The document summarizes the history and developments in ANCA testing for small vessel vasculitis. It discusses the key findings of a multicenter study that evaluated different ANCA assays and led to revised consensus recommendations in 2016. The study found variability in IIF performance but high diagnostic accuracy of antigen-specific immunoassays. It was concluded that immunoassays are the preferred initial screening method for ANCA-associated vasculitis, rather than IIF.

1. ANCA Testing in Small Vessel
Vasculitis – New Consensus
CJ

2. • 1969-Calabresi et al - perinclear staining pattern in inflammatory
conditions
• 1982-Davies et al. reported the occurrence of autoantibodies to
cytoplasmic constituents of granulocytes in the sera of a few patients with
glomerulonephritis
• 1985-van der Woude et al. - anti-cytoplasmic antibodies by IIF on
ethanol-fixed neutrophils and produced a characteristic cytoplasmic
fluorescence pattern (C-ANCA) in GPA

3. • 1989 – perinuclear staining P-ANCA in GPA
• 1988-89 – MPO , PR3 Autoantigens identified
• First international workshop on ANCA, Copenhagen 25–26 January 1988 –
standard procedure for IIF
• The nomenclature for these staining patterns, C-ANCA for the cytoplasmic pattern
and P-ANCA for the perinuclear pattern was agreed on at the second International
ANCA Workshop in 1989

4. PR3 & MPO ANCA

5. Atypical ANCAs
• ANCA’s targeting lactotransferrin, cathepsin G, neutrophil elastase
and bactericidal permeability-increasing protein
• Human neutrophil elastase (HNE)-ANCAs in patients with cocaine-
induced midline destructive lesions
• Autoantibodies directed against human lysosome-associated
membrane glycoprotein 2 (LAMP2) have also been described in AAV

6. ANCA Assays
• IIF – Indirect Immunofloroscence Assay
• Substrate - human donor peripheral blood leukocytes or
neutrophils
• Leukocytes are fixed with 96–99% ethanol at 41C for 5 min
• Patient serum is subsequently incubated in a dilution of
1:20, diluted in phosphate-buffered saline
• Autoantibodies are detected by a secondary anti-human
IgG antibody that is conjugated with fluorescein

7. • ANCA are finally detected by fluorescence
microscopy
• Two well-defined patterns can be
demonstrated:

8. Ethanol fixed
• Dissolve and redistribute
• C-ANCA –Cytoplasmic
• P-ANCA-Perinuclear
Formalin Fixed
• Substances fixed in their native
position
• Falk and Jenette reported that
the perinuclear staining pattern
was artifactual since formalin
fixation produced a cytoplasmic
staining pattern
• ANA VS P-ANCA – Do ANA on
HEP-2 Cells or on Formalin fixed
neutrophils

9. • Standardized, automated analysis of IFT for
ANCA detection provides advantages like
reduced consumption of samples and
reagents,
shortened analysis time and
less extensive handling of samples

10. • quantitative image analysis technique – ethanol fixation
• AKLIDES® (Medipan GmbH, Germany) automated IIF
analysis system - which is able to quantify fluorescence
intensity and interpret basic ANCA staining patterns on
combined ethanol-fixed and formalin-fixed human
neutrophils

11. Antigen specific assays
• ELISA

12. • Dot blot assay –
antigen-coated nitrocellulose strip
Qualitative test
biochip technology (EUROPLUS®, EUROIMMUN
AG, Germany) IFT is combined with a dot-blot
test for PR3-ANCA and MPO-ANCA

13. • Bead-based multiplex assay - capture antigens are bound to colour-coded
beads in suspension that are then analysed using flow cytometry
• The main advantage of this method is the ability to simultaneously detect
multiple autoantibodies relevant to vasculitis (ANCAs and anti-glomerular
basement membrane [GBM] antibodies) in a small serum sample
• The potential disadvantages are the same as with direct ELISA; that is, the
antigen-binding potential of the PR3 and MPO and its potential loss after
the coating process

14. • chemiluminescence technology -QUANTA-
Flash

15. • fluorescence enzyme immunoassays
• Addressable-laser-bead immunoassays
(ALBIA)

16. Pathogenecity of ANCA
• Xiao et al 2002-
• To test the pathogenic potential of antibodies alone, purified anti- MPO IgG or
control IgG was injected intravenously into Rag2–/– mice and wild-type mice
• Mice that received anti-MPO IgG but not mice that received control IgG developed
focal necrotizing and crescentic glomerulonephritis with a paucity of glomerular Ig
deposition
• Thus, anti-MPO IgG alone was able to cause pauci-immune glomerular necrosis
and crescent formation in the absence of functional T or B lymphocytes in Rag2–/–
mice and in the presence of an intact immune system in wild-type C57BL/6J mice.

17. • Chimeric mice were generated by injecting human haematopoietic stem cells into irradiated mice
• Matched chimera mice were treated with human IgG from patients with: anti-PR3 positive renal and lung
vasculitis; patients with non-vasculitic renal disease; or healthy controls
• Six-days later, 39% of anti-PR3 treated mice had haematuria, compared with none of controls. There was
punctate bleeding on the surface of lungs of anti-PR3 treated animals, with histological evidence of
vasculitis and haemorrhage
• Anti-PR3 treated mice had pauci-immune proliferative glomerulonephritis, with infiltration of human and
mouse leukocytes
• There were no glomerular changes in controls

19. ANCAs in other small-vessel
vasculitides.
• ANCAs are also found in 30–38% of patients with EGPA, a
disease characterized by asthma, eosinophilia and
granulomatous inflammation, and in 20–35% of patients
with anti-glomerular basement membrane (anti-GBM)
disease
• The majority of these ANCA-positive patients have MPO-
ANCAs

20. ANCAs in gastrointestinal disorders
• ANCAs are found in patients with
gastrointestinal disorders such as IBD, primary
sclerosing cholangitis and inflammatory liver
diseases (such as autoimmune hepatitis,
primary biliary cirrhosis and chronic viral
hepatitis)
• Atypical P ANCA or X-ANCA

21. ANCAs in systemic inflammatory and
malignant diseases.
• ANCAs have also been reported in systemic diseases
such as rheumatoid arthritis and systemic lupus
erythematosus (reviewed elsewhere)
• A rare association of AAV with malignant haemopathy
(mainly non-Hodgkin lymphoma and myelodysplasia)
has additionally been described

22. ANCAs and infection
• infective endocarditis
• hepatitis C infection and
• tuberculosis
• malaria,
• leprosy

23. Drug-induced AAV
DRUGS
levamisole-adulterated cocaine double positivity for MPO-ANCAs and
PR3-ANCAs
hydralazine MPO-ANCAs ,HNE-ANCAs, lactoferrin-
ANCAs and ANAs
propylthiouracil MPO-ANCAs , PR3-ANCAs and HNE-ANCAs
minocycline MPO, HNE, bactericidal permeability
increasing protein (BPI), lactoferrin or
cathepsin G,ANA
most patients with drug-induced AAV have MPO-ANCA

24. ANCA in non vasculitic conditions
• Inflammatory bowel disease -

25. CHCC in 1994

26. CHCC in 2012

27. IIF Vs ELISA in ANCA testing
• Hagen et al 1998

30. • When the results of the IIF test were combined with those of the
ELISAs (cANCA/anti-PR3 positive, pANCA/anti-MPO positive), the
diagnostic specificity increased to 99%
• The sensitivity of the combination of cANCA + anti-PR3 or pANCA
+ anti-MPO for WG, MPA or Irpgn was 73%, 67% and 82%,
respectively
• From this study it is concluded that the value of the IIF test for
ANCA detection can be greatly increased by the addition of a well
standardized antigen-specific ELISA

31. 1999 consensus document

33. • Sensitivity and specificity (against disease controls only) of assays for anti-MPO for
the diagnosis of systemic necrotizing vasculitides were 37.1% (confidence interval
26.6% to 47.6%) and 96.3% (CI 94.1% to 98.5%), respectively.
• When the pANCA pattern by IIF was combined with anti-MPO testing, the
specificity improved to 99.4%, with a lower sensitivity, 31.5%
• The combined ANCA testing system (anti-PR3/cANCA + anti-MPO/pANCA)
increased the sensitivity to 85.5% with a specificity of 98.6%

34. Rationale for a new consensus

35. • multicentre study
• Four European centres contributed samples and clinical data from
newly diagnosed patients with GPA (n = 186) and MPA (n = 65) and
relevant disease controls (n = 924)
• only newly diagnosed patients were included
• Eight different antigen-specific immunoassays (from seven
manufacturers, encompassing different technological platforms)
and four different IIF assays (including two automated assays) were
evaluated

37. • The results of the study revealed a large amount of variability between IIF
methods
• immunoassays for PR3-ANCAs and MPO-ANCAs had a high diagnostic
performance
• Consequently, dual IIF/antigen-specific immunoassay testing of each
sample is not necessary for maximal diagnostic accuracy
• These results indicate that the current international consensus on ANCA
testing for AAV needs revision.

38. • This study, did not reveal consistent differences between different assay
generations and formats.
• Hence, in contrast to expectations, the improvements in test characteristics were
independent of the assay principle
• Notably, some patients tested negatively by both IIF and immunoassay, or by
either immunoassay or IIF
• Depending on the assay, 11−17% of patients with AAV were negative by IIF and
9−16% by immunoassay.
• Hence, antigen-specific immunoassays might detect antibodies that are missed by
IIF and vice versa

40. New recommendations

42. • The likelihood ratio helps to describe the clinical value of a test result
• This ratio can be defined for different test result intervals of an assay and
is independent of the disease prevalence and pre-test probability
• A likelihood ratio of 1 indicates no difference in pre-test to post-test
probability, whereas likelihood ratios of >10 or <0.1 indicate large, often
clinically important differences in pre-test to post-test probability

43. • Detailed analysis EUVAS study confirmed that the likelihood ratio
for AAV increases with increasing levels of PR3-ANCAs and MPO-
ANCAs for all immunoassays included in the study
• The likelihood ratio for AAV was calculated to be 0.1, 1.2, 10.2, 64.6,
and ∞ for test result intervals of 0–12 CU, 12–24 CU, 24–78 CU, 78–
1,050 CU, and 1,050–3,500 CU, respectively, when using the
PR3-ANCA and MPO-ANCA QuantaFlash CLIA (Inova)

44. • post-test odds = pre-test odds × likelihood
ratio

48. Conclusion
• A 2016 multicentre EUVAS evaluation demonstrated the good diagnostic
performance of current antigen-specific immunoassays for ANCA
detection in patients with GPA and MPA, and the high variability in
performance of IIF
• high-quality immunoassays as the preferred first screening method for
GPA and MPA
• IIF is no longer deemed suitable as the first screening test, and adds little
additional benefit to antigen-specific assays in the diagnosis of AAV when
the pre-test probability for the disease is high

49. Thank you