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Antineutrophil Cytoplasmic Antibody

Systemic necrotizing vasculitides are inflammatory diseases of blood vessels, which are difficult to distinguish from infectious, connective tissue, and degenerative diseases.  Biopsies often reveal only nonspecific acute or chronic inflammation.  Standard laboratory tests such as hemoglobin, WBC, ESR, ANA, and urinalysis have poor predictive value for vasculitis, because none of them are specific for vasculitis. Abnormalities merely reflect inflammation and organ damage.  Negative results are often helpful in ruling out vasculitis.

In patients with clinical features of vasculitis, the presence of antineutrophil cytoplasmic antibody (ANCA) with specificity for myeloperoxidase (MPO) or proteinase 3 (PR3) has a high positive predictive value for a diagnosis of one of the ANCA-associated vasculitides, which include granulomatosis with polyangiitis (GPA, Wegener), microscopic polyangiitis (MPA), renal-limited vasculitis, and eosinophilic granulomatosis with polyangiitis (EGPA). The ANCA-associated vasculitides are characterized by pauci-immune necrotizing inflammation that predominantly involves small vessels. Prominent eosinophilia and asthma are the clinical features that distinguish eosinophilic granulomatosis with polyangiitis from the other forms of ANCA-associated vasculitis.

Traditionally, ANCA antibodies have been detected by indirect immunofluorescence microscopy. Two different immunofluorescent staining patterns have been identified. Cytoplasmic (C-ANCA) pattern is seen most often in patients with classical and limited granulomatosis with polyangiitis, while the perinuclear (P-ANCA) pattern occurs predominantly in patients with microscopic polyangiitis.

Antineutrophil cytoplasmic antibody is detected by an indirect immunofluorescent technique. Patient serum is diluted 1:4 and 1:8 and added to microscope slides coated with ethanol-fixed human neutrophils. If antibody is present, it binds to the neutrophils during incubation. Unbound serum proteins are washed away. Bound IgG antibodies are detected by the addition of a fluorescein isothiocyante (FITC)-labeled antihuman IgG reagent. The slides are then read by a clinical laboratory scientist using a fluorescent microscope. Autoantibody positive samples emit an apple-green fluorescence.

The presence of fluorescence in a coarse speckled cytoplasmic pattern with accentuation around the nuclear lobes is reported as C-ANCA. This pattern is produced by antibodies reacting with serine Protease 3 (PR3) in neutrophil cytoplasmic granules. If a patient sample is positive for C-ANCA, it is titered until an endpoint is reached (1:4, 8, 16, 32, etc.). The pattern and titer are reported.

A pattern of homogeneous fluorescent staining of nuclear lobes with perinuclear accentuation is interpreted as P-ANCA. This pattern is most often produced by reaction of autoantibody with myeloperoxidase (MPO) in the primary cytoplasmic granules of human neutrophils. If a patient sample is positive for P-ANCA, results are reported as positive, but a titer is not performed.

Ethanol fixed slides contain many other antigens that produce a P-ANCA pattern, including elastase, lactoferrin, cathepsin G, and cationic protein 57. Therefore, samples that test positive for P-ANCA may not always test positive for anti-MPO autoantibody.

The presence of an antinuclear antibody (ANA) may mimic a PANCA on ethanol-fixed neutrophils. Samples that test positive for P-ANCA should also be tested for ANA. Additionally, these samples can be tested with human neutrophils that have been fixed with formalin instead of ethanol. Formalin fixation disrupts perinuclear antigens and shifts the fluorescent staining pattern of P-ANCA from perinuclear to cytoplasmic.

Besides ANA, smooth muscle autoantibodies (anti-actin) may also react with ethanol-fixed human neutrophils producing a homogeneous cytoplasmic pattern. Some patients with inflammatory bowel disease have neutrophil reactive antibodies that produce an atypical P-ANCA pattern on ethanol-fixed slides. These antibodies are usually negative on formalin fixed neutrophil slides. Heterophile antibodies can produce various degrees of background staining that complicate interpretation of ANCA pattern.

Testing for ANCA by immunofluorescence assay should not be relied upon exclusively to establish the diagnosis of Wegener's granulomatosis or microscopic polyangiitis. Current guidelines recommend ordering specific tests for PR3 and MPO antibodies in addition to immunofluorescent tests for C-ANCA and P-ANCA. PR3 and MPO antibodies are detected by immunoassay.

The diagnostic accuracy of ANCA testing can be summarized as follows:

  • Approximately 90% of patients with active GPA are ANCA positive
  • Among patients who have GPA and positive ANCA, 85% have PR3 antibody
  • Approximately 90% of patients with MPA are ANCA positive
  • Most patients with MPA who are ANCA positive, have MPO antibodies
  • Majority of patients with renal limited vasculitis are ANCA positive
  • 75% of patients with renal limited vasculitis who are ANCA positive have MPO antibodies
  • Approximately 40% of patients with eosinophilic granulomatosis with polyangiitis are ANCA-positive

Most experts recommend that the diagnosis be confirmed by tissue biopsy prior to committing patients to long-term therapy.

The literature contains conflicting results regarding whether a persistently high ANCA titer or a rise in titer is predictive of subsequent flare in disease activity. If a patient was ANCA positive during a period of active disease, a persistently ANCA-negative status is consistent with, but not absolutely proof of, remission.

The clinical manifestations of eosinophilic granulomatosis with polyangiitis differ between ANCA-positive patients and ANCA-negative patients. For example, ANCA-positive patients with eosinophilic granulomatosis with polyangiitis are more likely than ANCA-negative patients to have glomerulonephritis, pulmonary hemorrhage, and mononeuritis multiplex. The consensus among experts is that ANCA-positive patients with eosinophilic granulomatosis with polyangiitis tend to have better treatment responses than ANCA-negative patients.

Test performance can be enhanced by only ordering ANCA tests for patients with at least one of the following clinical indications:

  • Glomerulonephritis, especially rapidly progressive
  • Pulmonary hemorrhage, especially pulmonary renal syndrome
  • Cutaneous vasculitis with systemic features
  • Multiple lung nodules
  • Chronic obstructive disease of the upper airways
  • Long standing sinusitis or otitis
  • Subglottic tracheal stenosis
  • Mononeuritis multiplex or other peripheral neuropathy
  • Retro-orbital mass

Reference value for cANCA and pANCA is negative.

Reference Values for MPO and PR3 antibodies performed on BioPlex  are <0.4 U (negative), 0.4-0.9 U (equivocal), > or =1.0 U (positive).

Specimen requirement is one red top or SST tube of blood.

References

Jennette JC. Overview of the 2012 revised International Chapel Hill Consensus Conference nomenclature of vasculitides. Clin Exp Nephrol 2013; 17: 603-6.

Locht H, Skogh T, Wiik A. Characterisation of autoantibodies to neutrophil granule constituents among patients with reactive arthritis, rheumatoid arthritis, and ulcerative colitis. Ann Rheum Dis 2000; 59: 898-903.

Hagen EC, Daha MR, Hermans J, et al. Diagnostic value of standardized assays for anti-neutrophil cytoplasmic antibodies in idiopathic systemic vasculitis. EC/BCR Project for ANCA Assay Standardization. Kidney Int 1998; 53:743.

 

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