCA in different groups of

small vessel vasculitis.

PR3 (%)

MPO (%)

Granulomatosis with
polyangiitis

66

24

Microscopic polyangiitis

26

58

Eosinophilic granulamtosis
with polyangiitis

10

60

enzyme linked immunosorbent assay (ELISA), multiplex

assays, or more recently by chemiluminescent immunoassay (CIA). Just as these autoantibodies are useful
tools for the diagnosis of small vessel vasculitis, they
are also useful markers for monitoring disease activity and therapy efficacy.5,6 Providing numerical laboratory results across a broad detection range is especially helpful for the management of these diseases.
Furthermore, when patients present with critical acute
symptoms having quantitative results can further aid in
making a differential diagnosis between other conditions including infection.

Limitations of IIF and ELISA for certain

patient subsets
Even though IIF assays are sensitive and widely
accepted as the first step method to be used in routine
practice for ANCA screening, they are time consuming
for technicians and inappropriate in emergency settings.
Moreover, some discrepancies between IIF and specific tests for identification of the target of these antibodies have been reported, mainly in patients undergoing
immunosuppressive treatment. Like any IIF assay, wide
intra- and inter-laboratory variability is also observed in
ANCA testing. This is due to variability from a number
of factors including; sources of neutrophils, neutrophil preparation and fixation, the conjugate reagent,
the reading system used and finally the subjectivity
and the expertise of the technician. Different PR3 and
MPO preparations and various methodologies are used
to detect these antibodies that specifically recognize
targets with conserved conformational epitopes and
overcome the challenges of IIF for patient monitoring.7, 8

TESTING FOR ANTINUCLEAR ANTIBODIES |

ANCA testing in emergency setting...Continued

ANCA testing in an emergency setting

Rapid and accurate results using the

BIO-FLASH chemiluminescent analyzer

ANCA associated vasculitidies are chronic, multi-systemic, disorders often affecting several organs, characterized by flares and remissions. However, despite this
chronic nature, ANCA testing can be requested in
emergency cases during acute stage flare-ups. Such
testing is critical in order to quickly support a differential diagnosis and initiate appropriate immunosuppressive therapies in order to avoid irreversible organ
damage.4, 9 Additionally, the clinical manifestations of
vasculitis can be severe including alveolar hemorrhage,
glomerulonephritis, scleritis, and necrotizing sinusitis.
Therefore, it is of the utmost importance to report ANCA
results in a timely manner to aid in prompt diagnosis
and treatment.

We have recently analyzed a chemiluminescence immunoassay developed on an automated analyzer for the
detection of PR3 and MPO-ANCA. A retrospective study
from 74 sera corresponding to 45 patients with ANCA
associated vasculitis (32 GPA, 13 MPA) and 62 sera from
healthy and control diseases (systemic lupus erythematosus, Sjgrens syndrome, healthy donors) was performed.
Sera were tested using the QUANTA Flash PR3 and MPO
assays on the BIO-FLASH chemiluminescent analyzer (INOVA Diagnostics). Results were compared to those
obtained using routine multiplex assays FidisTM Vasculitis
Panel (BMD) performed using the Luminex analyser in our
laboratory (Figure 1).

Since relapse of ANCA associated vasculitis occurs

in 30-60% of cases, ANCA testing is also required for
follow-up. According to disease stage and activity, serial
testing is required at intervals of several months (three
to six months). Furthermore, ANCA testing is usually
ordered to assess the side effects of immunosuppressive therapy, such as malignancies, or for differential
diagnosis with undercurrent infectious diseases.10,11

The sensitivity and the specificity of the QUANTA Flash

assay was 78.4% and 83.4%, respectively. The sensitivity and the specificity of routine technique was 73% and
80.1%, respectively. A good agreement was observed
for MPO (92.9% kappa =0.82) and for PR3 (96.4%
kappa=0.92). Four samples were PR3-ANCA positive by
QUANTA Flash and negative by multiplex. Eight samples
were MPO-ANCA positive by multiplex and negative

Figure 1. Comparison of PR3 and MPO-ANCA tested on BIO-FLASH and Fidis Vasculitis Panel.
Units are expressed as Calculated Units (CU) and Luminex Units (LU/mL)

10 | INOVA NEWS No. 8

BIO-FLASH

BIO-FLASH

by QUANTA Flash (Figure 2). Those samples were from

four patients with treated ANCA associated vasculitis
(two GPA, one mononeuritis simplex and one alveolar haemorrhage) and four patients with inflammatory/autoimmune diseases (Graves disease, psoriatic
rheumatism, giant cell arteritis and Sjgrens syndrome).
Four samples were PR3-ANCA positive by CIA and
negative by multiplex, coming from patients with
treated ANCA-associated vasculitis (two GPA, one MPA
and one EGPA).

PR3

Neg

Multiplex
Pos

Neg

70

Total
70

Pos

39

43

Total

74

39

113

MPO

Neg

Multiplex
Pos

Total

Neg

79

87

Pos

26

26

Total

79

34

113

Figure 2. Concordance analysis for PR3 and MPO-ANCA tested on

BIO-FLASH and Fidis Vasculitis Panel. Neg: negative; Pos: positive.

Utility of BIO-FLASH in an emergency

setting
For assessment of practicability of the BIO-FLASH in
an emergency setting, inexperienced residents at our
hospital were recruited to evaluate the functionality and ease of use of BIO-FLASH. After a short briefing lasting 15 minutes, the residents were able to
perform the analysis and retrieve results within 30
minutes. The QUANTA Flash PR3 and MPO assays on the
BIO-FLASH system display good sensitivity and specificity combined with high agreement to routine methods.
Moreover, the retrieval of results was rapid and easy to
obtain, making the technique suitable for emergency settings as well as for the follow-up of patients in
routine practice.

IN CONCLUSION
In conclusion:

ANCA testing by IIF is an important first step

when screening, positive results or cases of
strong clinical suspicion should be run on a
solid phase assay for confirmation.
When monitoring disease activity or therapy
efficacy, a methodology other than IIF should
be used to allow for numerical results.
Acute disease flares can be confounding and
adequate testing methods in an emergency
setting can impact patient outcomes.
A method such as BIO-FLASH which is rapid,
accurate, and can be performed by all levels of
laboratory staff is ideally suited for urgent as
well as monitoring requests for ANCA testing.

References
1.

Kallenberg CG. Pathogenesis of ANCA-associated vasculitis. Ann Rheum

Dis 2011 ; 70 Suppl1 : i59-63.
2. Watts RA, Scott DG. Recent developments in the classification and
assessment of vasculitis. Best Pract Res Clin Rheumatol. 2009;23:429-43.
3. Hagen EC, Daha MR, Hermans J, Andrassy K, Csernok E, Gaskin G et al.
Diagnostic value of standardized asays for anti-neutrophil cytoplasmic
antibodies in idiopathic systemic vasculitis. Kidney Int 1998; 53:743-53.
4. Vermeersch P, Vervaeke S, Blockmans D, et al. Determination of
anti-neutrophil cytoplamsic antibodies in small vessel vasculitis:
comparative analysis of different strategies. Clin Chim Acta 2008;397:7781.
5. Hellmich B, Flossmann O, Gross WL et al. EULAR recommendations for
conducting clinical studies and/or clinical trials in systemic vasculitis:
focus on anti-neutrophil cytoplasm antibody-associated vasculitis. Ann
Rheum Dis 2007; 66: 605-17.
6. Mukhtyar C, Guillevin L, Cid MC, Dasgupta B, De Groot K, Gross WL et
al. EULAR recommendations for the management of primary small and
medium vessel vasulitis. Ann Rheum Dis 2009; 68: 310-7.
7. Damoiseaux J, Dahnrich C, Rosemann A et al. A novel enzymelinked immunosorbent assay using a mixture of human native and
recombinant protinase-3significantly improves the diagnostic
potential for antineutrophil cytoplasmic antibody-associated vasculitis.
Ann Rheum Dis 2009;68:228-33.
8. Mahler M, Radice A, Yang W, Bentow C, Seaman A, Bianchi L, Sinico RA.
Development and performance evaluation of novel chemiluminescence
assays for detection of anti-PR3 anti-MPO antibodies. Clin Chim Acta
2012; 413: 719-26.
9. Seck SM, Dussol B, Brunet P, Burtey S. Clinical features and outcomes
of ANCA-associated renal vasculitis. Saudi J Kidney Dis Transpl. 2012
Mar;23(2):301-5.
10. Terrier B, Saadoun D, Sne D, Ghillani P, Amoura Z, Deray G, Fautrel B,
Piette JC, Cacoub P. Antimyeloperoxidase antibodies are a useful marker
of disease activity in antineutrophil cytoplasmic antibody-associated
vasculitides. Ann Rheum Dis 2009; 68:1564-1571.
11. Luxton G, Langham R. ANCA serology in the diagnosis and managment
of ANCA-associated renal vasculitis. Nephrology. 2008;13 (Suppl
2):S17-S23.

TESTING FOR ANTINUCLEAR ANTIBODIES | 11

A new look at
ANCA testing
with NOVA
View*
Gabriella Lakos, MD, PhD
Medical Director
INOVA Diagnostics
San Diego, CA
Carol Buchner, MT (ASCP)
Manager, IFA Development
INOVA Diagnostics
San Diego, CA

Introduction
The International Consensus Statement on Testing and
Reporting of Antineutrophil Cytoplasmic Antibodies
(ANCA)1, 2 states that the minimum requirements for
ANCA testing is that IIF should be performed on all sera
from new patients, since 10% of ANCA positive sera
in patients with Wegeners granulomatosis or microscopic polyangiitis can be demonstrated only by IIF.
(Wegeners granulomatosis has recently been renamed
as granulomatosis with polyangiitis (GPA)3). In spite
of these recommendations, laboratory strategy for the
detection of ANCA varies across laboratories according to geographical areas, traditions and local experience. New antigen-specific solid phase assays are highly
sensitive and specific,4 but the performance characteristics of available assays vary widely.5, 6

*NOVA View is not available for sale in the US

12 | INOVA NEWS No. 8

Challenges of IIF method for detection

of ANCA
The IIF method (whether it is used for the detection of
antinuclear antibodies (ANA), ANCA, or organ-specific antibodies) occupies a special not very popular
place in the laboratory. First, both slide preparation and
slide reading requires significant manual processes,
which is time consuming and prone to technical issues.
The manual nature of slide preparation and slide
reading, coupled with handwritten transcription of
results in the dark room is the primary source of sample
mixups and erroneous result reporting. Furthermore,
some (predominantly homogeneous) ANA samples
may produce patterns identical to those produced by
pANCA. This is one of the reasons why the interpretation of ANCA IIF images is challenging and requires the
review of results on both ethanol and formalin fixed
substrates.
After successful launch of the ANA module on NOVA
View, INOVA Diagnostics has developed a new application that allows the use of NOVA View automated
fluorescent microscope for ANCA testing. NOVA View
with ANCA software module and NOVA Lite ANCA
reagents are used together as a system to bring ANCA
IIF testing to a level that meets the expectations of
modern autoimmune laboratories.

NOVA View with ANCA module

The NOVA View system contains a fluorescent microscope with a motorized stage, LED light source, and a
CCD (charge-coupled device) digital camera. The NOVA
View software controls the microscope and the camera.
The system acquires high resolution digital images of
the substrate, archives them, and displays them on the
computer screen for the user for review. NOVA View
measures the light intensity of the images, and preliminarily categorizes the samples as negative or positive
according to a pre-programmed cutoff. The NOVA View
software also provides pattern recognition for positive
samples based on software algorithm. The ability of
the operator to manage (enlarge, overlay) the acquired
digital images facilitates interpretation. Importantly,
NOVA View does not report final results without
confirmation from a trained technician.
The NOVA Lite Ethanol ANCA kit with DAPI and NOVA
Lite Formalin ANCA kit with DAPI are enhanced versions
of the NOVA Lite ANCA kits, that are IVD licensed for

Use of the NOVA Lite ANCA DAPI kits with QUANTA-Lyser and NOVA View simplifies and
streamlines the IIF reading/interpretation workflow, by:
Reducing hands-on time in the slide reading process.
Supporting the interpretation of the samples with
objective information.
Eliminating the need for darkroom.

manual use. Cell density in the wells was increased and

standardized. The conjugate in the kit contains DAPI
(4,6-diamidino-2-phenylindole), a blue fluorescent dye
that stains the nuclei. As images are taken by NOVA
View with both the DAPI and the FITC filters, every
image is available in both colors which can be overlaid
to facilitate pattern identification, and can be used to
confirm the presence of cells on negative wells.

Eliminating transcription errors with

barcoded slides.
Separating image acquisition from image
interpretation.

displayed for the user. NOVA View identifies nuclear and

cytoplasmic staining patterns. In the case of non-characteristic or mixed patterns (such as in the presence of
certain ANAs), NOVA View reports positive result with
unrecognized patterns.

Each kit contains twenty, 12-well slides (Figure 1). Each

slide contains a two-dimensional barcode that designates the slide type (ethanol- or formalin-fixed, 12-well),
and has a unique identifier, allowing the identification of the slide and linking of patient information
to the wells (Figure 1). These slides can be processed
by automated slide processing instruments, such as
QUANTA-Lyser. QUANTA-Lyser is programmed to read
IFA slide barcodes to confirm the substrate type, and
to match patient information received from the
laboratory information system (LIS) to a specific well
location on the slide.
These unique features ensure sample traceability, and
deliver positive patient identification for IFA processing and analysis. QUANTA Link, a middleware software
facilitates communication between the LIS, QUANTALyser, and other lab instruments, thereby providing the
foundation of a fully integrated autoimmune laboratory.

ANCA module characteristics and user

interface
During slide reading, NOVA View analyzes at least 25 cells,
and acquires at least three representative digital images.
Very bright samples will undergo a second imaging,
when images are captured with reduced (optimal)
exposure time to facilitate pattern recognition. After
finishing the imaging process, the measured LIU and
the suggested result (negative/positive and pattern) are

Figure 1. 12-well barcoded ANCA slides containing

ethanol-fixed (E-ANCA) and formalin-fixed
(F-ANCA) granulocytes.

TESTING FOR ANTINUCLEAR ANTIBODIES | 13

A new look at ANCA testing... Continued

Figure 2 shows a representative screen shot of a pANCA
positive sample, as the user sees it. During the image
review process, the operator can enlarge and overlay
DAPI and FITC images (Figure 2). After review, the operator has two choices: either confirming the NOVA View
suggested result, or revising it (regarding both negativity/positivity and pattern) and then confirming. The
software contains five customizable buttons that the
users can pre-program according to their local reporting nomenclature (for example, pANCA, cANCA, atypical ANCA, Possible ANA, or See Comment). For each
sample, a comment can be entered that becomes part
of the reported results. Results can be transferred to the
LIS system or a patient report can be directly generated
by the NOVA View.

QUANTA Link and the Multi-Analyte Screen

QUANTA Link can greatly enhance the IIF experience with the NOVA View, and facilitates ANCA interpretation. One important feature of QUANTA Link is
the Multi-Analyte Screen. This feature brings together digital images generated on the same sample by
various assays. In the case of ANCA, images obtained
on ethanol-fixed and formalin-fixed substrates can be
displayed next to each other, and if HEp-2 ANA results
are available, this can also be added to the same screen.
Having these images on the same screen at the same
time allows the interpretation of ANCA IIF results with
high accuracy. Figure 3 shows an example that was
generated on a cANCA and speckled ANA double
positive sera sample.

Performance characteristics
During the establishment and validation of the ANCA
module performance, the cut-off LIU was determined
separately for ethanol- and formalin-fixed slides on 120
reference specimens.

Precision for both substrate types was examined

on three samples, run for five days in triplicate with
two runs per day. All results were within one grade
difference based on digital image reading.

Accuracy was assessed by determining the

endpoint titer of six positive samples based on LIU
value, digital image reading, and manual image
reading. Endpoints were within one dilution step
from each other in each comparison.

In a clinical validation study, samples from 80

healthy controls, 20 infectious disease patients,
and 21 MPO/PR3 positive patients were assayed.
Performance is shown in Table 1.

Figure 2. Representative user interface showing a DAPI/FITC

overlaid image of a pANCA positive sample.

Figure 3. Representative screenshot of the Multi-Analyte

Screen from QUANTA Link showing a cANCA and
speckled ANA double positive sample.

14 | INOVA NEWS No. 8

Table 1. Clinical performance of NOVA View with formalin-fixed and ethanol-fixed neutrophil substrate
% sensitivity or specificity (number of positive/tested samples)
Characteristics

Formalin-fixed neutrophil substrate.

Ethanol-fixed neutrophil substrate.

Relative sensitivity based on LIU cutoff

76.2% (16/21)

90.5% (19/21)

Relative sensitivity based on digital image reading

90.5% (19/21)

95.2% (20/21)

Relative specificity based on LIU cutoff

100.0% (80/80)

96.3% (77/80)

Relative specificity based on digital image reading

100.0% (80/80)

90.0% (72/80)

Agreement between LIU-based NOVA View results

and digital image reading

96.7% (117/121)

93.3% (112/120)

Agreement between digital image reading and

manual (microscopic) reading

90.9% (110/121)

93.4% (113/121)

IN CONCLUSION
In conclusion:

NOVA View is an automated solution for

autoimmune laboratories performing IIF assays.
It simplifies and streamlines the IIF reading/
interpretation workflow, and increases the
safety of IIF testing by sample traceability.
The newly developed ANCA module acquires
high resolution digital images on ANCA slides,
and provides the user with tools that facilities
image interpretation.
The ANCA module showed reliable and reproducible performance during evaluation studies.

References
1. Savige J, Gillis D, Benson E, Davies D, Esnault V, Falk RJ, Hagen EC, Jayne D,
Jennette JC, Paspaliaris B, Pollock W, Pusey C, Savage CO, Silvestrini R, van
der Woude F, Wieslander J, Wiik A. International Consensus Statement on
Testing and Reporting of Antineutrophil Cytoplasmic Antibodies (ANCA).
Am J Clin Pathol. 1999;111:507-13.
2. Savige J, Dimech W, Fritzler M, Goeken J, Hagen EC, Jennette JC, McEvoy
R, Pusey C, Pollock W, Trevisin M, Wiik A, Wong R; International Group for
Consensus Statement on Testing and Reporting of Antineutrophil Cytoplasmic Antibodies (ANCA). Addendum to the International Consensus
Statement on testing and reporting of antineutrophil cytoplasmic antibodies. Quality control guidelines, comments, and recommendations for
testing in other autoimmune diseases. Am J Clin Pathol. 2003;120:312-8.
3. Falk RJ, Gross WL, Guillevin L, Hoffman GS, Jayne DR, Jennette JC, Kallenberg CG, Luqmani R, Mahr AD, Matteson EL, Merkel PA, Specks U, Watts
RA; American College of Rheumatology; American Society of Nephrology;
European League Against Rheumatism. Granulomatosis with polyangiitis
(Wegeners): an alternative name for Wegeners granulomatosis. Arthritis
Rheum. 2011;63:863-4.
4. Mahler M, Radice A, Yang W, Bentow C, Seaman A, Bianchi L, Sinico RA.
Development and performance evaluation of novel chemiluminescence
assays for detection of anti-PR3 and anti-MPO antibodies. Clin Chim Acta.
2012;413:719-26.
5. Wang G, Csernok E, de Groot K, Gross WL. Comparison of eight commercial kits for quantitation of antineutrophil cytoplasmic antibodies (ANCA).
J Immunol Methods. 1997;208:203-11.
6. Holle JU, Herrmann K, Gross WL, Csernok E. Comparative analysis of
different commercial ELISA systems for the detection of anti-neutrophil
cytoplasm antibodies in ANCA-associated vasculitides. Clin Exp Rheumatol. 2012;30(1 Suppl 70):S66-9.

TESTING FOR ANTINUCLEAR ANTIBODIES | 15

Anti-PR3
antibodies
in ulcerative
colitis
Michael Mahler, PhD
Vice President Research & Development
INOVA Diagnostics, San Diego, CA
Gary L. Norman, PhD
Director Research & Development
INOVA Diagnostics, San Diego, CA
Marcos Lopez-Hoyos, MD, PhD
Director Clinical Laboratory Hospital Universitario
Marques de Valdecilla
Santander, Spain

Introduction
Antineutrophil cytoplasmic antibodies (ANCA) directed against proteinase 3 (PR3) or myeloperoxidase
(MPO) are used in the diagnostic workup of small vessel
vasculitis (SVV), such as granulomatosis with polyangiitis (GPA), and microscopic polyangiitis.1 Conventional
screening for anti-PR3- and MPO-antibodies starts
with the indirect immunofluorescence assay (IIF) on
ethanol-fixed human neutrophils followed by confirmatory ELISA.1, 2 Anti-PR3 antibodies generate a cytoplasmic staining pattern (cANCA) on ethanol-fixed human
neutrophils, whereas anti-MPO antibodies generate a
perinuclear staining (pANCA) pattern.1 Many laboratories also evaluate specimens on formalin-fixed human
neutrophils.3

16 | INOVA NEWS No. 8

Biomarkers for inflammatory bowel disease

An atypical pANCA pattern, sometimes referred to as
xANCA,4 is found in patients with inflammatory bowel
disease (IBD), mainly in patients with ulcerative colitis
(UC). When combined with anti-Saccharomyces cerevisiae antibodies (ASCA), atypical pANCA has been recommended as a way to help distinguish UC from Crohns
disease (CrD).3, 5-9 ASCA sero-positivity is a predominant
feature of CrD, while atypical pANCA is a marker of UC.3,7
Both ASCA and ANCA have been reported to predict
the development of IBD.10 Despite many studies, the
specificity of ANCA in IBD remains poorly defined.5, 11
The diagnosis of IBD including UC and CrD is largely
based on endoscopic and histological assessment of the
inflamed tissue.5, 12 While several antibody tests can assist
in the diagnosis of CrD including (ASCA) and pancreatic
major zymogen granule protein 2 (MZGP2),13-15 the only
serological biomarker for UC is atypical pANCA detected by IIF.3 However, IIF is time consuming and observer-dependent, has low throughput requiring highlytrained personnel, can generate significant variation16
and demonstrates limited specificity.6, 17 IIF is also unable
to provide information about ANCA antigen specificity.5,6
Several studies have aimed to identify the major target
antigen of atypical pANCA in IBD,11, 18 but major diseasespecific target antigens are are yet to be identified.
Table 1. Prevalence of autoantibodies in ulcerative colitis and
Crohns disease5, 14

Marker
pANCA
ASCA IgG
ASCA IgA
PR3-ANCA
MZGP2

Ulcerative colitis
40-60%
5-10%
5-10%
15-40%
0-5%

Crohns disease
5-20%
40-70%
40-70%
0-10%
20-30%

Detection methods for PR3-ANCA

Over the last decade, a variety of different methodologies have been developed and commercialized for
the detection of anti-PR3 antibodies, including IIF on
human neutrophils, ELISA,19 line immunoassays (LIA),20
capture21-22 and anchor ELISAs23-24 as well as multiplex
assays25-27 using native purified PR3 and, more recently,
recombinant PR3 antigen.28 Despite several comparative studies, it remains debatable as to which methodology for anti-PR3 antibodies detection provides the

 Chronic, lifelong disease

1-1.5 million people in USA
Substantial Morbidity and health care utilizationa
E

ASCA IgG/IgA
F

Crohns
Disease (CrD)
MZGP2

pANCA
Indeterminate
Colitis

UC-like
CD

Ulcerative
Colitis (UC)

PR3

Whole GI tract (terminal ileum)

Only large bowel

Discontinuous

Continuous

Transmural inflammation

Mucosal inflammation

Extraintestinal manifestations (skin,

eyes, joints) and colorectal malignancy

Extraintestinal manifestations (skin,

eyes, joints) and colorectal malignancy

Figure 1. Differentiation between ulcerative colitis (UC) and Crohn`s disease (CrD. The clinical difference between UC and CrD and the
potential role of PR3-ANCA as a marker to help in the diagnosis of UC is illustrated

highest clinical accuracy for the diagnosis of GPA.19, 29

Several studies published over the last decade suggested that the sensitivity of both capture as well as novel
anchor assays were superior to classical ELISA and even
to IIF.19, 23-24, 30-31
PR3-ANCA as a marker for UC
In contrast to historical data,32 more recent studies
reported anti-PR3 antibodies in a significant percentage of IBD patients.27 This raises the possibility that
akin to anti-PR3 antibodies as a marker for SVV, antiPR3 antibodies may also be a marker for IBD. Recently
it was suggested that anti-PR3 antibodies measured
by a novel chemiluminescent immunoassay (CIA) on a
random access auto-analyzer (BIO-FLASH) are useful
in the differential diagnosis of UC and CrD.33 Our observation that anti-PR3 antibodies can be detected in
sera from patients with IBD, with higher prevalence in
UC vs. CrD patients, suggests that anti-PR3 antibodies
testing could assist in discriminating UC from CrD, and
in discriminating IBD from other gastrointestinal conditions. The terms indeterminate colitis or IBD unclassified (IBD-U) categorize patients in whom the diagnosis
of UC or CrD is not clear.5, 17, 34 The differential diagnosis may be complicated in patients with irritable bowel

syndrome, celiac disease, or other colorectal diseases, with

features indistinguishable from those seen in patients
with IBD.5
Studies of anti-PR3 antibodies in IBD are limited and
have been based on relatively small cohorts of UC
patients.27, 35-36 When twelve anti-PR3 antibody assays
were compared using 22 IBD sera, the reported prevalence of anti-PR3 antibodies ranged from 4% to 43%,
raising concerns as to the reliability of the assays used in
these studies.27, 35-36 The two assays with highest sensitivity (BINDAZYME Anti-PR3 39% and Rainbow ELISA
PR3 43%) also showed the lowest specificity (88%). In a
recent study it was found that anti-PR3 antibodies are
found in patients with IBD, albeit at lower titers than in
patients with GPA. This was demonstrated using three
separate and independent fully automated platforms
for autoantibody detection. The prevalence of the
antibodies differed between the methods used, but
this was mainly related to the way the manufacturerspecific cut-off was defined (focused on high specificity
for GPA).

TESTING FOR ANTINUCLEAR ANTIBODIES | 17

Anti-PR3 antibodies in ulcerative colitis...Continued

Differentiation between PR3-ANCA positive
UC and GPA patients
The fact that anti-PR3 antibodies are found in UC could
be interpreted as compromising the specificity of antiPR3 antibodies for GPA. Anti-PR3 antibodies in patients
with GPA are often associated with a cANCA pattern on
ethanol-fixed neutrophils, while in UC patients an atypical pANCA is most often observed. The latter is most
likely explained by reactivity to other antigens that have
been reported in the past to be associated with UC.37
Interestingly, recent studies have described patients
with overlapping features of UC and GPA.38-40 To what
extent anti-PR3 antibody positive patients with UC will
develop full-blown SVV over the course of their disease,
needs to be assessed in large longitudinal studies. It is
widely appreciated that different autoimmune diseases can overlap in some patients, which was recently
described as poly-autoimmunity.41 Therefore, additional studies are required to determine whether there is an
overlap between the two chronic inflammatory diseases. While GPA typically affects the upper respiratory
tract and the kidneys, UC typically is an inflammatory
disease limited to the colon. Although 10% of patients
with SVV can present with ulcerations of the colon,42
isolated gastro intestinal tract involvement is infrequently seen in ANCA-positive patients with SVV.43

Abbreviations
ASCA, anti-Saccharomyces cerevisiae antibody; AUC, Area under the
curve; cANCA, cytoplasmic anti-neutrophil cytoplasmic antibodies;
CIA, chemiluminescence assay; CrD, Crohn`s disease; CU, calculated units; GPA, granulomatosis with polyangiitis; IBD, inflammatory bowel disease; IIF, indirect immunofluorescence assay; LIA, line
immunoassay; LR, likelihood ratio; pANCA, perinuclear anti-neutrophil cytoplasmic antibodies ROC, Receiver Operating Characteristic;
UC, ulcerative colitis; WG, Wegeners granulomatosis

18 | INOVA NEWS No. 8

IN CONCLUSION
In conclusion:

The only serological marker for UC is atypical

pANCA detected by IIF. However, IIF is observerdependent and is unable to provide information
about ANCA antigen specificity.
Anti-PR3 antibodies are present in a significant
percentage of IBD patients and may be a marker
of concurrent SVV-related disease.
Anti-PR3 antibodies measured by BIO-FLASH
CIA are useful in the differential diagnosis of
UC and CrD.

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Authors
Antonella Radice, PhD
Johannes Schulte-Pelkum, PhD
Lucile Musset, PhD
Makoto Miyara, MD, PhD
Gabriella Lakos, MD, PhD
Carol Buchner, MT (ASCP)
Michael Mahler, PhD
Gary L. Norman, PhD
Marcos Lopez-Hoyos, MD, PhD

Editor
Anna Eslami

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