1. No category

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1993 81: 1996-2002
Antineutrophil cytoplasmic autoantibodies: a review of the antigens
involved, the assays, and the clinical and possible pathogenetic
consequences
EC Hagen, BE Ballieux, LA van Es, MR Daha and FJ van der Woude
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REVIEW ARTICLE
Antineutrophil Cytoplasmic Autoantibodies: A Review of the
Antigens Involved, the Assays, and the Clinical and
Possible Pathogenetic Consequences
By E. Christiaan Hagen, Bart E.P.B. Ballieux, Leendert A. van Es, Mohammed R . Daha, and Fokke J. van der Woude
S
INCE THE ORIGINAL description of the clinical relevance of antineutrophil cytoplasmic antibodies (ANCA)
testing for patients with vasculitis and glomerulonephritis,
antibodies directed against different enzymes in granulocyte
granules have been the subject of many ongoing studies.'
Although the state of the art has been reviewed previously,2"
new findings over the last 2 years reflect the widespread interest in this new class of autoantibodies. The Fourth International Workshop on ANCA held in Liibeck, Germany in
May I992 was a clear demonstration of the recent progress
in antigen identification, the development of new ANCA assays, the association of ANCA subtypes with clinico-pathologic syndromes, and the description of in vivo and in vitro
models to study the pathogenic role of ANCA in vascular
inflammation.
In this review we summarize the present state of the art
with emphasis on the most recent findings.
ANCA-RELATED ANTIGENS
The polymorphonuclear granulocyte (PMN) contains two
main kinds of granules, the primary or a-granules and the
secondary granules. Enzymes within these granules are the
targets for ANCA. Therefore, the biochemistry of these granules will be briefly discussed. The a-granules are formed during the promyelocytic differentiation stage of the PMN. They
fuse with endosomes to form endolysosomes, cell structures
in which microbes are attacked by several granule constituents. The main a-granule proteins are the enzymes myeloperoxidase and a number of serine proteases, including neutrophil-elastase, cathepsin-G, and proteinase-3. Furthermore,
lysozyme and other microbicidal enzymes, such as bacterial
permeability increasing protein or cationic protein 57, and
defensins have been shown. These granules play an important
role in the digestion of both infectious and noninfectious
agents. Monocytes possess similar granules that contain myeloperoxidase, neutrophil elastase, and proteinase-3. The
secondary granules are formed later during cellular differentiation and contain lactoferrin and vitamin B- 12 binding
protein. A number of enzymes in the granules are now recognized to be target antigens for ANCA. Depending on the
nature, charge, and distribution of the antigen in the ethanol
fixed granulocyte, ANCA of various antigenic specificities
can be detected on the basis of different staining patterns in
the indirect immune fluorescence test. Two main patterns
From the Department of Nephrology, Universit-vHospital Leiden,
Leiden, The Netherlands.
Submitted August 5, 1992; accepted January 8, I993.
Address reprint requests to Fokke J. van der Woude, MD. PhD,
Department of Nephrology, PO Box 9600, 2300 RC Leiden. The
Netherlands.
0 I993 by The American Society of Hemutology.
0006-4971/93/8 IO8-003I$3.00/0
1996
are distinguished, cytoplasmic and perinuclear or nuclear
staining. The latter pattern is caused by an artifactual redistribution of proteins to the nucleus, caused by the ethanol
fixation and airdrying of the PMNs (the standard technique).
The cytoplasmic ANCA (cANCA) pattern is characterized
by diffuse fine granular staining of the cytoplasm with an
accentuation of staining in the central area of the cell between
the nuclear lobes. The main antigen associated with the
cANCA pattern is proteinase-3. These antibodies can be
found in patients with Wegener's granulomatosis or other
forms of systemic vasculitis. However, not all cANCA sera
may react with proteinase-3 in enzyme-linked immunosorbent assay (ELISA). In a large series of Wegener's patients
presented during the Fourth International Workshop on
ANCA, 80%of the cANCA-positive sera were shown to react
with purified proteinase-3.5Another investigation showed that
13 of 37 ANCA-positive sera from patients with Wegener's
granulomatosis did not react with purified proteinase-3 or
myeloperoxidase in ELISA.6 These findings contrast with
earlier data obtained using immune affinity-purified proteinase-3' and with a capture ELISA.8 These reports showed that
almost all cANCA-positive sera reacted with proteinase-3 in
ELISA. These discrepancies could be the result of differences
in isolation techniques, causing the loss ofantigenic epitopes,
or due to technical differences in the ELISA systems used.
However, on the basis of our experience within the EEC/
BCR workgroup on ANCA, we discovered that cANCA-positive sera may contain antibodies directed against antigens
other than proteinase-3. One of 12 cANCA-positive sera from
vasculitis patients showed a strong cANCA pattern in the
IIF-test, but did not react with any of five proteinase-3 preparations. Therefore, recent reports about a lower than 100%
correlation between cANCA-positive sera and antibodies to
proteinase-35 are probably a reflection of the real situation
and are not solely based on technical differences.
The second staining pattern recognized in the indirect immune fluorescence test is the perinuclear ANCA (pANCA)
staining. The pANCA pattern was described in 1989 by Falk
and Jennette' in patients with crescentic necrotizing glomerulonephritis and systemic vasculitis. The antigen most
commonly recognized by the sera from these patients was
myeloperoxidase. However, the staining pattern described is
an artifactual one. The myeloperoxidase, although present
in the same granules as proteinase-3, redistributes in the PMN
upon ethanol fixation and sticks to the nucleus. This artifactual redistribution results in the pANCA pattern observed
when antibodies against myeloperoxidase react with these
fixed cells. However, when PMNs are fixed with paraformaldehyde, the same sera show cytoplasmic staining. Antinuclear antibodies show a nuclear staining pattern both on
ethanol-fixed and on paraformaldehyde-fixed granulocytes.
To distinguish pANCA from antinuclear antibodies, it is advised to test the sera either on paraformaldehyde-fixed PMNs
Blood, Vol 8 1, No 8 (April 15). 1993: pp 1996-2002
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ANTINEUTROPHIL CYTOPLASMIC AUTOANTIBODIES
1997
Table 1. ANCA-Disease Association
Target Antigen
ANCA
Systemic vasculitis
1. Wegener's granulomatosis
2. Microscopic polyarteritis
3. Churg Strauss syndrome
4. Classic polyarteritis nodosa
5. Other forms of systemic vasculitis
Rheumatic diseases
1. Rheumatoid arthritis
2. Systemic lupus erythematosis
Inflammatory bowel disease
1. Ulcerative colitis
2. Crohn's disease
Other diseases
1. Chronic liver diseases
2. Acute/chronic infection
3. HIV infection
Remarks
High sensitivity and specificity of cANCA for
active disease, association with disease
activity
High sensitivity for active disease
cANCA, rarely pANCA
PR-3, rarely MPO
cANCA, pANCA
pANCA
Low percentage of ANCA
Rarely
PR-3, MPO
MPO
Rarely PR-3 or MPO
No PR-3 or MPO
GS-ANA/pANCA/atypicaI
ANCA
pANCA
Unknown, ANA, rarely
MPO, LF
Rarely MPO, LF
When complicated by vasculitis often anti-LF
antibodies
ANA can cause pANCA pattern in indirect immune
fluorescence test for ANCA
pANCA/atypical ANCA
pANCA/atypical ANCA
Cath-G, LF, unknown
Cath-G, LF, unknown
Low sensitivity or specificity
Lower frequency than ulcerative colitis
pANCA/atypical ANCA
ANCA?
cANCA
Cath-G, LF, unknown
Unknown
Unknown
Abbreviations: PR-3, proteinase-3; MPO, myeloperoxidase; LF, lactoferrin; Cath-G, cathepsin-(;.
or to use a different cellular substrate than PMNs, such as
liver sections or cultured HEp-2 cells. In addition to myeloperoxidase, antibodies directed against other enzymes of the
PMN may give rise to a similar staining pattern. Although
rare, antibodies have been described against lactofemn (in
patients with systemic lupus erythematosis and rheumatoid
arthritis), neutrophil elastase (in patients with vasculitis and
Wegener's granulomatosis), and cathepsin-G (in patients with
inflammatory bowel disease and chronic hepatic disorders).
The staining pattern(s) caused by these antibodies may be
perinuclear, or present an entirely different staining pattern.
During the Fourth International ANCA Workshop, it was
decided that staining patterns other than cANCA and pANCA
were to be noted as atypical until the antibodies responsible
for such staining and their antigenic targets are further characterized and their clinical relevance shown.
It is the aim of the EC/BCR Study Group for ANCA Assay
Standardization to standardize ANCA assays within Europe.
This group of 14 laboratories in 12 countries is working cooperatively to develop well-standardized ANCA assays and
to study both the sensitivity and specificity of such assays. In
the first phase of this study, solid-phase assays for antimyeloperoxidase antibodies using commercial antigen
preparations" gave comparable results. For the detection of
anti-proteinase-3 antibodies, various antigen preparations
were compared. There were discrepanciesbetween the various
assays. When a similax antigen (a-granules) was used by different laboratories, the results were discrepant as well.Io In
the next phases of the EEC study, proteinase-3 and myeloperoxidase preparations from selected sources will be provided
to all participating centers. The use of these defined antigens
for ANCA detection will also be evaluated in a large-scale
clinical study.
ANCA ASSAYS
Although the indirect immune fluorescencetest is of proven
value for clinical application, solid-phase assays to allow largescale testing, better quantification, and determination of the
ANCA specificity are necessary for further improvement of
clinical diagnosis. In a previous review we have given an outline of the evolution of ANCA solid-phase assays until 1991.4
Currently, to determine or confirm seropositivity for cANCA,
either an a-granule extract or purified proteinase-3 are used."
Proteinase-3 may be purified by several isolation methods,
including affinity chromatography using anti-proteinase-3
monoclonal antibodies, affinity chromatography on an Orange A column, reverse-phase high-pressure liquid chromatography, gel filtration, and cation exchange chromatography." The antigen, in a complexed form with al-antitrypsin
can also be obtained in large quantities from purulent sputum
and may be used successfully in ELISA systems in this form."
ANCA-DISEASE ASSOCIATION (TABLE 1 )
Wegeners granulomatosis. ANCA were first described
in patients with glomerulonephritis in association with a viral
infe~ti0n.l~
The original report on the association of cANCA
with Wegener's granulomatosis' has since been confirmed by
numerous investigator^.'^^^'^ At present, the sensitivity for
generalized, histologically proven Wegener's granulomatosis
is believed to be around SO%, with the specificity of these
tests reaching 97%. In a recent study, values obtained for
sensitivity and specificity using consecutive laboratory samples were not higher than 71% and 84%," respectively.
Moreover, there were 95 (57%)confirmed false-positiveresults
(54 pANCA and 41 cANCA). These latter results are in line
with those obtained in an earlier study.16 Although the ANCA
indirect immune fluorescence test is still useful in the diagnostic workup of patients with symptoms and signs com-
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1998
patible with Wegener’s granulomatosis or other forms of systemic vasculitis, the ANCA assay should not be regarded as
diagnostic proof for Wegener’s granulomatosis, and efforts
should always be undertaken to obtain histologic evidence
for the disease. Immunosuppressive treatment should not be
started solely on the presence of a positive ANCA test. However, during active Wegener’s granulomatosis, it is rare to
have a negative ANCA test when using immunofluorescence.
It has been claimed that the ANCA test may be of value
for monitoring disease activity during patient follow-up. The
ANCA titer levels may increase before clinical relapse of disease occurs.17,18
The differentiation between intercurrent infection under immune suppressive therapy and relapse of the
disease may be facilitated by an increase in the ANCA titer
level. Cohen Tervaert et al” closely monitored the ANCA
titer in patients with Wegener’s granulomatosis in a prospective study. They randomized patients with more than a
twofold increase in titer levels between a group receiving early
immunosuppressive treatment and a group receiving treatment when clinical symptoms occurred.17 The group treated
early developed fewer relapses and overall used less immunosuppressive drugs than the group treated when symptoms
of disease occurred. However, Kerr et all9 investigated serial
sera obtained from a group of 53 patients. They could demonstrate a titer level increase before disease relapse in only
24% of patients. Other patients remained ANCA positive
while in complete remission, or ANCA negative while still
having active disease. These data and our own experience
warrant great caution in initiation of treatment based solely
on ANCA titers. The risk of overtreatment with cyclophosphamide and steroids is clearly present.
Other forms of systemic vasculitis. ANCA has been associated with microscopic polyarteritis as well.‘4 This form
of systemic vasculitis, defined by crescentic necrotizing glomerulonephritis in association with systemic small vessel
vasculitis, but without granulomas” is associated with both
cANCA and pANCA. Anti-proteinase-3 antibodies were reported in 46%of patients with microscopic polyarteritis, and
antimyeloperoxidase in 50%.” Microscopic polyarteritis is
not uniformly accepted as a diagnostic entity. Therefore, the
frequency of pANCA and cANCA in these patients varies
with the definition used. In the series of Hauschild et al,5 for
example, the presence of pANCA was 4 times more frequent
than cANCA.
ANCA have not been studied in large series of patients
with the Churg-Strauss syndrome, a clinical entity defined as
the combination of asthma, eosinophilia, and necrotizing
vasculitis, possibly with crescentic necrotizing glomerulonephritis or necrotizing granulomas. In small series of patients, the presence of antimyeloperoxidase antibodies has
been
In some cases of classical polyarteritis nodosa, the presence
of ANCA has been shown. O’Donoghue et a122areported
ANCA in 27%ofpatients with polyarteritis nodosa; Hauschild
et aI5showed data from 36 patients, 4 having cANCA (1 with
anti-proteinase-3), 3 with pANCA (2 anti-cathepsin-G, 1
antielastase). ANCA, therefore, seems to be an infrequent
feature in classical polyarteritis nodosa.
HAGEN ET AL
ANCA have only rarely been described in patients with
giant cell arteritis. In a recent report,23giant cell arteritis and,
to a lesser extent, polymyalgia rheumatica were associated
with cytoplasmic staining of formalin-fixed neutrophils, but
not with ethanol-fixed neutrophils. The antigen involved in
these cases is not known.
ANCA have not been described in patients with other forms
of systemic vasculitis, such as thromboangitis obliterans,
Takayasu’s disease, M. Behqet, or mixed cryoglobulinemia.
ANCA in rheumatic diseases. The first description of antineutrophil antibodies by Wilk et aIz4 concerned patients
with rheumatoid arthritis (RA) and the Felty’s syndrome.
These antibodies were described as granulocyte-specific antinuclear antibodies (GS-ANA) and were directed against the
nucleus of the PMN. The antigen recognized by GS-ANA is
still unknown. However, several studies have shown the
presence of ANCA with different staining patterns in sera
from RA patients, with various antigenic specificities. Braun
et a125found a pANCA pattern in 20% of RA cases, and 50%
of cases of Felty’s syndrome. Antibodies against other neutrophil constituents were found as well. Other investigators
have shown pANCA in RA patients with varying frequency.
We have described pANCA and atypical ANCA in 36% of
patients with active seropositive RA, and in 43% of patients
with RA complicated by vasculitis. In contrast to the cytoplasmic or perinuclear pattern, the sera showed an atypical
staining, with both perinuclear and diffuse cytoplasmic fluorescence. In the RA plus vasculitis patients, there was a
significant correlation between a positive atypical ANCA test
by indirect immune fluorescence and the presence of antilactoferrin antibodies in ELISA.26
In systemic lupus erythematosis, antinuclear antibodies
may be hard to distinguish from pANCA when ethanol-fixed
PMNs are used. However, in lupus patients, a number of
sera were reported to be pANCA positive and antinuclear
antibody negative (HEp-2 cells).27Some ofthese sera reacted
with myeloperoxidase,26whereas other sera reacted with lactoferrin.*’ The correlation of the titer of these antibodies to
the activity of the disease has not been established.
Inflammatory bowel disease. An increasing amount of
scientificdata has accumulated since the description of ANCA
in patients with ulcerative colitis (UC) and Crohn’s disease
(CD).29The original report showed that pANCA were present
mainly in UC and not in CD. During the Fourth International
Workshop, new data on these clinical entities were presented.
Positivity for pANCA can be found in 40% to 70% of sera
from patients with UC. The staining pattern of ethanol fixed
neutrophils differs from the perinuclear pattern of antimyeloperoxidase ANCA. The staining is cytoplasmic when formalin-fixed neutrophils are used.30A similar staining pattern
can be observed in 5% to 35% of patients with CD. There is
conflicting data about the relation between disease activity
and the presence or absence of ANCA. Most investigators
did not find a r e l a t i ~ n , whereas
~ ~ ~ ~ ’ Rump et a13’ showed that
ANCA disappeared after steroid treatment. Several studies
were performed to identify the antigen(s) related to the inflammatory bowel disease ANCA. In general, only few sera
reacted with proteinase-3 or myeloperoxidase in ELISA. Antibodies against lactoferrin were more frequent (10% to
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ANTINEUTROPHIL CYTOPLASMIC AUTOANTIBODIES
1999
pathogenesis of the histologic lesions encountered in Wege25%).32-34
Lesavre et a1” found anti-cathepsin-(; antibodies
ner’s granulomatosis and related diseases:
in 68% of patients with UC, but reactivity was also observed
(1) Myeloperoxidase may directly bind to cultured human
with a wide variety of chronic liver diseases. Mulder et a130
endothelial cells and remain antigenic. Savage et a148
described as yet unknown antigens with a molecular size of
showed that myeloperoxidase bound to endothelial
67 Kd and 63/54 Kd.
cells is recognized by pANCA-containing sera. Seven
From these data we can conclude that pANCA, directed
pANCA-positive sera led to complement-mediated
against both known and unknown antigens, can be found in
cytotoxicity after the addition of rabbit complement.
CD and UC, but more frequently in the latter. During the
It is thought that the initial binding of (positively
Workshop, it was decided that the ANCA found in inflamcharged) myeloperoxidase to the (negatively charged)
matory bowel disease should not yet be nominated as a special
endothelial cell surface can be explained by charge inANCA entity, but instead that more work needs to be perteractions.
formed to identify and define the target antigens involved.
(2) Proteinase-3 is possibly produced by endothelial cells;
Other diseases. pANCA have also been described in sevMayet et
reported that proteinase-3 could be shown
eral kinds of chronic liver d i ~ e a s e .The
~ ~ -antigens
~~
involved
to be present in the cytoplasm of untreated cultured
remain unclear, although Lesavre et a135found anti-cathependothelial cells using both monoclonal mouse and
sin-G in 57% to 88% of sera.
polyclonal
human antibodies in confocal laser scanning
Two reports have shown ANCA staining in patients with
microscopy. Stimulation with TNF-a led to a markedly
human immunodeficiency virus (HIV) infection. Klaassen
increased antigen expression in the Golgi region and
et ala described both cANCA and pANCA in a cross-sectional
a time-dependent translocation to the cell surface. Instudy of HIV-infected individuals. The antigens recognized
cubation of endothelial cells stimulated with TNF-a
could not be identified. Savige et a14’ confirmed these data,
with cANCA-positive sera led to enhanced procoagand found reactivity against proteinase-3 and myeloperoxiulatory activity and adhesion of neutrophils. The indase in some of the positive sera.
creased neutrophil adhesion of granulocytes to cultured
ANCAs in patients with acute infection (2 of 2 2 ) were
endothelium induced by ANCA can be inhibited by
found by Mege et al.42An earlier report showed ANCA in
monoclonal antibodies to CD 18, suggesting that inpatients with chronic airway infections. Similar findings have
tegrin molecules play an important role in this phebeen reported in patients with cystic fibrosis and purulent
nomenon .50
airway disease or in patients with pneumonia or em~yema.4~
(3)
ANCA
may activate neutrophils and thereby act as
These findings have not been confirmed by other centers.
proinflammatory
mediators. In 1973, Dale et a15’ reThe antigenic targets for such ANCAs are unknown currently.
ported that total blood pools and turnover rates were
significantly higher in patients with active Wegener’s
PATHOGENESIS AND ANCA
granulomatosis as compared with normals or patients
Although we have speculated previously’-4on the pathoon therapy. Falk et a15*showed that ANCA-positive
genetic role of ANCA, it should be made clear that there is
sera, ANCA-IgG, heterologous antimyeloperoxidase,
no definitive proof as yet for a causative role of ANCA in
and myeloperoxidase-ANCA-positive F(ab), fragany disease. In rare instances, patients with Wegener’s granments are able to stimulate the release of reactive oxulomatosis (the disease in which the occurrence of ANCA
ygen species. This stimulation is facilitated by priming
has been documented most convincingly) may present with
the neutrophils with TNF-a and is also reflected by
all known manifestations of the disease without ever develdegranulation and c h e m ~ t a x i s . ~ ~ ~ ’ ~
oping detectable cANCA titers. The association of pANCA
(4) Anti-proteinase-3 antibodies prevent the inactivation
with active disease of whatever nature is less firm.
of proteinase-3 by its natural inhibitor Q 1-antitrypsin.
Although the pathogenesis of Wegener’s granulomatosis
On the other hand, these antibodies directly decrease
remains unclear, infections of the upper airways are thought
the proteolytic activity of proteinase-3 towards large
to precipitate disease
As a result of the respiratory
s ~ b s t r a t e sThe
. ~ ~ net effect in vivo of these antibodies
tract infection, large quantities of proteinase-3 may occur in
remains uncertain, although it is conceivable that antithe sputum,12 leading to an anti-proteinase-3 immune reproteinase-3 antibodies may play a role in the widesponse in susceptible individuals. Further, cytokines such as
spread tissue necrosis observed in Wegener’s granutumor necrosis factor-a (TNF-a) are now known to translomatosis, through the interference with complex
locate ANCA-reactive proteins from the primary granules to
formation of proteinase-3 with a 1-antitrypsin.
the neutrophil cell
thereby exposing the antigen to
( 5 ) T lymphocytes of Wegener’s granulomatosis patients
the patient’s immune system. Moreover, there may be an
may proliferate in response to proteinase-3. We have
association between certain HLA class allotypes and the susreported that T lymphocytes from patients with Weceptibility for ANCA-associated disease^,^^,^' suggesting that
gener’s granulomatosis proliferate in response to proonly susceptible individuals may develop ANCA after an inteins derived from the azurophilic granule and profectious or inflammatory stimulus.
teinase-3 isolated from sputum.55 This proliferative
There are now five independent lines of evidence generated
effect was not seen in normal patient control cells.
from in vitro experiments that suggest that ANCA or ANCAThese results were later confirmed by others.56 It has
not been elucidated how T lymphocytes are involved
antigen related immunity may directly play a role in the
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
2000
HAGEN ET AL
in the production of autoantibodies and/or how tissue
destruction with granuloma formation occurs in Wegener’s granulomatosis patients.
There is little data on in vivo studies of the pathogenic
effect of ANCA. Mathieson et aI5’ reported on the Occurrence
of antimyeloperoxidase antibodies after the administration
of HgC12 to Brown Norway rats; however, these animals develop autoantibodies against several antigens and it is therefore hard to attribute a specific role of the antimyeloperoxidase antibodies in the pathogenesis of the disease
manifestations in this animal model. Kiser et a15’ succeeded
in increasing vascular permeability in the dermal vasculature
of the anesthesized and spontaneously hypertensive rat by
intradermal injection of polyclonal rabbit antimyeloperoxidase antibodies. It is not clear if this is a direct effect or one
mediated by antimyeloperoxidase-induced leukocyte activation.
Finally, Brouwer et aP9reported on a renal perfusion model
with myeloperoxidase in Brown Norway rats immunized with
myeloperoxidase. After 10 days, giant cell formation and
vasculitis could be observed in the glomeruli after perfusion
with myeloperoxidase, HzOz and antimyeloperoxidase antibodies. Damage to the glomerular basement membrane by
the myeloperoxidase enzymatic activity possibly explains
these findings; this model might reflect a pathogenetic mechanism relevant for ANCA-related diseases in humans.
CONCLUSION
Several antigens recognized by ANCA in sera from patients
with systemic vasculitis or glomerulonephritis have now been
defined. However, new classes of ANCA in diseases such as
inflammatory bowel disease and chronic liver disease have
recently been identified. The nature of the target antigens in
these diseases is poorly understood and their clinical relevance
is thus far unclear. The clinical spectrum of ANCA has become more extensive, and sometimes more confusing. In the
near future, antigen-specific ELISAs may clarify the issue of
disease specificity of ANCA.
ACKNOWLEDGMENT
The excellent work done by the organizers of the Fourth International ANCA Workshop held in Liibeck, Germany, May 28-30,
1992 was greatly appreciated by the participants. The abstract Book
of the 4th International Symposium on ANCA is still available on
request from Prof W. L. Gross, Rheumaklinik Bad Bramstedt, OskarAlexander-Strasse 26, 2357 Bad Bramstedt, Germany.
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