In patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), laboratory analyses available for the detection of ANCAs include indirect immunofluorescence (IIF), monospecific immunoassays, and line immunoassays (LIAs).

Recognizing that well-defined guidelines with respect to mode of laboratory testing for ANCAs do not exist, which can lead to diagnostic and patient management issues, the researchers have described 4 case reports that highlight the difficulties involved in ANCA analysis in patients suspected of having AAV. Findings from the case series have been published in the journal Biochemia Medica (Zabreb).

Currently, the immunoassays are designed primarily for the diagnosis of individuals with AAV—that is, granulomatosis with polyangiitis and microscopic polyangiitis. According to the 2017 International Consensus Guidelines on ANCA testing, immunoassays are recommended over IIF for the detection of proteinase 3 (PR3)-ANCAs and myeloperoxidase (MPO)-ANCAs. Based on the 1999 International Consensus on ANCA testing, however, IIF should be used to screen for ANCAs in general, and samples that yield positive ANCAs should then be tested with immunoassays for detection of PR3 and MPO antibodies.

The main drawback associated with using IIF to detect ANCAs is the interference of antinuclear antibodies (ANAs), which can lead to a false-positive ANCA result. Such preanalytical interference may result in significant errors in decision-making and delayed reports. By presenting these 4 case reports, the researchers sought to describe the difficulties involved in ANCA testing by IIF in patients with coexisting autoimmune disorders, based on interference by ANAs, and the significance of immunoassay-based monospecific assays as the main testing method to use in such individuals.

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Each of the 4 cases described by the authors demonstrated “positive findings on ANA screening by IIF and ANA profile by LIA.” Further, all of the 4 patients subsequently tested positive for perinuclear ANCAs on IIF; however, they “turned negative on vasculitis profile by LIA.”

A common finding recognized in all 4 patients based on ANA screening was the presence of a coexisting ANA, with a nuclear pattern in the first 3 cases. The 4 cases all exhibited false-positive ANCA testing results by IIF, which turned negative when tested by LIA.

The following 4 cases were analyzed:

  • A 24-year-old female who presented with joint pain, fever, abdominal pain, and occasional dry cough
  • A 18-year-old female who presented with generalized body swelling, generalized body pain, and fever
  • A 16-year-old female who presented with generalized body swelling, fever, reduced appetite, and abdominal pain
  • A 16-year-old female who presented with intermittent nasal bleeding and complaints of feeling a mass in her nose

“ANCA is a very sensitive and specific biomarker for diagnosing AAV,” the authors noted. “However, the correct methodology of . . . detection plays a very important role to ensure correct diagnosis,” they emphasized. “Probability of atypical ANCA in diseases other than AAV should also be considered in case of ambiguous results,” they concluded.


Mahto M, Rai N, Das PR, Karmakar S, Bhushan D. Interference of anti-nuclear antibodies on determination of anti-neutrophil cytoplasmic antibodies in patients suspected of vasculitis: a case series. Biochem Med (Zagreb). Published online August 5, 2023. doi:10.11613/BM.2023.031001