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[Diagnostic aspects in the determination of antineutrophil cytoplasmic antibodies]
M Manes1, G Bonfant, P Belfanti
1Servizio di Nefrologia e Dialisi, Ospedale Regionale della Valle D'Aosta.
Insights
A novel four-step flowchart accurately identifies anti-neutrophil cytoplasmic antibody (ANCA) positivity and specific antigen targets. This method effectively distinguishes true ANCA from antinuclear antibodies (ANA), improving diagnostic precision.
Area of Science:
- Immunology
- Clinical Chemistry
- Autoimmunity
Background:
- Anti-neutrophil cytoplasmic antibodies (ANCAs) are crucial diagnostic markers for systemic vasculitis.
- Distinguishing true ANCAs from antinuclear antibodies (ANAs) can be challenging due to overlapping immunofluorescence patterns.
- Accurate identification of ANCA and their specific antigenic targets is essential for precise diagnosis and management.
Purpose of the Study:
- To propose and validate a four-step flowchart for defining ANCA positivity and identifying specific antigenic targets.
- To differentiate between cytoplasmic (C-ANCA), perinuclear (P-ANCA), and atypical (X-ANCA) patterns using indirect immunofluorescence (IIF).
- To distinguish true P-ANCAs from ANAs and characterize the antigenic targets of identified ANCAs.
Main Methods:
- A four-step diagnostic flowchart integrating indirect immunofluorescence tests (IIF) and solid-phase assays (EIA).
- Step 1: IIF on ethanol-fixed granulocytes (IIF-E) for initial ANCA screening and pattern determination (C-ANCA, P-ANCA, X-ANCA).
- Step 2: IIF on formalin-fixed granulocytes (IIF-F) to differentiate P-ANCA from ANA.
- Step 3: IIF on monkey liver sections (IIF-M) to assess simultaneous ANA and P-ANCA positivity.
- Step 4: Solid-phase assays (EIA) with purified proteins to identify specific antigenic targets (e.g., PR3, MPO, LF).
Main Results:
- Out of 173 patients screened, 17 C-ANCAs were identified, with specific antigens including PR3, MPO, Lys, and Cat G.
- Of 21 P-ANCA positive sera, 15 showed cytoplasmic patterns; EIA revealed MPO (6), LF (2), and unknown antigens (5), with two cases positive for MPO & LF.
- IIF-M analysis of IIF-F negative sera identified one ANCA+/ANA+ case (LF antigen) and three ANCA+/ANA- cases (unknown antigens), alongside two false positives.
Conclusions:
- The proposed four-step flowchart provides a detailed and effective method for analyzing ANCA positivity and antigenic targets.
- This approach successfully differentiates true ANCAs from ANAs, enhancing diagnostic accuracy.
- The flowchart utilizes readily available commercial kits, making it a practical tool for clinical laboratories.
Abstract:
We propose a four step flow-chart to define ANCA positivity and antigenic target. 1st step: indirect immunofluorescence test on ethanol-fixed human granulocytes (IIF-E), as screening test. Different staining patterns can be observed: a granular cytoplasmic fluorescence (C-ANCA), a smooth or fine granular perinuclear fluorescence (P-ANCA) and an intermediate pattern (X-ANCA). Antinuclear antibodies (ANA) may mimic P-ANCA. 2nd step: IIF test on formalin-fixed human granulocytes (IIF-F) differentiates true P-ANCA from ANA: most of P-ANCA show cytoplasmic pattern, whereas ANA are negative. 3rd step: IIF test on monkey liver sections (IIF-M) investigates simultaneous ANA and P-ANCA positiveness. P-ANCA positive sera show an exclusive reactivity with neutrophils infiltrating the portal tract, whereas ANA react with hepatocytes nuclei. 4th step: to characterize antigenic target, a solid phase assay, using purified proteins as substrates, is performed. We found 17 C-ANCA (6 PR3, 3 MPO, 1 Lys, 1 Cat G and 6 unknown antigens) out of 173 patients screened with IIF-E. 21 P-ANCA positive sera have been investigated by IIF-F test: 15 showed a cytoplasmic pattern; EIA test gave the following results: 6 MPO, 2 LF, 5 unknown antigens; 2 cases were positive for two antigens, MPO & LF. Using IIF-M on the 6 IIF-F negative sera, we observed: 2 false positives (ANCA-/ANA-), one ANCA+/ANA+ (antigen LF), 3 ANCA+/ANA- (unknown antigens). The flow chart suggested allows to analyse in detail ANCA, using easily available commercial kits.