ANCA Testing in Clinical Practice: A Comprehensive Guide for the Internist

Dr Neeraj Manikath , claude.ai

Abstract

Anti-neutrophil cytoplasmic antibody (ANCA) testing has become indispensable in diagnosing ANCA-associated vasculitides (AAV), yet its appropriate use remains poorly understood, leading to misinterpretation and diagnostic errors. This review provides practical guidance on patient selection, test interpretation, and clinical integration of ANCA testing for internal medicine practitioners.

Introduction

ANCA-associated vasculitides—comprising granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA)—represent life-threatening systemic diseases requiring prompt recognition. While ANCA testing has revolutionized AAV diagnosis, inappropriate ordering and interpretation plague clinical practice. Studies suggest that over 80% of ANCA requests may be inappropriate, contributing to false-positive results that outnumber true positives in low-prevalence populations.

Understanding ANCA: Biology and Pathophysiology

ANCAs are autoantibodies targeting antigens within neutrophil cytoplasmic granules and monocyte lysosomes. The two clinically significant targets are:

Proteinase 3 (PR3): A serine protease predominantly associated with GPA
Myeloperoxidase (MPO): A heme-containing enzyme more common in MPA and EGPA

These antibodies directly activate primed neutrophils through Fc receptor engagement, triggering degranulation, reactive oxygen species production, and neutrophil extracellular trap formation. This cascade amplifies vascular inflammation and recruits additional inflammatory cells, propagating the vasculitic process.

The Two-Tiered Testing Strategy

Current international guidelines recommend a two-step approach combining indirect immunofluorescence (IIF) with antigen-specific immunoassays.

Step 1: Indirect Immunofluorescence

IIF using ethanol-fixed neutrophils produces three patterns:

Cytoplasmic (C-ANCA): Diffuse granular cytoplasmic staining, typically PR3-antibodies
Perinuclear (P-ANCA): Perinuclear rim staining, typically MPO-antibodies
Atypical patterns: Various non-specific patterns

Pearl: The P-ANCA pattern results from an artifact of ethanol fixation, which causes cationic MPO to migrate toward the negatively charged nuclear membrane. Using formalin fixation eliminates this redistribution.

Step 2: Antigen-Specific Immunoassays

Enzyme-linked immunosorbent assay (ELISA) or newer techniques like chemiluminescence, fluorescence enzyme immunoassay, and multiplex flow immunoassay directly detect antibodies against PR3 or MPO.

Oyster: While IIF was historically considered essential, recent evidence suggests antigen-specific immunoassays alone may suffice in high-quality laboratories using modern techniques. The 2017 international consensus statement acknowledged that high-quality immunoassays with established performance may replace IIF as the initial screening test.

When to Test: Clinical Scenarios

Definite Indications

Rapidly progressive glomerulonephritis (RPGN): Acute kidney injury with active urinary sediment (dysmorphic red blood cells, red cell casts) without other obvious cause
Pulmonary-renal syndrome: Diffuse alveolar hemorrhage combined with glomerulonephritis
Chronic destructive upper respiratory disease: Saddle nose deformity, subglottic stenosis, chronic sinusitis with bone destruction, otitis media with sensorineural hearing loss
Systemic vasculitis with compatible organ involvement: Mononeuritis multiplex, palpable purpura, cutaneous ulcers, or scleritis in appropriate clinical context
Unexplained multisystem disease: Constitutional symptoms with lung nodules/infiltrates plus renal involvement or other compatible organ manifestations

Situations Warranting Caution

Hack: Use the "two-organ rule"—ANCA testing is most appropriate when at least two organs are involved in a pattern consistent with small-vessel vasculitis. Single-organ disease presentations have much lower pre-test probability.

When NOT to Test

Testing should be avoided in:

Isolated constitutional symptoms without organ involvement
Chronic fatigue or non-specific arthralgias
Isolated positive ANA or rheumatoid factor
"Screening" in asymptomatic patients
Isolated COPD exacerbation
Isolated interstitial lung disease without other features
Chronic kidney disease without active sediment

Oyster: A common pitfall is ordering ANCA in patients with inflammatory bowel disease or autoimmune hepatitis. While P-ANCA can be positive in these conditions, these represent atypical ANCAs against different antigens (not MPO), and testing adds no diagnostic value.

Interpreting ANCA Results: The Bayesian Approach

Sensitivity and Specificity

For active, systemic AAV:

PR3-ANCA: Sensitivity 65-90% (highest in GPA), specificity >95%
MPO-ANCA: Sensitivity 50-80% (highest in MPA), specificity >95%

Pearl: Sensitivity varies dramatically by disease activity and extent. Limited GPA (formerly Wegener's granulomatosis without renal involvement) may have PR3-ANCA positivity in only 60-70% of cases, while severe, active disease approaches 90% sensitivity.

Positive Predictive Value: The Critical Concept

The positive predictive value (PPV) depends heavily on pre-test probability. In a low-prevalence setting:

High clinical suspicion: PPV approximately 90%
Low clinical suspicion: PPV may be <10%

Hack: Before ordering ANCA, ask yourself: "Would I treat this patient for vasculitis if the ANCA is positive?" If the answer is no without additional evidence, reconsider testing.

False Positives

False-positive results occur in:

Drug-induced vasculitis (levamisole-contaminated cocaine, hydralazine, propylthiouracil, minocycline)
Infections (endocarditis, tuberculosis, HIV)
Malignancies
Other autoimmune diseases (rheumatoid arthritis, systemic lupus erythematosus)
Inflammatory bowel disease (atypical P-ANCA)

Negative ANCA in True AAV

Oyster: Approximately 10-20% of biopsy-proven AAV patients are ANCA-negative. This is more common in:

Limited disease extent
EGPA (up to 60% may be ANCA-negative)
Drug-induced vasculitis
Patients previously treated with immunosuppression

A negative ANCA never excludes AAV in the appropriate clinical context.

Which Test: Practical Laboratory Considerations

The 2017 International Consensus

The consensus statement recommends:

High-quality antigen-specific immunoassays (PR3-ANCA and MPO-ANCA) as the primary screening method
IIF to be used in specific situations: negative immunoassay with high clinical suspicion, or confirming positive immunoassays when clinical suspicion is low
Results reported as PR3-ANCA or MPO-ANCA positive/negative rather than C-ANCA/P-ANCA terminology

Pearl: The shift from pattern-based (C-ANCA/P-ANCA) to antigen-specific reporting reflects superior diagnostic performance and clinical utility of direct antigen detection.

Modern Immunoassay Platforms

Newer platforms offer advantages:

Fluorescence enzyme immunoassays: Higher throughput, objective quantification
Chemiluminescence immunoassays: Increased sensitivity
Addressable laser bead immunoassays: Simultaneous detection of multiple antigens

Hack: When results are discordant (positive IIF, negative specific assay, or vice versa), always correlate with clinical features. If clinical suspicion remains high despite negative antigen-specific testing, consider repeating testing or proceeding with tissue biopsy.

Special Situations

Monitoring Disease Activity

Serial ANCA testing for monitoring remains controversial:

Rising titers correlate poorly with impending relapse in individual patients
Persistently positive ANCA does not mandate treatment changes in clinically quiescent disease
Use clinical assessment as primary monitoring tool

Pearl: ANCA titers at diagnosis do not predict disease severity or prognosis. Treatment decisions should never be based on antibody levels alone.

Drug-Induced ANCA-Associated Vasculitis

Certain medications trigger ANCA-positive vasculitis:

Propylthiouracil: Up to 20-30% of treated patients develop ANCAs; <5% develop clinical vasculitis
Hydralazine: Dose and duration dependent
Levamisole-contaminated cocaine: Creates unique pattern of anti-PR3, anti-MPO, and anti-elastase antibodies

Hack: Always obtain detailed medication history, including over-the-counter drugs and substance use, before attributing ANCA positivity to primary AAV.

Double-Positive (PR3 and MPO) ANCA

Rare cases show positivity for both PR3-ANCA and MPO-ANCA:

Consider drug-induced vasculitis (especially levamisole)
May represent cross-reactivity rather than true dual specificity
Clinical phenotype usually resembles single-positive disease

ANCA in Renal-Limited Disease

Isolated renal vasculitis (pauci-immune crescentic glomerulonephritis without systemic features):

Represents approximately 30% of AAV cases
ANCA positivity approximately 80-85%
Kidney biopsy essential for diagnosis

Integration with Histopathology

Oyster: ANCA testing does not replace tissue biopsy. The gold standard for AAV diagnosis combines:

Compatible clinical features
Positive ANCA (PR3 or MPO)
Histopathological confirmation when feasible

Biopsy reveals necrotizing vasculitis with minimal or absent immune deposits ("pauci-immune"). However, in specific scenarios—severe diffuse alveolar hemorrhage or immediately life-threatening disease—treatment may begin based on clinical and serological findings pending biopsy confirmation.

Pearl: The 2022 ACR/EULAR classification criteria for AAV incorporate ANCA testing as weighted items but require a clinical diagnosis of vasculitis as an entry criterion. These are classification, not diagnostic, criteria.

Common Pitfalls and How to Avoid Them

Testing too broadly: Implement clinical pathways requiring specific indication before ANCA ordering
Over-interpreting low-positive results: Consider clinical context and confirm with alternative methods
Treating ANCA positivity rather than disease: Clinical and histological correlation mandatory
Assuming negative ANCA excludes disease: Biopsy remains essential in appropriate clinical settings
Using ANCA for disease monitoring: Clinical assessment superior to serial antibody measurement

Practical Algorithm

Clinical suspicion for AAV
↓
High pretest probability (≥2 organs, compatible features)?
↓
Yes → Order PR3-ANCA and MPO-ANCA
↓
Positive → Proceed with tissue biopsy when feasible + appropriate specialist referral
Negative → Reconsider diagnosis OR proceed with biopsy if high clinical suspicion

Future Directions

Emerging technologies promise improved diagnostic accuracy:

Point-of-care testing for rapid diagnosis
Improved standardization of immunoassay calibration
Novel biomarkers (anti-lysosome-associated membrane protein 2, anti-plasminogen antibodies)
Proteomics and epitope mapping for precision diagnosis

Conclusion

ANCA testing represents a powerful diagnostic tool when applied judiciously in appropriate clinical contexts. The key principles for internal medicine practitioners include:

Test only when clinical features suggest AAV
Understand that positive predictive value depends on pre-test probability
Use antigen-specific assays (PR3-ANCA, MPO-ANCA) as primary tests
Never treat antibodies—treat patients with confirmed disease
Negative ANCA does not exclude AAV when clinical suspicion is high
Tissue diagnosis remains the gold standard when feasible

By adhering to these principles, clinicians can maximize the diagnostic utility of ANCA testing while minimizing false-positive results and associated patient harm.

Key Take-Home Points

ANCA testing should be reserved for patients with clinical features suggesting small-vessel vasculitis
Modern antigen-specific immunoassays (PR3-ANCA, MPO-ANCA) are preferred over pattern-based IIF
Positive results require clinical and ideally histopathological correlation
Negative ANCA occurs in 10-20% of true AAV cases
Serial ANCA monitoring does not reliably predict relapse
Tissue biopsy remains essential for definitive diagnosis

Search This Blog

Internal medicine

ANCA Testing in Clinical Practice when where and in whom and by what method