Expert & reliable performance requires technologists & a lab adept at IFA testing AND using a 3-substrate sytem (see link to patterns, below). Significant percentages of patients with certain diseases develop IgG-specific auto-antibodies (auto-Ab) produced at the memory-cell level against constituents of the two peripheral-blood leukocytes (WBCs), neutrophiles and monocytes. When to use this test and the interpretation of what it means is totally dependant (1) on testing lab & reagent quality, (2) on the spectrum of findings in a particular case AND (3) how the particular diagnostic specialist factors/weighs the test results among all other factors. ANCA positivity tends to imply presence of a disease with a blood-vessel-inflammatory (vasculitis) component, especially (85% of cases) if there are significant (rather than negative or low levels) antibodies against MPO or PR3. See this complex vasculitis decision tree @ ARUP & the ARUP file about ANCA (which has a link to the Chapel Hill vasculitis nomenclature), and the Inova ANCA reaction patterns charts & decision trees for ANA, ANCA, and liver HERE & our decision tree HERE.

The IFA (indirect fluorescence Ab) ANCA test is a visual test using a fluorescent microscope (patient's Ab-containing serum vs. antigen-containing substrate carefully non-activated⁸ neutrophiles substrate ethanol-fixed on a glass slide).  And the IFA method is the most sensitive method⁴ for detecting ANCA; therefore, it picks up the most "false positives". IgG antibody (Ab) class ANCA is a nonspecific finding (no exact indicator of a certain disease), as such ("ANCA positive"), and seen in the following test PATTERNS:

• CYTOPLASMIC (c-ANCA): IgG specific (there are some IgM and IgA C-ANCAs⁴). Of no clinical importance, but for IFA techs,
  1. typical cytoplasmic c-ANCA: with the typical, granular/homogeneous (MPO & PR3 targets are the azurophilic¹⁰ granules), somewhat restricted, central inter-lobar accentuation; tends PR3 strongly¹⁰ positive & Wegener's type.
  2. atypical cytoplasmic c-ANCA: with more diffuse, flat, nongranular cytoplasmic positivity without central interlobular accentuation which is NOS & has many possible causes, including MPO^{9, 10}. The PR3 tends to be weakly positive or negative¹⁰.
• PERINUCLEAR (p-ANCA):
  1. typical perinuclear p-ANCA: has ethanol-induced artifactual dislocation & ionic attraction to perinuclear (MPO & PR3 targets are azurophilic¹⁰ granules) positivity with "nuclear extension" (fuzziness") & coverts to a c-ANCA pattern on formalin fixed substrate; tends MPO specific⁹, strongly¹⁰ positive & small vessel vasculitic.
  2. atypical p-ANCA:
     • very perinuclear: lacks nuclear extension; tends neg/low MPO¹⁰ & IBD ANCA (seen in about 79% IBD-UC [therefore, some call it uc-ANCA] & only about 20% IBD-CD).
     • less perinuclear: lacks nuclear extension but not quite "very" perinuclear; is most common IFA ANCA pattern⁹. Tends neg/low MPO¹⁰.
  3. antinuclear: (these are strongly degraded or negative in the formalin fixed substrate slides⁸)
     • ANA: substrate lymphocyte or eosinophil nuclei positive¹⁰; suggests SLE-type-categories systemic autoimmune disease. Reflex IFA test on Hep-2 clarifies.
     • gs-ANA: is a p-ANCA that scatters into nuclear lobes⁹; seen in 20% of cases of RA⁸; substrate lymphocyte nuclei not positive. Any reflex IFA test on Hep-2 tests negative. Also called anti-neutrophili nuclear antibody (ANNA) when determined by EIA or ELISA.
• ATYPICAL ANCA: this includes all other ANCA patterns, is uncommon, and is usually a combination of c & p positivity; some have called it x-ANCA; some may be due to variability in substrate processing or heat inactivation of patient serum samples⁹.

When ordered: The ANCA test is most useful in patients with blood in sputum (hemoptysis), blood in urine (hematuria), possible indications of systemic vasculitis (as are skin purpura, blood in stool [hematochezia], and retinal hemorrhages) and currently maybe more commonly used in inflammatory bowel disease (IBD), auto-immune hepatitis (AIH), and autoimmune biliary (PBS) disease diagnosis. [warning] 

The test quickie capsule:

• c-ANCA positivity is usually directed at PR3 and used to support Dx of Wegener's when PR3 positive;
• p-ANCA positivity is usually directed to MPO and used to support Dx of immuno-vasculitis, glomerulonephritis, C-S syndrome, polyarteritis nodosa (SLE, RA, and IBD-UC, etc. MPO low/neg). A proprietary DNase-sensitive (treatment with DNase makes the ANCA disappear) p-ANCA is said to favor IBD-UC but may just be an EIA way to do what the formalin slide IFA does.

Situations having negative or undetected levels of Ab:

• true negative test
• poor samples = lipemic, hemolysed, icteric, or bacterially contaminated may be false negative or titers lower than actual.
• rheumatoid vasculitis cases may be ANCA negative
• almost all of the below when low-grade, inactive, in remission
• NOTE: some advise that an ANCA determination not be "called" negative unless IFA, PR3, and MPO tests are, all 3, negative.

• c-ANCA: (95% of positives are specific for PR-3 [proteinase 3] by EIA or ELISA test methods)
  • Wegener's granulomatosis (WG): NOTE that IFA is 90-98% sensitive for active, classical WG (and negativity in this situation is rare). [About 90% are PR-3 positive and less than 10% MPO positive⁴] So, a negative almost rules out active, classical WG. Serial negatives confirm the rule out. Only 65-70% of limited WG are positive, and less than 35% of any types of WG in remission are positive. Re-institution of immunosuppressive therapy is not recommended on sole basis of rising titers following a period of remission. About 10% WG have anti-GBM Abs. Histopathology: look for vasculitis, geographic necrotizing granulomatous lesions, and hyperchromatic giant cells; look for histologic variants of bronchocentric WG [LMC-04-3602], eosinophilic WG, BOOP-like WG, and alveolar hemorrhage and capillaritis WG (a more fulminant type).
  • small vessel vasculitis/microscopic polyarteritis nodosa (MPA)/microscopic polyangiitis [30% of cases PR3 positive].
  • Churg-Strauss syndrome (asthmatics; usually blood eosinophilia).
  • "false positives" [in that they are not WG]:
    1. Kawasaki disease
    2. small vessel vasculitis in  cystic fibrosis
    3. atrial myxoma
    4. rheumatoid arthritis (need not be vasculitic)
    5. polyangiitis overlap syndrome
    6. idiopathic crescentic glomerulonephritis
    7. classic polyarteritis nodosa (but positivity so rare in this disease that great caution urged in making a diagnosis in the face of a positive ANCA)
    8. alveolar hemorrhage syndromes with or without glomerulonephritis
    9. peripheral neuropathy
    10. infections (TB, HIV, endocarditis)
    11. nasal septal perforation
    12. drug-induced WG-like disease (retinoids, alpha methyldopa, propylthiouracil)
    13. MGUS (monoclonal gammopathy of uncertain significance)
    14. neoplastic diseases.
  • technical false positives⁴:
    • calling cytoplasmic positivity "positive"  when it does not accentuate between the nuclear lobes
    • calling cytoplasmic positivity "positive"  when it does not occur in 95% or more of polys
    • calling cytoplasmic positivity "positive"  when it is not limited to polys
    • calling cytoplasmic positivity "positive"  when it is a heterogeneous type of fluorescence.
• usual p-ANCA: :
  1. Churg-Strauss syndrome (C-Ss) of allergic granulomatosis (a syndrome of asthmatics)
  2. necrotizing and crescentic glomerulonephritis (n/cGN)
  3. small vessel vasculitis/microscopic polyarteritis nodosa (MPA)/microscopic polyangiitis [60% of cases MPO positive]
  4. "false positives" [not  C-Ss, n/cGN, or MPA]
  5. systemic polyarteritis nodosa (but positivity so rare in this disease that great caution urged in making a diagnosis in the face of a positive ANCA)
  6. Goodpasture's syndrome/anti-GBM-positive renal disease
  7. 11%  of cases of giant-cell arteritis
  8. Felty's syndrome
  9. atypical pneumonia cases
  10. post-streptococcal glomerulonephritis
  11. systemic lupus erythematosus
  12. mixed connective tissue disease (MCTD)
  13. about 25% of WGs have this MPO positivity
  14. MPO ANCA develops in many people on hydralazine therapy
• atypical p-ANCA:
  1. auto-immune hepatitis (AIH): 50% of cases¹ [biopsies tend to show a very "lupoid" heavy plasma cell infiltrate ; all cases of AIH will be positive for ASM, AMA, or an atypical p-ANCA].
  2. ulcerative colitis (IBD-UC): some 70% of cases¹.
  3. systemic lupus erythematosus (SLE).
  4. Crohn's disease: <10% of IBD-CD¹.
  5. primary biliary cirrhosis (PBC).
  6. primary sclerosing cholangitis (PSC), 70% of cases¹.

References:

1. James A. Goeken, MD, immunopathologist & director of immunopathology lab at the U. of Iowa Hospitals and Clinics (Sept. 2000, personal communications).
2. CAP TODAY, June 1998 issue
3. "Vasculitis" Seminars in Diagnostic Pathology, Feb. 2001
4. Clinical Diagnosis & Management by Lab. Methods [text], JB Henry, page 992-993, 2001 edition.
5. HAPS web site
6. Savige J, Goeken JA, et. al., "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", AJCP 111:507-518, 2003 (HERE).
7. Wiik A, "Editorial: Rational use of ANCA in the diagnosis of vasculitis". Rheumatology 41 (5): 481-483, 2002 [HERE].
8. Bradwell AR, et. al., Atlas of Autoantibody Patterns on Tissues, 103 pages, 1997.
9. Bradwell AR, et. al., Advanced Atlas of Autoantibody Patterns, 113 pages, 1999.
10. Detrick B, et. al., Manual of Molecular and Clinical Laboratory Immunology, 7th Ed., 2006, 1340 pages.
11. Carl Schroder, MT, 40-year expert & veteran IFA expert with ARUP & then Inova; presentation at LMC 6/21/2011.

Other names for this exact or approximate test  are:   

1. ANCA
2. A-ANCA
3. C-ANCA
4. C-ANCA, atypical
5. P-ANCA
6. UC-ANCA
7. x-ANCA
8. PR3
9. MPO
10. gs-ANA
11. ANNA