ANCA nelle vasculiti (AAV) Dott.ssa Maria Parmeggiani Azienda Ospedaliera Arcispedale Santa Maria Nuova IRCCS Reggio Emilia

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1 ANCA nelle vasculiti (AAV) Dott.ssa Maria Parmeggiani Azienda Ospedaliera Arcispedale Santa Maria Nuova IRCCS Reggio Emilia RHEU SIM-W-10/2017

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3 Systemic Vasculitides (Chapel Hill - Classification) Large vessel vasculitis Giant cell (temporal) arteritis Takayasu s arteritis Medium-sized vessel vasculitis Polyarteritis (= panarteritis) nodosa Kawasaki s disease Small vessel vasculitis Wegener s granulomatosis Churg-Strauss syndrome Mikroscopic polyangiitis Henoch-Schönlein purpura Essential cryoglobulinaemic vasculitis cutaneous leukocytoclastic angiitis } ANCA-associated vasculitides

4 Vasculite ANCA associata Malattia autoimmune sistemica a causa sconosciuta che colpisce i piccoli vasi Malattia rara Nessuna prevalenza di genere Età media 50 anni

5 Polimorfonucleati (PMN) Risposta immune precoce (infiltrato cellulare) Vasta scelta di killer (proteine a potere battericida, enzimi proteolitici ) Alcuni di questi diventano target di risposte autoimmuni in alcune malattie infiammatorie

6 ANCA Gruppo eterogeneo di anticorpi con specificità e correlazioni cliniche diverse: PR3 e MPO sono da più di 25 anni i target più importanti per la produzione di ANCA e clinicamente più rilevanti per la diagnosi delle VASCULITI SISTEMICHE

7 Proteinasi 3 PR3 is a linear polypeptide containing 228 amino acids, PM kd, encoded by a gene on chromosome 19, close to the elastase and azurocidin sites. ANCA reactivity depends on the native conformation of the protein. The crystal structure was published in 1996 by Fuginaga et al. The protein is progressively expressed from the early myelocyte maturation stage of bone marrow development The ability of IgG PR3-ANCA to bind to and inhibit PR3 enzyme activity seems to depend on the stage at which serum was taken from the patient, since inhibition is preferentially seen during stages of active WG Allan Wiik Mod Reumatol, 2009

8 Mieloperossidasi The MPO-encoding gene is localized on chromosome 17 Special precautions need to be taken after coating MPO onto ELISA plates to avoid altering the structure of the MPO by exposing it to light and thus the reactivity with native MPO For unknown reasons, MPO-ANCA may produce a C-ANCA pattern in some cases Allan Wiik Mod Reumatol, 2009

9 Antigeni minori Sono nei granuli primari (BPI, Azurocidina, catepsina G, elastasi), nei granuli secondari (Lysozima e lattoferrina) Target in altre malattie infiammatorie (IBD), connettiviti, infezioni. ANCA rari in vasculiti sistemiche, possibili in vasculiti indotte da farmaci Producono il pattern di fluorescenza chiamato atipico (aanca)

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11 Vasculitis PR3-ANCA (%) MPO-ANCA (%) Wegener's Granulomatosis a Microscopic Polyangiitis Churg-Strauss Syndrome Renal-limited Vasculitis Drug-induced Vasculitis b Reports from Europe and the United States on ANCA in different forms of AAV vary considerably. Thus PR3-ANCA has been reported to be present in around 40% to 95% of patients with WG and in 9% to 30% of patients with CSS. The reason for these discrepancies may rely on the selected cohorts studied, disease extent and activity at the time of serum sampling, the criteria used for setting the diagnoses, and the way positive cutoff values for the assays used have been set. About 75% of all AAV are positive to at least one of the parameters Wiik AS. Rheum Dis Clin N Am 2010;36:

12 Metodi per la determinazione degli ANCA Immunoflorescenza indiretta (IFA) Immunoenzimatica Diretta (prima generazione) A cattura (seconda generazione) Anchor (terza generazione) Altri

13 Am J Clin Pathol Apr;111(4): International Consensus Statement on Testing and Reporting of Antineutrophil Cytoplasmic Antibodies (ANCA) Savige J1, Gillis D, Benson E, Davies D, Esnault V, Falk RJ, Hagen EC, Jayne D, Jennette JC, Paspaliaris B, Pollock W, Pusey C, Savage CO, Silvestrini R, van der Woude F, Wieslander J, Wiik A. Antineutrophil cytoplasmic antibody (ANCA) tests are used to diagnose and monitor inflammatory activity in the primary systemic small vessel vasculitides. ANCA is best demonstrated in these diseases by using a combination of indirect immunofluorescence (IIF) of normal peripheral blood neutrophils and enzyme-linked immunosorbent assays (ELISAs) that detect ANCA specific for proteinase 3 (PR3) or myeloperoxidase (MPO). For ANCA testing in "new" patients, IIF must be performed on all serum samples. Serum samples containing ANCA, any other cytoplasmic fluorescence, or an antinuclear antibody (ANA) that results in homogeneous or peripheral nuclear fluorescence then should be tested in ELISAs for PR3-ANCA and MPO-ANCA. Optimally, ELISAs for PR3-ANCA and MPO-ANCA should be performed on all serum samples.

14 The International Consensus Statement advocates screening by IIF and confirmation of IIF positivity in PR3-ANCA and MPO-ANCA ELISAs. There are no published studies comparing this protocol with screening by ELISA and confirmation of the results by IIF. Am J Clin Pathol 2003;120:

15 IFA Primo step per la determinazione degli ANCA (semiquantitativo) PMF fissati su vetrini

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19 IFA Diluizione di screening dei sieri: 1/20 (1/40) Antisiero polivalente (IgG, IgA, IgM) come tracciante fluorescente In concomitanza ad ANCA positivo eseguire sempre ANA (HEp-2). ANA pos non esclude P- ANCA 5% dei campioni sono positivi solo con ELISA (eseguire sempre anche PR3-ANCA e MPO- ANCA) In accordo con le raccomandazioni del consensus internazionale 1999

20 IFA IFA pattern è di non facile interpretazione (lettura dei vetrini da parte di personale esperto) Richiede alto consumo di tempo/uomo L uso di entrambi i fissativi (+metanolo) è di aiuto alla corretta interpretazione dei pattern lfa non è altamente specifica per PR3 o MPO (aanca) Cautela nell interpretazione dei pattern solo IFA positivi

21 Lettura IFA Automatizzata Recente introduzione di sistemi di acquisizione, conservazione e interpretazione digitale ad alta risoluzione delle immagini: Facilita lo screening (routine ad alta numerosità) Supporto nell interpretazione (Pos vs Neg, second opinion) > standardizzazione (intensità del segnale) Necessaria ancora l interpretazione finale del risultato da parte di personale esperto.

22 Tecniche immunoenzimatiche Secondo step (identificazione della Ag) Quantificazione del titolo anticorpale non lettore dipendente (IFA) Possibilità di automazione Vede solo le Ag coattate nel pozzetto Diretta A cattura Anchor

23 Ag Elisa Devono essere molecole conformazionalmente intatte quindi: Rigorose tecniche di purificazione Condizioni stringenti di coating Possono essere: Native Ricombinanti

24 The most important factor in such techniques is the use of strictly purified native antigens that are conformationally well preserved or recombinant antigens that can replace these native antigens without loss of reactivity with ANCAs. Further details of ANCA testing are available in the vast existing literature. 2 Wiik AS. Rheum Dis Clin N Am 2010;36:

25 Il limite di sensibilità è dovuto al mascheramento degli epitopi dell Ag che non si rendono completamente disponibili al legame con l Ab. Elisa diretta ( 1 generazione)

26 Elisa a cattura (2 generazione) L Ab di cattura rende più disponibile l AG aumentando la sensibilità del test

27 Results: In patients histologically and clinically known as WG, the detection of ANCA by IFT varied between 52 and 83% among the participating centres. PR3-ANCA positivity with the different ELISAs ranged from 53 to 80% in direct ELISA and from 72 to 76% in capture ELISA. While most capture ELISAs successfully detected PR3-ANCA, there were significant differences between IFT and direct ELISA results between laboratories. ROC curve analysis demonstrated that in five of six laboratories the overall diagnostic performance of capture ELISA was superior to IFT and direct ELISA, respectively. Conclusion: Capture ELISA is a highly sensitive assay for detection of PR3-ANCA in WG and should be used in conjunction with compatible clinical picture and histological evidence.

28 Anchor (3 generazione) The new assay uses the same native human purified PR3, but an indirect coating to the polystyrene Well using a spacer between the polystyrene Well and the antigen, which presents the antigen better. This technology is also called anchor.

29 A novel high sensitivity ELISA for detection of antineutrophil cytoplasm antibodies against proteinase-3. Hellmich B, Csernok E, Fredenhagen G, Gross WL. RESULTS: In prospectively analysed consecutive patients, anchor ELISA showed the highest sensitivity for a diagnosis of WG of 96.0% (95% CI: ), followed by IFT 92.0% ( ), capture ELISA 72.0 ( ) and direct ELISA 60.0 ( ). Specificity was high for all methods and ranged from 98.5 ( ) to 95.5% ( ). Receiver operating characteristics curve analysis revealed that the overall diagnostic performance of the anchor ELISA was significantly superior compared to the direct ELISA and the capture ELISA in patients with generalized WG, and also compared to IFT and immunoblotting in patients with localised WG. CONCLUSION: Anchor ELISA is a novel highly sensitive and specific method for the detection of PR3-ANCA in patients with WG, which may replace the need for a combined analysis with IFT and ELISA in the future. Clin Exp Rheumatol Jan-Feb;25(1 Suppl 44):S1-5.

30 Standard Necessità di Standardizzazione dei dosaggi usati per la determinazione/quantificazione degli ANCA attraverso un riferimento internazionale che permetta di fornire al clinico dati più omogenei e sempre più significativi dal punto di vista diagnostico

31 APMIS June 2009 (1-184) Special Issue : Proceedings of the 14th International Vasculitis and ANCA Workshop Such reference reagents are now available from CDC in Atlanta (details can be seen on

32 PR3 e MPO Standardizzati: calibrati contro CDC reference sera Risultati espressi in UI/ml

33 The international consensus statement on testing and reporting on ANCA as published in 1999/2003 does need revision. We recommend that both the IIF technique and an antigen-specific test in which the antigen is either captured or anchored on the solid phase are used. When ANCA testing is performed for the diagnosis and/or the prognosis of other nonvasculitic inflammatory disorders, we recommend IIF testing on both ethanol- and formalin-fixed neutrophils.

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36 EULAR recommendations for the management of primary small and medium vessel vasculitis 2. We recommend that anti-neutrophilic cytoplasmic antibody (ANCA) testing (including indirect immunofluorescence and ELISA) should be performed in the appropriate clinical context (level of evidence 1A, grade of recommendation A) ANCA testing should be performed by indirect immunofluorescence to detect the labelling characteristic (cytoplasmic or perinuclear). The international consensus statement on testing for ANCA recommends testing all serum samples positive for ANCA by immunofluorescence for proteinase 3 (PR3) and myeloperoxidase (MPO). A positive test for cytoplasmic (C) ANCA targeted to PR3, or perinuclear (P) ANCA against MPO has a high sensitivity and specificity for the diagnosis of ANCAassociated vasculitis. We stress that the absence of a positive test does not rule out a diagnosis; and patients with less severe disease, especially those with isolated granulomatous disease of the upper or lower respiratory tract, may not have a positive ANCA. ANCA testing should be performed in accredited laboratories that participate in external quality control programmes and undergo regular review of laboratory management and staff performing the assays. Ann Rheum Dis 2009;68: doi: /ard

37 Monitoraggio Recently published guidelines by the British Society for Rheumatology recommend therapy for at least 24 months. Early cessation of therapy is associated with an increased risk of relapse. The role of serial ANCA testing to guide therapy is controversial. Some studies have shown that patients in whom the ANCA titres persist, rise fourfold or become positive have a higher incidence of relapse, while other studies have not shown this association. Ann Rheum Dis 2009;68: doi: /ard

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39 Relapsing disease in granulomatosis with polyangiitis (Wegener s) and microscopic polyangiitis Authors John H Stone, MD, MPH Ronald J Falk, MD Section Editors Richard J Glassock, MD, MACP Gerald B Appel, MD Deputy Editor John P Forman, MD, MSc Si raccomanda il follow-up più ravvicinato per i pazienti con titoli di ANCA in aumento per monitorare la comparsa di segni clinici Non si consiglia la ripresa della terapia sulla sola base del rialzo degli ANCA.

40 Considerando che : Le vasculiti ANCA associate sono malattie rare Gli ANCA, se richiesti in modo indiscriminato sulla popolazione, danno un grande numero di falsi positivi Al contrario richiesti in pz con alto sospetto di vasculite il n dei falsi positivi si riduce notevolmente Vol 368 July 29, 2006

41 Si conclude che: I risultati vanno interpretati alla luce dei dati clinici. Gli ANCA vanno rischiesti solo quando esiste un forte sospetto clinico Una stretta collaborazione fra clinico e laboratorista è fortemente raccomandata RHEU SIM-W-10/2017