SpecialIssue

Antineutrophil Cytoplasmic Antibodies Tests:

What Should We Know?
Jaruda Kobkitjaroen M.D., Ronnachai Viriyataveekul M.D.
Department of Clinical Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.

Siriraj Med J 2010;62:179-181
E-journal: http://www.sirirajmedj.com

I



ntroduction
Antineutrophil cytoplasmic antibodies (ANCA) are
serological markers for supporting the diagnosis
of ANCA-associated small-vessel vasculitis (AASV),
namely Wegener’s granulomatosis (WG), microscopic
polyangiitis (MPA), Churg-Strauss syndrome (CSS) and






● Chronic destructive disease of the upper airways
● Long-standing sinusitis or otitis
● Subglottic tracheal stenosis
● Mononeuritis multiplex or peripheral neuropathy
● Retro-orbital mass

pauci-immune crescentic glomerulonephritis. ANCA also Although histopathological findings remain the
help in the diagnosis of other non-vasculitic inflam- gold standard for the diagnosis of AASV, in some cir-
matory disorders such as inflammatory bowel disease cumstances tissue biopsy cannot be obtained or do not
(IBD), primary sclerosing cholangitis (PSC) and autoim- yield diagnostic findings, ANCA testing constitute an
mune hepatitis (AIH). ANCA tests are currently deter- important adjunct to the diagnosis.
mined by an indirect immunofluorescence (IIF) assay Determination of ANCA is also useful for diag-
using polymorphonuclear (PMN) cells as a substrate and nosing other inflammatory disorders, i.e. IBD, PSC and
an ELISA for detection of antibodies against specific AIH2-8. ANCA have been reported 50-80% of patients
antigens; proteinase3 (PR3) and myeloperoxidase (MPO). with ulcerative colitis (UC) and 5-30% of patients with
Different ANCA patterns are demonstrated by IIF. Some Crohn disease (CD). The combination of ANCA test by
patterns are associated with specific antigens, while IIF and anti-Saccharomyces cerevisiae antibody (ASCA)
some patterns have multiple specificities. Positive C- test by ELISA often helps differentiate between UC and
ANCA or P-ANCA in combination with positive PR3- CD. ANCA occurs in 60-90% of patients with PSC and
ANCA or MPO-ANCA, respectively, occurs in AASV. 50-90% of patients with AIH.
Positive ANCA with antigen specificities other than The role of ANCA test for monitoring patients to
PR3 and MPO occurs in other inflammatory disorders. detect disease relapse is still under debate. The corre-
The interpretation of ANCA test is somewhat compli- lation of ANCA titers and disease activity varied in
cated both in laboratory and clinical aspect. Under- different studies9-11. Nevertheless, a negative result during
standing the interpretation of ANCA test leads to appro- a follow-up indicates that the disease is still in remis-
priate request of ANCA test. In this review, we sum- sion. Reappearance or increase of ANCA titer indicates
marized the general information of ANCA test, inclu- that the disease likely relapses.
ding indications, methods and interpretation of the test
and proposed the appropriate ANCA test request. Methods for ANCA detection
Indirect immunofluorescence (IIF) is the standard
Indications of ANCA tests method for ANCA test at present. This method is
The ANCA test is mainly indicated to diagnose mainly used for ANCA pattern screening. The use of
AASV, e.g. Wegener’s granulomatosis (WG), microsco- ethanol-fixed PMN as a substrate for IIF was 12recom-
pic polyangiitis (MPA), Churg-Strauss syndrome (CSS) mended by the first ANCA workshop in 1989 . Four
and pauci-immune crescentic glomerulonephritis. The patterns of ANCA demonstrated on ethanol-fixed
International Consensus Statement on testing and repor- PMN have been described. First, cytoplasmic pattern
ting of ANCA published in 1999 stated the use of (C-ANCA) is defined as diffuse granular cytoplasmic
ANCA testing in1 patients suspected of small vessel staining with central interlobular accentuation. Second,
vasculitis (SVV) . Clinical manifestations suggestive of perinuclear pattern (P-ANCA) is defined as perinuclear
AASV that ANCA test is warranted are as follows2 staining with nuclear extension. Third, C-ANCA (aty-
● Glomerulonephritis, especially rapidly progres- pical) pattern is defined as cytoplasmic staining without
sive glomerulonephritis central interlobular accentuation or cytoplasmic granu-
● Pulmonary hemorrhage, especially pulmonary- larity. The forth pattern called atypical ANCA pattern is
renal syndrome defined as homogeneous cytoplasmic staining in com-
● Cutaneous vasculitis, especially with systemic bination with nuclear staining.
features The use of formalin-fixed PMN helps differen-
● Multiple lung nodules tiate ANCA pattern, in particular distinguish between P-
Siriraj Med J, Volume 62, Number 4, July-August 2010 179
TABLE 1. Interpretation of ANCA results.
IIF IIF ANA ELISA
(EOH) (HCHO) (PR3&MPO) Interpretation
1 + + - + ANCA specific to PR3, MPO Fig 1a-1d
2 + -/weakly+ - - ANCA specific to other Ag† (rarely PR3, MPO) Fig 2a-2b
3 + + + + ANCA specific to PR3, MPO with ANA Fig 3a-3c
4 + -/weakly+ + - ANA induced ANCA pattern or Fig 4a-4f
ANCA specific to other Ag† (rarely PR3, MPO) with ANA
5 - - + - ANA
6 - - - + ANCA specific to PR3, MPO (5%)
EOH = ethanol fixed PMN, HCHO = formalin-fixed PMN, + = positive, - = negative, Ag = antigens
† Not associated with AASV

ANCA specific to formalin-resistant antigens (mostly
PR3 and MPO) and P-ANCA specific to formalin-sensi-
tive antigens (antigens other than PR3 and MPO). P-
ANCA specific to formalin-resistant antigens shows
cytoplasmic staining on formalin-fixed PMN (positive),
while P-ANCA specific to formalin-sensitive antigens
shows no fluorescence staining on formalin-fixed PMN
(negative).
The employment of other substrates such as Hep-
2 cells is helpful for better interpretation of ANCA IIF
test. Hep-2 cells substrate, a substrate of antinuclear
antibodies (ANA) test, helps suggest the presence of
ANA which can mimic both C-ANCA and P-ANCA
patterns.
antigen, bactericidal/permeability-increasing protein,
ELISA is another method widely used for ANCA
detection. This method has an advantage in characteri-
zation of ANCA specificity. The detection of ANCA
with PR3 and MPO specificity provides adjunctive
information for the diagnosis of AASV. ELISA for
PR3- and MPO-ANCA are widespread commercially
available. Moreover, ACNA specific to antigens other
than PR3 and MPO can be detected by some commer-
cial ELISA kits13.
Interpretation of ANCA tests in laboratory aspect
The C-ANCA pattern usually corresponds to PR3
antigen. C-ANCA with MPO specificity can be found
in less percentage. The P-ANCA pattern corresponds to
a number of antigens, i.e. MPO, which is the major
lactoferrin, lysozyme, elastase and cathepsin G. P-
ANCA with PR3 specificity can be rarely found. C-
ANCA (atypical) and atypical ANCA patterns have
been shown to target multiple antigens2,7.
To correctly interpret the ANCA tests, it is neces-
sary to understand the relationship of ANCA results from
IIF and ELISA as shown in Table 1. The clinician should
consult laboratory specialist for correct interpretation.
Interpretation of ANCA tests in clinical aspect
The ANCA tests will be helpful when used in

Fig 3. Immunofluorescence patterns. P-ANCA specific to MPO

with positive ANA. (a) ethanol-fixed PMN. (b) formalin-fixed
Fig 1. Immunofluorescence patterns. C-ANCA with PR3 PMN. (c) ANA
specificity on (a) ethanol-fixed PMN and (b) formalin-fixed
PMN. P-ANCA with MPO specificity on (c) ethanol-fixed
PMN and (d) formalin-fixed PMN.

Fig 4. Immunofluorescence patterns. ANA-induced ANCA

Fig 2. Immunofluorescence patterns. P-ANCA with lactoferin pattern. (a-c) P-ANCA pattern induced by coarse speckled ANA.
specificity on (a) ethanol-fixed PMN and (b) formalin-fixed PMN. (d-f) C-ANCA pattern induced by anticytoplasmic antibodies.

180
 antigens. Negative ANCA by IIF should also be tested
by ELISA for PR3- and MPO-ANCA because 5% of
patients with AASV have positive PR3- or MPO-ANCA
alone.
For patients with clinical manifestations mini-
mally suggestive of AASV and patients with suspected
other ANCA-associated inflammatory disorders, ANCA
test by IIF on ethanol-fixed PMN should be firstly
requested. Positive ANCA on ethanol-fixed PMN regar-
dless of patterns should be subsequently tested by IIF
assays including formalin-fixed PMN and ANA. ELISA
Fig 5. Appropriate ANCA test request. for ANCA specific to PR3, MPO or other antigens
EOH = ethanol fixed PMN, HCHO = formalin-fixed PMN should be requested, according to ANCA results from
IIF as mention above. Negative ANCA on ethanol-fixed
PMN has no further ANCA test required (Fig 5).
proper clinical setting. The results of ANCA test should
be interpreted with caution. ANCA results from IIF
alone give some suggestion according to different ANCA
pattern. C-ANCA and P-ANCA is suggestive, but not
diagnostic, of AASV. C-ANCA (atypical) and atypical
ANCA is not typically associated with AASV. ANCA
results from both IIF and ELISA give more information REFERRENCES
to support the diagnosis. Positive C-ANCA with positive 1. Savige J, Gillis D, Benson E, Davies D, Esnault V, Falk RJ, et al. Inter-
PR3-ANCA or positive P-ANCA with positive MPO- national Consensus Statement on Testing and Reporting of Antineutro-
ANCA strongly supports the diagnosis of AASV. Positive phil Cytoplasmic Antibodies (ANCA). Am J Clin Pathol. 1999 Apr;111
PR3- or MPO-ANCA alone can be found in AASV. 2. (4):507-13. Savige J, Davies D, Falk RJ, Jennette JC, Wiik A. Antineutrophil cyto-
Positive any of ANCA patterns with negative PR3- and plasmic antibodies and associated diseases: a review of the clinical and
MPO-ANCA occurs in infections, malignancies and other Cambridge
laboratory features. Kidney Int. 2000 Mar;57(3):846-62.
inflammatory disorders6. The negative ANCA result 3. G, Rampton DS, Stevens TR, McCarthy DA, Kamm M,
Leaker B. Anti-neutrophil antibodies in inflammatory bowel disease:
does not exclude AASV because 10-20% of patients prevalence and diagnostic role. Gut. 1992 May;33(5):668-74.
with AASV have been found ANCA negative14. The 4. Seibold F, Weber P, Klein R, Berg PA, Wiedmann KH. Clinical signi-
positive ANCA result in the absence of suggestive ficance of antibodies against neutrophils in patients with inflammatory
bowel disease and primary sclerosing cholangitis. Gut. 1992 May;33(5):
clinical manifestation does not give any diagnosis. 657-62.
ANCA patterns found in patients with non-vas- 5. berg Mulder AH, Horst G, Haagsma EB, Limburg PC, Kleibeuker JH, Kallen-
culitic ANCA-associated inflammatory disorders; UC, bodiesCG.in autoimmune
Prevalence and characterization of neutrophil cytoplasmic anti-
liver diseases. Hepatology. 1993 Mar;17(3):411-7.
CD, PSC and AIH are usually described as P-ANCA or 6. Savige J, Dimech W, Fritzler M, Goeken J, Hagen EC, Jennette JC, et al.
atypical ANCA. These antibodies recognized many Addendum to the International Consensus Statement on testing and repor-
different antigens, but occasionally PR3 and MPO2,6,9. comments,
ting of antineutrophil cytoplasmic antibodies. Quality control guidelines,
and recommendations for testing in other autoimmune disea-
Adherence to the International Consensus Statement, the 7. ses. Am J Clin Pathol. 2003 Sep;120(3):312-8.
demonstration of distinctive peripheral rim (or cytoplas- Radice A, Sinico RA. Antineutrophil cytoplasmic antibodies (ANCA).
Autoimmunity. 2005 Feb;38(1):93-103.
mic or atypical) fluorescence pattern on ethanol fixed 8. Papp M, Norman GL, Altorjay I, Lakatos PL. Utility of serological
PMN and the absence of PR3- and MPO-ANCA by markers in inflammatory bowel diseases: gadget or magic? World J
Gastroenterol. 2007 Apr 14;13(14):2028-36.
ELISA helps in the diagnosis of1 non-vasculitic ANCA- 9. Tervaert JW, van der Woude FJ, Fauci AS, Ambrus JL, Velosa J, Keane
associated inflammatory disorders . WF, et al. Association between active Wegener’s granulomatosis and
anticytoplasmic antibodies. Arch Intern Med. 1989 Nov;149(11):2461-5.
Appropriate ANCA test request 10. van der Woude FJ, Rasmussen N, Lobatto S, Wiik A, Permin H, van
Es LA, et al. Autoantibodies against neutrophils and monocytes: tool for
For patients with clinical manifestations highly diagnosis and marker of disease activity in Wegener’s granulomatosis.
suggestive of AASV, as described in the topic of indi- 11. Lancet. 1985 Feb 23;1(8426):425-9.
cations of ANCA tests, IIF assays including ethanol- Kerr GS, Fleisher TA, Hallahan CW, Leavitt RY, Fauci AS, Hoffman
GS. Limited prognostic value of changes in antineutrophil cytoplasmic
fixed PMN, formalin-fixed PMN and ANA should be antibody titer in patients with Wegener’s granulomatosis. Arthritis Rheum.
requested simultaneously. Positive ANCA on both 12. 1993 Mar;36(3):365-71.
ethanol-fixed PMN and formalin-fixed PMN strongly Wiik, A. Delineation of a standard procedure for indirect immunofluo-
rescence detection of ANCA. APMIS Suppl. 1989;6:12-3.
suggest PR3 or MPO specificity, depending on ANCA 13. Talor MV, Stone JH, Stebbing J, Barin J, Rose NR, Burek CL. Antibo-
pattern, thus ELISA for PR3- and MPO-ANCA is cytoplasmic dies to selected minor target antigens in patients with anti-neutrophil
required in some cases for confirmation. Positive ANCA 42-8. antibodies (ANCA). Clin Exp Immunol. 2007 Oct;150(1):
on ethanol-fixed PMN but negative on both formalin- 14. Stone JH, Talor M, Stebbing J, Uhlfelder ML, Rose NR, Carson KA,
fixed PMN and ANA should be subsequently tested by etconsecutiveal. Test characteristics of immunofluorescence and ELISA tests in 856
patients with possible ANCA-associated conditions. Arthritis
ELISA for ANCA specific to PR3, MPO and other Care Res. 2000 Dec;13(6):424-34.

Siriraj Med J, Volume 62, Number 4, July-August 2010 181