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WIV NIS, 06/06/2015
Lieve Van Hoovels
ANCA BACK TO BASICS
ANCA – Back to basics
International guidelines: revision
Laboratory diagnosis
Role in follow-up
Role in diagnosis
ANCA definition
1. ANCA definition Anti-neutrophil cytoplasmic antibodies
Proteinase 3 Myeloperoxidase BPI
Elastase
Lysozyme
Cathespin G
Lactoferrin
Azurocidin
β-glucuronidase
hLAMP-2
2. Role in diagnosis Chapel Hill Consensus Conference 2012
Jennette et al. Arthritis Rheum 2013; 65: 1-11
MPA GPA
EGPA
2. Role in diagnosis
Jennette et al. Arthritis Rheum 2013; 65: 1-11
ANCA-Associated Vasculitis (AAV)
NCGN MPA GPA EGPA
Renal-limited Systemic manifestations
2. Role in diagnosis
Jennette et al. Arthritis Rheum 2013; 65: 1-11
ANCA-Associated Vasculitis (AAV)
NCGN MPA GPA EGPA
Renal-limited Systemic manifestations
Abscent Granulomatous airway lesions
Jennette et al. Arthritis Rheum 2013; 65: 1-11
NCGN MPA GPA EGPA
Renal-limited Systemic manifestations
Abscent
Asthma Eosinophilia Abscent
Granulomatous airway lesions
ANCA-Associated Vasculitis (AAV)
2. Role in diagnosis
Classification criteria idiopathic vasculitis
2. Role in diagnosis
• Histopathology of the lesions 1,2
• Size of the vessles involved 1,2
• Clinical symptoms 1
• ANCA
1 American College of rheumatology
2 Chapel Hill Consensus Conference
Fries et al. Arthritis Rheum 1994; 33: 1135-1136
Jennette et al. Arthritis Rheum 1994; 37: 187-192
Classification criteria idiopathic vasculitis
2. Role in diagnosis
• Histopathology of the lesions 1,2,3
• Size of the vessles involved 1,2,3
• Clinical symptoms 1,3
• ANCA 3
1 American College of rheumatology
2 Chapel Hill Consensus Conference
3 European Medicines Agency
Fries et al. Arthritis Rheum 1994; 33: 1135-1136
Jennette et al. Arthritis Rheum 1994; 37: 187-192
Watts et al. Ann Rheum Dis 2007; 66: 222-227
PR3-ANCA MPO-ANCA Negative
NCGN § 20% 70% 10%
MPA 40% 50% 10%
GPA 75% 20% 5%
EGPA * 5% 40% 55%
ANCA-Associated Vasculitis (AAV)
2. Role in diagnosis
§ 90% hLAMP-2 positive
* EAPG with GN is > 75% ANCA positive
Jennette et al. Annu Rev Pathol Mech Dis 2013; 8: 139-160
Cohen Tervaert, Damoiseaux. Clin Rev Allergy Immunol 2012; 43: 211-219; Lyons et al. NEJM 2012; 367: 214-223
“Genetics, clinical manifestations and response to therapy are more related to ANCA serotype than to clinical subtype”
2. Role in diagnosis ANCA-Associated Vasculitis (AAV)
Genetics
Lyons et al. NEJM 2012; 367: 214-223
HLA-DP SERPINA1 PRTN3
HLA-DQ
2. Role in diagnosis
Clinical manifestations
Lionakiet al. Arthritis Rheum 2012; 64: 3452-3462
2. Role in diagnosis
Respons to therapy
Lionaki et al. Arthritis Rheum 2012; 64: 3452-3462
2. Role in diagnosis
Hilhorst et al. J Am Soc Nephrol 2015; 26: ISSN: 1046-6673/2610
“Genetics, clinical manifestations and response to therapy are more related to ANCA serotype than to clinical subtype”
2. Role in diagnosis ANCA-Associated Vasculitis (AAV)
Patient classification based on ANCA subtype? Contra:
ANCA negative AAV
No difference in treatment strategies
IIF Antigens
Rheumatoid arthritis P-ANCA atypical
- lactoferrin - cathepsin G - elastase - lysozyme - unknown
SLE
Sjögren’s syndrome
Juvenile chronic arthritis
Reactive arthritis
Sclerodermia
Antiphospholipid syndrome
Rheumatic disorders
2. Role in diagnosis
Savige. Best Prac Res Clin Rheum 2005; 19: 263-276
AI gastrointestinal disorders IIF Antigens
Ulcerative colitis (60-80%) (pANCA) atypisch
- (PR3) - BPI - lactoferrin - cathepsin G - elastase - lysozyme
Crohn’s disease (10-30%)
Primary sclerosing cholangitis + UC (85-95%)
Primary sclerosing cholangitis – UC (15-20%)
Chronic AI hepatitis (70%)
pANCA + en ASCA - => PPV UC= 82-100% pANCA – en ASCA + => PPV CD= 75-96%
2. Role in diagnosis
Savige. Best Prac Res Clin Rheum 2005; 19: 263-276
Gaffo et al. Rheum Dis Clin North Am 2010; 36: 491-506
Others
Categorie
Vasculitis Cryoglobulinemic Giant cell arteritis Behçet syndrome Polyarteritis nodosa Goodpasture’s syndrome
Infections Mycobacterium tuberculosis HIV Hepatitis C Endocarditis
Others Sarcoïdosis Interstitial lung fibrosis Medication (oa. propylthiouracil) Mucoviscidosis Cocaine (levamisole) …
2. Role in diagnosis
Tomasson et al. Rheumatology 2012; 51: 100-109
3. Role in follow-up ANCA titer ~ clinical relaps
Verstockt et al. Clin Exp Rheum 2015; 33: S72-S76
3. Role in follow-up ANCA titer ~ clinical relaps
Kemna et al. J Am Soc Nephrol 2015; 26: 537-542
3. Role in follow-up
For ANCA positive patients at diagnosis of AAV: • Serial ANCA measurement in AAV patients without renal involvement: limited value
• In patients with renal involvement: clinical relapse is unlikely without rise in ANCA level a rise in ANCA level should warn the clinician for relapse
ANCA titer ~ clinical relaps
Kemna et al. J Am Soc Nephrol 2015; 26: 537-542
1999/2003 International consensus statement on testing and reporting ANCA
Gating policy
- Glomerulonephritis, especially RPGN - Pulmonary hemorrhage, especially pulmonary renal syndrome - Cutaneous vasculitis with systemic features - Multiple lung nodules - Chronic destructive disease of the upper airways - Long-standing sinusitis or otitis - Subglottic tracheal stenosis - Mononeuritis multiplex or other peripheral neuropathy - Retro-orbital mass
*
* When there is no other obvious cause
Clinical manifestations suggestive of AAV
4. Laboratory diagnosis
Savige et al. Am J Clin Pathol 1999; 111: 507-13; Arnold et al. J Clin Pathol 2010; 63: 678-680
1999/2003 International consensus statement on testing and reporting ANCA
Clinical manifestations suggestive of AAV
4. Laboratory diagnosis
IIF ANCA= screening assay PPV IIF ANCA non selected population < 5% PPV IIF ANCA correct clinical context > 90%
Savige et al. Am J Clin Pathol 1999; 111: 507-13 ; Savige et al. Am J Clin Pathol 2003; 120: 312-8
IIF ANCA
4. Laboratory diagnosis
Nomenclature P-ANCA
Perinuclear staining, with or without nuclear extension
ethanol formol
4. Laboratory diagnosis
Savige et al. Am J Clin Pathol 1999; 111: 507-513
Nomenclature P-ANCA
ethanol formol
4. Laboratory diagnosis
ANA interference
Nomenclature C-ANCA
ethanol formol
Granular, cytoplasmatic fluorescence with central or interlobular accentuation
4. Laboratory diagnosis
Savige et al. Am J Clin Pathol 1999; 111: 507-513
Nomenclature atypical ANCA
ethanol ethanol
Atypical ANCA Atypical C-ANCA
4. Laboratory diagnosis
Savige et al. Am J Clin Pathol 1999; 111: 507-513
Combination of cytoplasmic and perinuclear staining
Cytoplasmic without interlobular accentuation
IIF ANCA
P-ANCA C-ANCA
>MPO > PR3 Atypical ANCA Cathepsine G
BPI Lactoferrin Lysozyme
Elastase …
4. Laboratory diagnosis
Savige et al. Am J Clin Pathol 1999; 111: 507-513
min. ethanol fixed min. dilution 1/20
IIF ANCA automatisation
4. Laboratory diagnosis
IIF ANCA automatisation
4. Laboratory diagnosis
Sensitivity ~ visual scoring systems
Csernok et al. Nat Rev Rheumatol 2014; 10: 494-501
1999/2003 International consensus statement on testing and reporting ANCA
Savige et al. Am J Clin Pathol 1999; 111: 507-13 ; Savige et al. Am J Clin Pathol 2003; 120: 312-8
IIF ANCA= screening assay min. ethanol fixed granulocyten min. dilution 1/20
Antigen specific test: MPO/PR3 Specificity ELISA MPO + and IIF P-ANCA= 98,6% Specificity ELISA PR3 + and IIF C-ANCA= 87-99%
4. Laboratory diagnosis
Clinical manifestations suggestive of AAV
1999/2003 International consensus statement on testing and reporting ANCA
Savige et al. Am J Clin Pathol 1999; 111: 507-13 ; Savige et al. Am J Clin Pathol 2003; 120: 312-8
Antigen specific test: MPO/PR3 Specificity ELISA MPO + and IIF P-ANCA= 98,6% Specificity ELISA PR3 + and IIF C-ANCA= 87-99% = depends on ELISA test
Trevisin et al. Immunopathology 2008; 129: 42-53
4. Laboratory diagnosis
IIF ANCA= screening assay min. ethanol fixed granulocyten min. dilution 1/20
Clinical manifestations suggestive of AAV
4. Laboratory diagnosis
Dot-blot
Line-blot
Bead-based multiplex testing
CytoBead technology
CLIA
FEIA
Csernok et al. Nat Rev Rheumatol 2014; 10: 494-501
4. Laboratory diagnosis
1st generation 2nd generation 3rd generation
Direct ELISA Capture ELISA Anchor ELISA
Holle et al. Clin Exp Rheumatol 2012; 30: S66-69; Csernok et al. Nat Rev Rheumatol 2014; 10: 494-501
Capture and anchor ELISAs are superior to direct ELISAs
Max. sensitivity = IIF + ELISA
Poor interassay standardization (even if expressed in IU)
4. Laboratory diagnosis
4. Laboratory diagnosis ANCA reporting
• Minimum: ANCA IIF pattern MPO/PR3 quantification arbitrary units or IU (cut-off)
• LR: Test dependent (standardization!) Have to be determined locally
Vermeersch et al. Clin Chem 2009; 55: 1886-88
Savige et al. Am J Clin Pathol 1999; 111: 507-13 ; Savige et al. Am J Clin Pathol 2003; 120: 312-8
4. Laboratory diagnosis
IIF ANCA= screening assay
Antigen specific test: MPO/PR3
Clinical manifestations suggestive of AAV
1999/2003 International consensus statement on testing and reporting ANCA
Histology
Necrotizing vasculitis
Pauci-immuun
Revision necessary
5. International guidelines
• Expert opinion vs. evidence based guidelines
Revision necessary
5. International guidelines
• Expert opinion vs. evidence based guidelines
• Test algorithm : new generation ELISA ↔ IIF
• Necessity of IIF formol
Revision necessary
5. International guidelines
Van der Molen et al. Ned Tijdschr Klin Chem Labgeneesk 2014; 39: 19-24
Conclusions: A strategy based on screening for ANCA with ELISA or FEIA (without prior IIF) is a valuable alternative to screening with IIF and confirming with ELISA or FEIA.
Ongoing multicentre international study
Untill revision: IIF as screening assay is ‘the best we have’
Vermeersch et al. Clin Chim Acta 2008; 397: 77-81
Revision necessary
5. International guidelines
• Expert opinion vs. evidence based guidelines
• Test algorithm : new generation ELISA ↔ IIF
• Necessity of IIF formol
• Role of new antigens (hLAMP-2)
• Reporting of LR
ANCAs valuable serological marker for diagnosis of AAV
International ANCA standardization and evidence based revision of international guidelines is necessary
Untill revision of international guidelines: screening by IIF
For positive IIF: quantitative PR3/MPO ELISA
ANCA titer follow up: restricted use
6. Conclusion